create a hypothesis related to whether plants have genetic material

The Early History of Genetics

No-one can say why the same peculiarity in different individuals…is sometimes inherited and sometimes not so: why the child often reverts in certain characters to its grandfather, or other much more remote ancestor; why a peculiarity is often transmitted from one sex to both sexes, or to one sex alone, more commonly but not exclusively to the like sex. —Charles Darwin, On the Origin of Species

Our ancestors started agriculture with a certain sense that traits are inherited from parents to progeny. Centuries of breeding domestic animals and plants showed that useful traits could be accentuated by controlled mating, and as we have discussed in chapter 2, domestication and artificial selection gave rise to most modern crops. However, there was no rational way to predict the outcome of a cross between two parents or understanding how and why certain traits show up while others remain hidden (see figure 5.1). Until the nineteenth century, the prevailing theory was the blending inheritance , which was similar to the mixing of two different color paints: progeny were expected to have traits that were a blend of those of its two parents.

Darwin was puzzled about the mechanism of heredity and how the substance that carries parental traits into offspring could possess two seemingly contradictory qualities: its flexibility to give rise to variations and the stability required to maintain the species. He proposed the pangenesis theory to explain the mechanism of heredity. This theory suggests that an organism continually produces a specific type of small organic particle called a gemmule that accumulates in the gonads and then is transmitted to the gametes. When gametes form embryos after fertilization, their gemmules from the parents mix like the paint of two colors. Interestingly, pangenesis was in direct contradiction with Darwin’s theory of (bio)evolution. If the principles of pangenesis were correct, then the natural variations that spontaneously arise within any species would have been an exception among most individuals. Therefore, as a result of mating between an exceptional variant with “normal” individuals (and mating of its progenies with normal members for generation after generation), the variations would gradually disappear. However, Darwin’s extensive research suggested that variations in different birds of the Galápagos Islands were maintained over generations. Eventually, Darwin realized that pangenesis does not explain evolution, and these two hypotheses—(bio)evolution and pangenesis—were contradictory. But he could not solve the riddle.

The greatest challenge to pangenesis came from the German zoologist August Weismann (1834–1914). Weismann experimented with mice to see if cutting off their tails generation after generation would cause any change in their offspring. However, the progeny of those mice had normal tails. He proposed the germplasm theory, which suggests that multicellular organisms consist of two types of cells: (1) germ cells, which are present in the gonads (ovaries and testes) and contain and transmit heritable information from parents to progeny, and (2) somatic cells, which carry out ordinary bodily functions. Thus the gametes (egg cells and sperm cells) produced by the germ cells serve as carriers of heredity information, and other cells of the body do not function as agents of heredity. This hypothesis discredited the ideas of inheritance of acquired characteristics as proposed by Lamarckism and pangenesis. In this way, the distinction between the hardwired inherited traits contained within the germ cells and the soft-wired traits acquired by the somatic cells was established.

However, it remained to be known how much biological contribution the mother and father make to their offspring and what rules govern heredity.

5.1 Gregor Johann Mendel: The Mathematics of Heredity

During Darwin’s lifetime, a clergyman in a monastery in Moravia, Gregor Johann Mendel (1822–84), studied the laws of genetics by crossing pea plants. Moravia, where Mendel (figure 5.2) was born and educated, was the center of plant crossing since the eighteenth century. As discussed in the previous chapter, various pastors took on crossing and hybridization experiments for improving crops and domestic animals. They included crossing and breeding experiments in natural science curricula in the classes they taught at schools. Thus Mendel’s curiosity and his experiments aimed at understanding the laws of heredity come as no surprise.

From 1856 to 1863, Mendel grew pea plants in the five-acre garden of the monastery and conducted about 29,000 crossing experiments. The pea proved to be the ideal plant for investigating heredity. The pea is a selfing plant, but it is also easy to perform artificial pollination on it. It has a short life cycle of two and a half months and can be grown in large numbers with very few resources. Thus it is possible to study several generations of this plant within a short period. Mendel selected seven contrasting characteristics of the pea plant (see figure 5.3) in his experiment: stem length (tall or dwarf), flower color (purple or white), pod shape (inflated or constricted), pod color (green or yellow), seed shape (round or wrinkled), seed color (yellow or green), and flower position (axil or terminal). For many years, Mendel developed purebreds by self-pollination. All the offspring produced by the selfing of purebreds are the same. For example, tall plants give rise to 100 percent tall progeny, and dwarf plants produce 100 percent dwarf progeny.

Subsequently, he used purebreds of peas for generating hybrids and for studying the pattern of inheritance of various characteristics. He observed that the crossing of the purebred purple-flowered plant with the white-flowered plant produced only purple flowers in the hybrid (first hybrid generation, or F1). He repeated these experiments on the seven pairs of pea plants that exhibited contrasting traits and found every time that all the hybrids of the F1 generation showed traits from one parent, while the contrasting trait from the other parent remained hidden. He did not observe the mixing of two contrasting traits. Based on these observations, Mendel proposed the first principle of heredity, known as the law of dominance, postulating that within any (multicellular) organism, at least two factors determine a given trait, of which only the dominant factor appears in the hybrid, and the recessive factor remains hidden or masked by the dominant trait.

In the second step, Mendel crossed F1 hybrids and analyzed their progeny (second hybrid generation, or F2) and found that 75 percent of F2 plants contained purple flowers and 25 percent of F2 plants contained white flowers (see figure 5.4). Thus after skipping the F1 generation, the white flower color reappeared in the F2 generation, but the distribution of dominant (purple) versus recessive (white) traits in their flowers was 3:1 (refer back to figure 5.1). Mendel repeated these experiments on all seven traits and always got a ratio of 3:1 between dominant and recessive traits in F2 progeny.

For the first time in history, it became known that the contrasting parental traits do not mix like paints of two colors; instead, they are maintained and transmitted as independent entities. During the gamete formation, the two factors separate and are distributed equally in the gametes. The fertilization between egg and sperm cells that both contain the recessive factor gives rise to the progeny containing both recessive factors, and thus the corresponding trait reappears. Mendel suggested the second law of heredity as the following:

• The individual has two copies of each factor. Each parent contributes one factor of each trait shown in the offspring.
• Some traits can mask others, but the traits don’t blend. The two members of each pair of factors segregate from each other during gamete formation.

Today, these factors are known as the alleles of a gene, which behave like alternatives to each other. Most recessive alleles cause/indicate a functional deficiency. If one allele of the pair works properly, then it hides the other’s deficiency. If both alleles of a gene are effective/functional or if at least one allele of the pair is functional, then in both cases, we see a dominant trait.

Subsequently, Mendel studied the inheritance of two different traits simultaneously. For example, he crossed homozygous plants producing yellow, smooth peas with plants producing homozygous green, wrinkled peas. As expected, the F1 generation peas were yellow and smooth; only dominant traits showed up. Mendel made crosses between the F1 individuals and found that of the sixteen plants in the F2 generation, nine produced yellow, smooth peas (resembling the dominant ancestor); one produced green, wrinkled peas (resembling the recessive ancestor); three produced smooth, green peas; and three produced yellow, wrinkled peas (see figure 5.5). In the F2 generation, he found a new combination of smooth, green and yellow, wrinkled peas in equal ratio. The overall result suggested that two different traits (such as the color and shape of a pea) are transmitted independently of each other to future generations. Mendel postulated the third law of heredity (a.k.a. the law of independent assortment), suggesting that different traits—like seed shape and seed color—are inherited independently of each other.

Thus Mendel’s experiment revealed the role of sexual reproduction in generating variations within the same species. Today, Mendel’s three laws are known as the fundamental principles of genetics. Mendel was not aware of the physical and chemical properties of the genetic material, but his discoveries led to the first concrete understanding of how the genetic material behaves.

Understanding the behavior of heredity required a mathematical approach rooted in deduction, the fragmentation of big questions into small workable hypotheses, and a good model system for studying heredity. Mendel, a trained meteorologist, had the ability to look at the data and apply math to it. In contrast, Darwin and most biologists of his time implemented the cataloging and description of the morphology of plants and animals. Today, Mendel is the father of genetics, but during his lifetime, his contributions were not valued by his peers. In later years, Mendel did the monastery’s administrative work, and his three principles of genetics were forgotten for thirty-five years.

Mendel’s work was no less important than Darwin’s. In fact, his work explained Darwin’s theory of evolution in concrete terms. Many people now wonder why Mendel’s discoveries were ignored, while Darwin became one of the most popular scientists during his lifetime. Perhaps, to some extent, Darwin benefited from his family background. Both his father and grandfather were well-known doctors, and he graduated from the University of Cambridge. He developed his academic network at an early age while working with John Henslow and then got an opportunity to join the HMS Beagle as a naturalist (a volunteer who was not paid a salary). In contrast, Mendel was born into a peasant family. After his initial education in the village, he became a monk and pursued further education in the monastery. During his youth, he struggled to become a high school teacher and carried out experiments with pea plants in the garden. He did not get much opportunity to discuss theories with other scientists, as he was not a part of academia; none took cognizance of his work. Also, most of his contemporary scientists were naturalists involved in cataloging and describing plant and animal diversity; they lacked mathematical understanding. Therefore, most academics and scientists ignored Mendel’s published papers. We have no way of knowing if Darwin ever knew about Mendel’s research work.

5.2 The Rediscovery of Mendel’s Laws and the Birth of Genetics

In 1900, Hugo de Vries, Carl Correns, and Erich von Tschermak independently rediscovered Mendel’s work. Thus Mendel’s three laws of heredity resurfaced after being buried after thirty-five years, but even then, these principles were not easily accepted. The first few who recognized the importance of Mendel’s laws include Cambridge University biologist William Bateson. Bateson zealously lectured on the laws of heredity in European and American institutions to popularize Mendel’s work and became known as “Mendel’s bulldog.” Bateson established the first genetics laboratory in Cambridge. Bateson proposed the term genetics for a new branch of biology rooted in the Mendelian approach and the study of inheritance and the structure function of genetic material. During this period, Vavilov had joined Bateson’s laboratory and thus became the first geneticist in the USSR. Botanist Wilhelm Johannsen proposed the word gene for the Mendelian units of heredity (factor) and called two different versions of a gene-defining trait alleles , since hybrid plants show only dominant traits and appear similar to purebreds even though their genetic configuration is different. Hence in 1909, Johannsen proposed the terms genotype for the genetic configuration and phenotype for the manifested trait—that is, its outward appearance. Hugo de Vries proposed the term mutant for organisms with rare traits (whose numbers are less than 1 percent in a population or have a defect). In this way, gradually, terminology and vocabulary for genetics increased, and slowly it was established as a subdiscipline of biology.

5.3 Exceptions and Extension of Mendelian Genetics

In the early twentieth century, many scientists repeated Mendel’s experiments and began analyzing their crossing experiments using a framework provided by Mendelian genetics. Often, the laws of heredity successfully explained their results, but less frequently, exceptions were encountered, which added new dimensions to and knowledge of heredity. Here we discuss several of such examples and how they contributed to furthering knowledge in the field.

5.3.1 Partial or Incomplete Dominance and Codominance

One exception to Mendel’s first law is partial dominance. When scientists crossed the white- and red-flower varieties of the “four o’clock,” they found pink flowers in the F1 hybrids. Thus in this case, the presence of a dominant allele did not completely mask the recessive allele, and the phenotype of the heterozygous F1 hybrid differs from its homozygous dominant parent. Furthermore, the selfed progeny of heterozygous F1 hybrid segregated in a ratio of 1 red: 1 white: 2 pink. However, the pink flower in the hybrid is not a result of the mixing of red and white but is due to the diminished quantity of red pigment. In fact, both allele coding for red and white are transmitted from one generation to another as discrete factors, but the red flowers are produced by plants where both factors are functional. When only one of the two works, only half the pigment is formed, and the pink color is seen.

Another exception is codominance, where both alleles (factors) determining a trait are functional. For example, in people with AB blood group, both A and B alleles are equally active and produce A and B antigens. A third allele, i, is also found in some people that do not produce any antigen due to mutations. Therefore, people with AA or Ai have blood group A; those with BB or Bi have blood group B, and others with ii have blood group O (no antigen).

Mendel proposed the basic rules of genetics based on the assumption that there are two alleles (dimorphic) of any gene. While it stands true for an individual, multiple alleles for the same trait exist within any population of a plant or animal. Many genes have several common alleles (they are polymorphic), which may show a complicated pattern of dominance and can be placed in a hierarchy. The interactions of these various alleles cannot be understood without concerted efforts of crossing and establishing a dominance series. For example, if we look carefully at the whole lentil grain, there are many patterns of small or big dots on the light-colored surface. If a lentil with a clear surface is crossed with one with a dotted surface, then the F1 hybrid shows a dotted trait. However, a crossing of the dotted lentil with spotted lentil plants produces an F1 hybrid showing a spotted pattern (here the dotted trait behaves recessively). Based on the results from such crossings, the dominance hierarchy of different alleles can be prepared, and then the results can be explained using Mendel’s laws. But without understanding this hierarchy, the results cannot be explained. It is noteworthy to mention that in such cases, the dominance relations affect only the correspondence between genotype and phenotype; however, the alleles still segregate and unite randomly and follow the Mendelian pattern. The difference lies in the complex relationship between genotype and phenotype.

5.3.2 Lethal Mutations

There are many essential genes found in a living organism, known as the housekeeping genes , that are required for that organism’s survival. Some of these genes are active in all cells of an organism, whereas others act specifically in a particular type of cell, tissue, or organ. Mutations in housekeeping genes can be lethal. Sometimes heterozygotes exhibit a phenotype or a disease, which leads to recessive homozygotes dying (and thus a progeny class is eliminated). In these cases, we do not find the segregation of F2 progeny according to Mendel’s laws.

In contrast, the gametes (egg or sperm cells) contain only one allele, and thus the gametes carrying a nonfunctional allele of housekeeping genes are destroyed. As a result, only the gametes carrying the functional alleles are transmitted to the next generation. In such cases, the results of the crossing do not fit into the Mendelian hypothesis, and only the functioning allele is seen in the progeny. However, close observation can reveal decreased pollination and lower seed formation. In other instances, after successful pollination and fertilization, the embryo does not develop because both alleles of a housekeeping gene required for embryonic development are mutants. Similarly, some plants die after seed germination due to a lack of functional alleles (e.g., albino mutants that lack photosynthesis capacity). There are also instances where both alleles are necessary for the normal development of an organism. For instance, in healthy individuals, both alleles of the fibroblast growth receptor gene function, but people with a mutant allele suffer from the most common form of dwarfism (known as achondroplasia, with a normal-length body but shortened limbs).

5.3.3 Pleiotropy

Sometimes a single gene affects several unrelated phenotypic traits in the same organism, and this phenomenon is known as pleiotropy. For example, specific mutations in the hemoglobin B gene cause changes in the shape of red blood cells (from round to sickle shaped) that causes sickle cell anemia (termed this to differentiate it from dietary deficiency and other causes of anemia). Due to their shape, these cells obstruct the smooth flow of blood and have a short life compared to normal cells. As a result, people carrying the mutant gene become anemic. In addition to anemia, an enlarged spleen, muscle and heart pains, a weak immune system, resistance to malaria, and early death are associated with the mutations in the hemoglobin B gene.

5.3.4 Penetrance or Expressivity in Phenotype

Researchers also noticed that certain traits are expressed fully only under a certain environment. Thus two new terms were added:

• Penetrance is whether a trait is expressed or not.
• Expressivity is the degree to which a trait is expressed (fully or partially).

We see many such examples in our day-to-day lives. For instance, genetically identical hydrangeas growing in soils of different acidity (different environments) produce flowers of a different color. Many houseplants change color if their exposure to light changes or the temperature changes. We also find different pigmentation in cats, dogs, and other animals, where the colder body parts are darker while the warm body parts are lighter.

5.3.5 Epistasis

In many instances, a single phenotype is controlled by the interaction of two or more genes. This phenomenon is known as epistasis. The epistatic genes may code for proteins involved in different steps of a biosynthetic pathway or may have an additive function. In such cases, we see alterations in F2 segregation ratios. Examples of such interactions can explain the various sizes and shapes of squashes, the different skin colors of onions, or the diversity in flower colors of many plants.

Unlike animals, plants contain many duplicate genes, and some of these have redundant and/or additive functions. Thus when both alleles of a gene are mutated and are nonfunctional, we see no phenotype due to the presence of a duplicated gene that functions properly. In such cases, a phenotype is only visible if both genes (all four alleles) become nonfunctional. In contrast, the opposite is also true: sometimes, a phenotype is only visible when two or more duplicate genes are simultaneously functional in the organism. For example, for zucchini, two genes (four alleles in total) determine the fruit size. If both genes work or at least one allele of both works, then a very small disk-shaped fruit is produced. If only one gene (or one of its alleles) is functional, then a big, rounded fruit is produced. If both genes (all four alleles) become nonfunctional, then a long fruit is produced. Here crossing between the long and flat breeds of zucchini will give rise to an F1 hybrid bearing round fruits. And the selfing of F1 progeny will result in the segregation of this trait in F2 progeny as 9 disk shaped (A_B_):6 round (A_bb or aaB_):and 1 long (aabb).

5.3.6 Genetic Linkage and Chromosome Theory

Mendel was extremely fortunate with the plant model he selected for deciphering the basic laws of heredity. He chose seven traits of pea plants that gave a consistent pattern of dominance and recessiveness between two contrasting alleles. Many of Mendel’s contemporaries could not find such a great experimental model or traits, and due to many deviations, their efforts were derailed. Even Bateson, the biggest supporter of Mendelian genetics, could not find an easy path and got entangled with unexpected results not explainable by Mendel’s laws. In 1905, Bateson and Reginald C. Punnett made a dihybrid cross between purebreds of sweet pea plants, where the dominant parent produced purple flowers and long pollen grains (PP LL), and the recessive parent produced red flowers and round pollens (pp ll). Bateson and Punnett observed that the F1 hybrid had purple flowers and long pollen grains (Pp Ll), as expected.

However, the F2 population generated by the selfing of F1 hybrids showed a skewed ratio that did not match what was expected based on Mendel’s second law of independent assortment of two independent traits. The data clearly showed that the characteristics of pollen size and flower color did not transmit independent of each other from parents to offspring. The expected ratio of the dihybrid cross was 9:3:3:1, but the results did not fit this pattern. As shown in table 5.1, the gene coding for flower color and pollen shape appears to be linked to some extent: the two phenotypic classes (purple flower + long pollen and red flower + round pollen) are larger than expected, and the number of recombinants is less than expected (1) . Bateson was puzzled and could not explain these results in terms of epistasis either. However, Bateson and Punnett proposed that the F1 hybrid produced more P L and p l gametes due to some sort of physical coupling between the dominant alleles P and L and between the recessive alleles p and l (1) .

Table 5.1: The results of the di-hybrid cross in sweet peas observed by Bateson and Punnett)

American scientist Thomas Hunt Morgan chose the fruit fly (Drosophila melanogaster) to study genetics. The fruit fly has several advantages for this study, including a short life cycle (forty to fifty days), easy propagation within milk bottles, and being amenable to the crossing. Also, with the help of a microscope, changes in the structure of the fruit fly can be easily identified. Thus it was a cheap and accessible model for the study of genetics and since then has served as an important model organism to study eukaryotic genetics. When Morgan crossed a female fly containing a purple eye (pr) and a normal wing (vg; both dominant traits: pr/pr.vg/vg) with a male fly containing a red eye (pr+) and the vestigial wing (vg+; both recessive traits: pr+/pr+.vg+/vg+), he got an F1 hybrid with the purple eye and the normal wing (pr/pr+.vg/vg+), as expected. Then he made a backcross between the F1 hybrid female (pr/pr+.vg/vg+) and the recessive male parent (pr+/pr+.vg+/vg+) (2) . In these experiments, the recessive male can only produce one type of gamete, one that carries recessive red eye and vestigial wing traits. Thus the alleles contributed by the F1 female specify the F2 progeny. If both traits are sorted independently of each other, the expected ratio of these traits in the F2 progeny would be 1:1:1:1, representing both parental genotypes and two new recombinant classes.

As shown in table 5.2, he found unexpected results: a much larger number of offspring with purple eyes and normal wings or red eyes and vestigial wings was obtained in the F2 progeny. Like Bateson, he also observed a linkage between two independent traits: the linkage was not absolute, and the two types of recombinant class of progeny were in a similar proportion.

Table 5.2: The results of fruit fly backcross observed by Morgon

The progress in the field of cytology [1] helped us understand the phenomenon of linkage between genes that were observed by Bateson and Morgan independently. From the seventeenth century onward, scientists studied the structure of different organisms through microscopes and understood that organisms are made of one or more cells. The simplest forms of life, such as bacteria, are made of only one cell (unicellular), and various animals and plants are made of many cells (multicellular). Therefore, the unit of the structure and the function of life are the cell. In the late nineteenth century, high-resolution microscopes became available, which allowed the identification of various subcellular structures. It was natural that scientists started probing the location of genetic material within the cell. In 1879, Walter Fleming noticed a fine, threadlike structure in the center of salamander cells that shrunk and assumed a clear shape during cell division that was named chromosomes. It was clear from a series of studies that the number of chromosomes is constant for a species, and chromosome numbers vary between species. Typically, multicellular organisms have two copies of each chromosome (two sets of chromosomes) within the somatic cells, and the mother and father each contribute one set of chromosomes to their offspring. For example, human somatic cells have 46 chromosomes (23 pairs; diploid). In contrast, one set of chromosomes (23 chromosomes) is present in the gametes (eggs and sperm cells).

At the time of cell division, chromosomes first double in number and then divide equally into two daughter cells. In this way, two cells are made from one mother cell, and both daughter cells have the same number of chromosomes as their mother cell. In contrast, the cell division of a mother germ cell gives rise to gametes that only contain one set of chromosomes (haploid) and thus only one copy of each chromosome. During sexual reproduction, the fusion of male and female gametes produces a diploid embryo, and the two sets of chromosomes are restored in progeny. The behavior of chromosomes during the cell division of the germ cell grossly reminds us of Mendel’s laws.

By the beginning of the twentieth century, it was recognized that chromosomes play a role in transmitting genetic traits from parents to offspring. Incidentally, the seven traits chosen by Mendel for his crossing experiments are encoded by seven genes that reside in seven different chromosomes, and thus he did not observe linkage. Therefore, Mendel did not see any contradiction in his experiments. However, besides gene linkage, Bateson and Morgan made another important observation that the linkage between two genes was not absolute and that a few recombinants are present in the F2 population. Morgan hypothesized that during germ cell division (meiosis), homozygous chromosomes interchange small regions before being divided into daughter cells, and thus the linkage between two genes located on the same chromosome occurs, resulting in the formation of recombinants (2) . Thus based on the findings of cell biology and results from his laboratory, Morgan provided an explanation of the linkage between genes (2) . He proposed the following:

• Genes occur in a linear order on chromosomes, and their location on chromosomes is fixed.
• Genes on the same chromosome are linked together and do not exhibit an independent assortment.
• Genes can be exchanged between chromosomes during meiosis.
• The closer genes are located on a chromosome, the less likely they will separate and recombine in meiosis.
• Chromosomes undergo segregation and independent assortment. Therefore, genes on different chromosomes follow the law of independent assortment.

5.4 Chromosome Mapping

Several experiments in Morgan’s laboratory revealed that the closer the two genes are, the stronger their association. As the distance between the two genes increases, the probability of crossing over increases, and the number of newly combined progeny (recombinant) increases in the same proportion. Thus the distance between two genes can be estimated by the number of recombinants present among the total progeny. By this method, the distance between genes can be measured in centimorgan (cM). Thus a distance of 1 cM between the two genes meant that in such cases, 1 percent of F2 progeny (of dihybrid cross) would be recombinant. Morgan’s laboratory generated a vast amount of fruit fly crossing data. Eventually, one of his students, Alfred Henry Sturtevant, analyzed this data and constructed the first genetic map of fruit flies’ chromosomes one by one. Subsequently, this method was applied to many other animals and plants to generate their chromosome maps.

Morgan did not directly observe the reciprocal exchange of segments between homologous chromosomes, but the assumptions he made about crossing over during meiosis proved to be correct. The crossing over (see figure 5.6) was experimentally confirmed almost two decades later by Barbara McClintock. But by then, scientists had constructed genetic maps of many organisms by following Morgan’s lead. Genetic maps proved to be very useful for breeders. Genes whose phenotypes are visible served as a marker for selecting other nearby/adjacent genes that had no visible phenotype but contributed to important agronomic traits. Similarly, if one desired gene is linked to another undesirable gene, with the help of a genetic map, it became possible to estimate how many crossings will be required to break this linkage.

5.5 Polyploidy

The study of the cells of various animals and plants revealed that the number of chromosomes within a species is fixed. Moreover, most animals are unable to tolerate a slight change in chromosome numbers. Usually, embryos that have lost or gained one or more chromosomes are unable to survive. Unlike animals, plants have a tremendous capacity to harbor multiple sets of chromosomes; this phenomenon is known as polyploidy. Many plant species have two (diploid), three (triploid), four (tetraploid), six (hexaploid), or eight (octoploid) copies of the entire set of chromosomes. Many polyploid crops have come into existence by spontaneous hybridization events between two closely related species naturally. For example, there are 28 chromosomes within emmer wheat ( Triticum dicoccoides ), 14 of which are from diploid einkorn wheat ( Triticum Urartu ; AA genome donor) and 14 from a diploid goat grass related to Aegilops speltoides (the BB-genome donor). Therefore, emmer wheat is a tetraploid (AABB) species. Another natural hybridization event between T. dicoccoides and Aegilops tauschii (the DD-genome donor) gave rise to hexaploid modern bread wheat ( Triticum aestivum ; AABBDD). Thus hexaploid common bread wheat has 42 chromosomes, of which 28 are from the emmer (AABB) and 14 are from A. tauschii .

Often the increased ploidy has a direct effect on the structure of the plant in the form of an increase in the size of the leaf, fruit, or grain—thus it adds to the agronomic value of a crop. Multiplication of chromosomes in emmer and common bread wheat had resulted in an increase in grain size and greater tolerance for adverse environmental conditions. Similarly, as discussed in an earlier chapter, the octoploid ananassa strawberry (8 sets of chromosomes) is much larger than diploid wild strawberry varieties. Sometimes, breeders also create sterile hybrids by crossing two closely related species of different ploidy levels that disrupt the formation of seeds in hybrids. The seedless watermelon (a triploid) is one such example that was made by crossing a tetraploid with a diploid variety.

5.6 Summary

In later decades of the nineteenth century, a tremendous amount of geological and biological data suggested that both the earth and the living species inhabiting it have changed over time; in the process of adapting to new environments, new species emerge from the previously existing species, and some existing species also disappear. Mendel extended the knowledge by postulating three fundamental laws of heredity—which provided a correct theoretical explanation for Darwin’s theory of evolution—to describe the origin of species in the natural world. Many new discoveries in cell biology led to the birth of genetics, a subdiscipline of biology that studies heredity and the physical and structural properties of genetic material. By 1900, cells and chromosomes were sufficiently understood to give Mendel’s abstract ideas physical context. It was discovered that chromosomes contain the genes that code the various traits of an organism and that during germ cell division, the reciprocal exchange of chromosomal segments further adds to genetic diversity within a species. The observation of recombination frequencies was exploited to construct chromosomal maps and decipher the location of genes on various chromosomes and their physical relationship with one another. Chromosomal maps provided breeders with the tools for designing a rational experiment for creating improved varieties of plants and animals.

Bateson, W., & Saunders, E. R. (1902). Experimental studies in the physiology of heredity. In Reports to the Evolution Committee of the Royal Society. Harrison & Sons. ( ↵ Return 1 ) ( ↵ Return 2 )

Morgan, T. H. (1909). What are “factors” in Mendelian explanations? American Breeders Association Reports , 5, 365–68. http://www.esp.org/foundations/genetics/classical/thm-09.pdf ( ↵ Return 1 ) ( ↵ Return 2 ) ( ↵ Return 3 )

Further Readings

Landmarks in the History of Genetics. http://www.dorak.info/genetics/notes01.html

Mendel, J. G. (1865). Versuche über Pflanzenhybriden Verhandlungen des naturforschenden Vereines in Brünn, Bd. IV für das Jahr. Abhandlungen. 3–47 [for English translation, see http://www.mendelweb.org/Mendel.plain.html ].

Mitosis and Meiosis Simulation (video for cell division of two types). https://www.youtube.com/watch?v=zGVBAHAsjJM

Winther, R. (2001). August Weismann on germ-plasm variation. Journal of the History of Biology , 34(3), 517–55. https://doi.org/10.1023/A:1012950826540

• The following are major discoveries in cell biology that played important role in the progress of genetics: 1839: M. J. Schleiden and T Schwann develop the cell theory. 1866: E. H. Haeckel (Häckel) hypothesizes that the nucleus of a cell transmits its hereditary information. 1869: Friedrich Miescher isolates DNA for the first time. 1879: Walter Flemming observes mitosis. 1902: W. S. Sutton and T. Boveri independently propose the chromosome theory of heredity: a full set of chromosomes are needed for normal development; individual chromosomes carry different hereditary determinants; and independent assortments of gene pairs occur during meiosis. ↵

History and Science of Cultivated Plants Copyright © 2021 by Sushma Naithani is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Chapter 18. Mendelian Genetics

Chapter Outline

• 18.1 Mendel’s Experiments
• 18.2 Mendel’s Principles of Inheritance
• 18.3 Exceptions to Mendel’s Principles of Inheritance

Introduction

Genetics is the study of heredity. Johann Gregor Mendel (1822–1884) set the framework for genetics long before chromosomes or genes had been identified, at a time when meiosis was not well understood ( Figure 18.2 ). Mendel selected a simple biological system and conducted methodical, quantitative analyses using large sample sizes. Because of Mendel’s work, the fundamental principles of heredity were revealed. We now know that genes, carried on chromosomes, are the basic functional units of heredity with the capability to be replicated, expressed, or mutated. Today, the postulates put forth by Mendel form the basis of classical, or Mendelian, genetics. Not all genes are transmitted from parents to offspring according to Mendelian genetics, but Mendel’s experiments serve as an excellent starting point for thinking about inheritance.

18.1 | Mendel’s Experiments

Learning Objectives

By the end of this section, you will be able to:

• Describe the scientific reasons for the success of Mendel’s experimental work.
• Describe the expected outcomes of monohybrid crosses involving dominant and recessive alleles.

Johann Gregor Mendel (1822–1884) was a lifelong learner, teacher, scientist, and man of faith. As a young adult, he joined the Augustinian Abbey of St. Thomas in Brno in what is now the Czech Republic. Supported by the monastery, he taught physics, botany, and natural science courses at the secondary and university levels. In 1856, he began a decade-long research pursuit involving inheritance patterns in honeybees and plants, ultimately settling on pea plants as his primary model system . In 1865, Mendel presented the results of his experiments with nearly 30,000 pea plants to the local Natural History Society. He demonstrated that traits are transmitted faithfully from parents to offspring independently of other traits and in dominant and recessive patterns. In 1866, he published his work, Exp eriments in Plant Hybridization [1]  in the proceedings of the Natural History Society of Brünn.

Mendel’s work went virtually unnoticed by the scientific community that believed, incorrectly, that the process of inheritance involved a blending of parental traits that produced an intermediate physical appearance in offspring; this hypothetical process appeared to be correct because of what we know now as continuous variation. Continuous variation results when many genes work together to determine a characteristic, such as human height or eye color. Offspring appear to be a “blend” of their parents’ traits when we look at characteristics that exhibit continuous variation.

Mendel worked with traits that were inherited in distinct classes, such as violet versus white flowers. These traits display discontinuous variation . Mendel’s choice of these kinds of traits allowed him to see that the traits were not blended in the offspring, nor were they absorbed, but rather that they kept their distinctness and could be passed on. In 1868, Mendel became abbot of the monastery and exchanged his scientific pursuits for his pastoral duties. He was not recognized for his extraordinary scientific contributions during his lifetime. In fact, it was not until 1900 that his work was rediscovered, reproduced, and revitalized by scientists on the brink of discovering the chromosomal basis of heredity.

18.1.1 Mendel’s Model System

Mendel’s seminal work was accomplished using the garden pea, Pisum sativum , to study inheritance. This species naturally self-fertilizes, such that pollen encounters ova within individual flowers. The flower petals remain sealed tightly until after pollination, preventing pollination from other plants. The result is highly inbred, or “true-breeding,” pea plants. These are plants that always produce offspring that look like the parent. By experimenting with true-breeding pea plants, Mendel avoided the appearance of unexpected traits in offspring that might occur if the plants were not true breeding. The garden pea also grows to maturity within one season, meaning that several generations could be evaluated over a relatively short time. Finally, large quantities of garden peas could be cultivated simultaneously, allowing Mendel to conclude that his results did not come about simply by chance.

18.1.2 Mendelian Crosses

Mendel performed hybridizations , which involve mating two true-breeding individuals that have different traits. In the pea, this is done by manually transferring pollen from one pea plant to the stigma of another pea plant. In plants, pollen carries the male gametes (sperm) to the stigma, a sticky organ that traps pollen and allows the sperm to move down the pistil to the female gametes (ova) below. To prevent the pea plant that was receiving pollen from self-fertilizing and confounding his results, Mendel painstakingly removed all of the pollen-producing anthers from the plant’s flowers before they had a chance to mature.

Plants used in first-generation crosses were called P , or parental generation, plants ( Figure 18.3 ). Mendel collected the seeds that resulted from each cross and grew them the following season. These offspring were called the F1 , or the first filial ( filial = offspring, daughter or son), generation. Once Mendel examined the characteristics in the F1 generation of plants, he allowed them to self-fertilize. He then collected and grew the seeds from the F1 plants to produce the F2 , or second filial, generation. Mendel’s experiments extended beyond the F2 generation to the F3 and F4 generations, and so on, but it was the ratio of characteristics in the P−F1−F2 generations that were the most intriguing and became the basis for Mendel’s principles.

18.1.3 Garden Pea Characteristics Revealed the Basics of Heredity

In his 1865 publication, Mendel reported the results of his crosses involving seven different characteristics, each with two contrasting traits. A trait is defined as a variation in the physical appearance of a heritable characteristic. The characteristics include: tall vs. short plant height, wrinkled vs. round seeds, green vs. yellow seeds, violet vs. white flowers, etc. ( Table 18.1 ). To fully examine each characteristic, Mendel generated large numbers of F1 and F2 plants, reporting results from 19,959 F2 plants alone.

As an example, let us look at Mendel’s results for the flower color trait. First, Mendel confirmed that he had plants that bred true for white or violet flower color. Regardless of how many generations Mendel examined, all self-crossed offspring of parents with white flowers had white flowers, and all self-crossed offspring of parents with violet flowers had violet flowers. In addition, Mendel confirmed that, other than flower color, the pea plants were physically identical.

Once these validations were complete, Mendel applied pollen from a plant with violet flowers to the stigma of a plant with white flowers. After gathering and sowing the seeds that resulted from this cross, Mendel found that 100 percent of the F1 hybrid generation had violet flowers. Conventional wisdom at that time would have predicted the hybrid flowers to be pale violet or for hybrid plants to have equal numbers of white and violet flowers. In other words, the contrasting parental traits were expected to blend in the offspring. Instead, Mendel’s results demonstrated that the white flower trait in the F1 generation had completely disappeared.

Importantly, Mendel did not stop his experimentation there. He allowed the F1 plants to self-fertilize and found that, of F2- generation plants, 705 had violet flowers and 224 had white flowers. This was a ratio of 3.15 violet flowers per one white flower, or approximately 3:1. When Mendel transferred pollen from a plant with violet flowers to the stigma of a plant with white flowers and vice versa, he obtained about the same ratio regardless of which parent, male or female, contributed which trait. This is called a reciprocal cross —a paired cross in which the respective traits of the male and female in one cross become the respective traits of the female and male in the other cross. For the other six characteristics Mendel examined, the F1 and F2 generations behaved in the same way as they had for flower color. One of the two traits would disappear completely from the F1 generation only to reappear in the F2 generation at a ratio of approximately 3:1 ( Table 18.1 ).

Table 18.1 The Results of Mendel’s Garden Pea Hybridizations

18.2 | Mendel’s Principles of Inheritance

• Describe the three principles of inheritance.
• Explain the relationship between phenotype and genotype.
• Develop a Punnett square to calculate the expected proportions of genotypes and phenotypes in a monohybrid cross.
• Explain the purpose and methods of a test cross.
• Draw and interpret a pedigree.

Mendel generalized the results of his pea-plant experiments into three principles that describe the basis of inheritance in diploid organisms. They are: the principle of segregation, the principle of dominance, and the principle of independent assortment. Together, these principles summarize the basics of classical, or Mendelian, genetics.

18.2.1 The Principle of Segregation

Since the white flower trait reappeared in the F2 generation, Mendel saw that the traits remained separate (not blended) in the plants of the F1 generation. This led to the principle of segregation , which states that individuals have two copies of each trait, and that each parent transmits one of its two copies to its offspring.

We now know that the traits that are passed on are a result of genes that are inherited on chromosomes during meiosis and fertilization. The fact that the genetic factors proposed by Mendel were carried on chromosomes was proposed in 1902 by Walter and Sutton and Theodor Boveri ( Figure 18.4 ) as the Chromosomal Theory of Inheritance .

Different versions of genes are called alleles . Diploid organisms that have two identical alleles of a gene on their two homologous chromosomes are homozygous for that trait. Diploid organisms that have two different alleles of a gene on their two homologous chromosomes are heterozygous for that trait.

The physical basis of the principle of segregation is the first division of meiosis, in which the homologous chromosomes with their different versions of each gene are segregated into daughter nuclei. Since each gamete receives only one homolog of each chromosome, it follows that they receive only one allele for each trait. At fertilization, the zygote receives one of each homologous chromosome, and one of each allele, from each parent.

18.2.2 The Principle of Dominance

Upon compiling his results for many thousands of plants, Mendel concluded that the characteristics could be divided into dominant and recessive traits. Dominant traits are those that are expressed in a hybridization. Recessive traits become latent, or disappear, in the offspring of a hybridization but reappear in the progeny of the hybrid offspring. Thus, the violet-flower trait is dominant and the white-flower trait is recessive.

The principle of dominance states that in a heterozygote, only the dominant allele will be expressed. The recessive allele will remain “latent” but will be transmitted to offspring by the same manner in which the dominant allele is transmitted. The recessive trait will only be expressed by offspring that have two copies of this allele ( Figure 18. 5 ). Individuals with a dominant trait could have either two dominant versions of the trait or one dominant and one recessive version of the trait. Individuals with a recessive trait have two recessive alleles.

In Mendel’s experiments, the principle of dominance explains why the F1 heterozygous offspring were identical to one of the parents, rather than expressing both alleles. For a gene that is expressed in a dominant and recessive pattern, homozygous dominant and heterozygous organisms will look identical. The recessive allele will only be observed in homozygous recessive individuals. Some examples of human dominant and recessive traits are shown in Table 18.2 .

Table 18.2 Examples of dominant and recessive traits in humans.

The principles of segregation and dominance could be deduced by simple crosses that follow only one genetic trait. These crosses are called monohybrid crosses . Before we discuss the principle of independent assortment, let’s look at some tools and terminology used for monohybrid crosses.

18.2.3 Phenotypes and Genotypes

Several conventions exist for referring to genes and alleles. For the purposes of this chapter, we will abbreviate genes using the first letter of the gene’s corresponding dominant trait. For example, green is the dominant trait for pea pod color, so the pod-color gene would be abbreviated as G (note that it is customary to italicize gene designations). Furthermore, we will use uppercase and lowercase letters to represent dominant and recessive alleles, respectively. Therefore, we would refer to the genotype of a homozygous dominant pea plant with green pods as GG , a homozygous recessive pea plant with yellow pods as gg , and a heterozygous pea plant with green pods as Gg .

The two alleles for each given gene in a diploid organism may be expressed and interact to produce physical characteristics. The observable traits expressed by an organism are referred to as its phenotype . An organism’s underlying genetic makeup, which alleles it has, is called its genotype . Mendel’s hybridization experiments demonstrate the difference between phenotype and genotype. When true-breeding plants in which one parent had yellow pods and one had green pods were cross-fertilized, all of the F1 hybrid offspring had green pods. Although the hybrid offspring had the same phenotype as the true-breeding parent with green pods, we know that the genotype of the parent was homozygous dominant ( GG ), while the genotype of the F1 offspring was heterozygous ( Gg ). We know this since the yellow pod allele reappeared in some of the F2 offspring ( gg ).

18.2.4 Using Punnett Squares for Monohybrid Crosses

Punnett squares , devised by the British geneticist Reginald Punnett, can be used to predict the possible outcomes of a genetic cross or mating and their expected frequencies. To demonstrate a monohybrid cross, consider the case of true- breeding pea plants with yellow versus green pea seeds. The dominant seed color is yellow; therefore, the parental genotypes were YY for the plants with yellow seeds and yy for the plants with green seeds, respectively. To prepare a Punnett square, all possible combinations of the parental alleles are listed along the top (for one parent) and side (for the other parent) of a grid, representing their meiotic segregation into haploid gametes. Then the combinations of egg and sperm are made in the boxes in the table to show which alleles are combining. Each box then represents the diploid genotype of a zygote, or fertilized egg, that could result from this mating. Because each possibility is equally likely, genotypic ratios can be determined from a Punnett square. If the pattern of inheritance (dominant or recessive) is known, the phenotypic ratios can be inferred as well. For a monohybrid cross of two true-breeding parents, each parent contributes one type of allele. In this case, only one genotype is possible. All offspring are Yy and have yellow seeds ( Figure 18. 6 ).

A self-cross of one of the Yy heterozygous offspring can be represented in a 2 × 2 Punnett square because each parent can donate one of two different alleles. Therefore, the offspring can potentially have one of four allele combinations: YY , Yy , yY , or yy ( Figure 18. 6 ). Notice that there are two ways to obtain the Yy genotype: a Y from the egg and a y from the sperm, or a y from the egg and a Y from the sperm. Both of these possibilities must be counted. Recall that Mendel’s pea- plant characteristics behaved in the same way in reciprocal crosses. Therefore, the two possible heterozygous combinations produce offspring that are genotypically and phenotypically identical despite their dominant and recessive alleles deriving from different parents.

Because fertilization is a random event, we expect each combination to be equally likely and for the offspring to exhibit a ratio of YY : Yy : yy genotypes of 1:2:1 ( Figure 18. 6 ). Furthermore, because the YY and Yy offspring have yellow seeds and are phenotypically identical, we expect the offspring to exhibit a phenotypic ratio of 3 yellow:1 green. Indeed, working with large sample sizes, Mendel observed approximately this ratio in every F2 generation resulting from crosses for individual traits.

Using a Test Cross to Determine Genotype

Beyond predicting the offspring of a cross between known homozygous or heterozygous parents, Mendel also developed a way to determine whether an organism that expressed a dominant trait was a heterozygote or a homozygote. Called the test cross , this technique is still used by plant and animal breeders. In a test cross, an organism with the dominant phenotype is crossed with an organism that is homozygous recessive for the same characteristic. If the dominant- expressing organism is a homozygote, then all F1 offspring will be heterozygotes expressing the dominant trait. Alternatively, if the dominant expressing organism is a heterozygote, the F1 offspring will exhibit a 1:1 ratio of heterozygotes and recessive homozygotes ( Figure 18. 7 ). The test cross further validates Mendel’s postulate that pairs of unit factors segregate equally.

Concept Check

In pea plants, round peas (R) are dominant to wrinkled peas (r). You do a test cross between a pea plant with wrinkled peas (genotype rr) and a plant of unknown genotype that has round peas. You end up with three plants, all which have round peas.

• From this data, can you tell if the round pea parent plant is homozygous dominant or heterozygous?
• If the round pea parent plant is heterozygous, what is the probability that a random sample of 3 progeny peas will all be round?

18.2.5 Using Pedigrees to Study Inheritance Patterns

Many human diseases are inherited genetically. A healthy person in a family in which some members suffer from a recessive genetic disorder may want to know if he or she has the disease-causing gene and what risk exists of passing the disorder on to his or her offspring. Of course, doing a test cross in humans is unethical and impractical. Instead, geneticists use pedigree analysis to study the inheritance pattern of human genetic diseases.

Each row of a pedigree represents one generation of the family. Women are represented by circles; males by squares. People who had children together are connected with a horizontal line and their children are connected to this line with a vertical line. See Figure 18. 8 for an example of a pedigree for a human genetic disease.

People with the recessive genetic disease alkaptonuria cannot properly metabolize two amino acids, phenylalanine and tyrosine. Affected individuals may have darkened skin and brown urine, and may suffer joint damage and other complications.

In this pedigree, individuals with the disorder are indicated in blue and have the genotype  aa . Unaffected individuals are indicated in yellow and have the genotype  AA  or  Aa . Note that it is often possible to determine a person’s genotype from the genotype of their offspring. For example, if neither parent has the disorder but their child does, both parents must be heterozygous. Two individuals on the pedigree have an unaffected phenotype but unknown genotype. Because they do not have the disorder, they must have at least one normal allele, so their genotype gets the “ A? ” designation.

What are the genotypes of the individuals labeled 1, 2, and 3?

18.2.6 Principle of Independent Assortment

Mendel’s principle of independent assortment states that genes do not influence each other with regard to the sorting of alleles into gametes, and every possible combination of alleles for every gene is equally likely to occur. The independent assortment of genes can be illustrated by a dihybrid cross, a cross between two true-breeding parents that express different traits for two characteristics. Consider the characteristics of seed color and seed texture for two pea plants, one that has green, wrinkled seeds ( yyrr ) and another that has yellow, round seeds ( YYRR ). Because each parent is homozygous, the principle of segregation indicates that the gametes for the green/wrinkled plant all are yr , and the gametes for the yellow/round plant are all YR . Therefore, the F1 generation of offspring all are YyRr ( Figure 18.9 ).

For the F2 generation, the principle of segregation requires that each gamete receive either an R allele or an r allele along with either a Y allele or a y allele. The principle of independent assortment states that a gamete into which an r allele sorted would be equally likely to contain either a Y allele or a y allele. Thus, there are four equally likely gametes that can be formed when the YyRr heterozygote is self-crossed, as follows: YR , Yr , yR , and yr . Arranging these gametes along the top and left of a 4 × 4 Punnett square gives us 16 equally likely genotypic combinations. From these genotypes, we infer a phenotypic ratio of 9 round/yellow:3 round/green:3 wrinkled/yellow:1 wrinkled/green ( Figure 18.9 ).

The physical basis for the principle of independent assortment also lies in meiosis I, in which the different homologous pairs line up in random orientations. Each gamete can contain any combination of paternal and maternal chromosomes (and therefore the genes on them) because the orientation of tetrads on the metaphase plane is random.

Testing the Hypothesis of Independent Assortment

To better appreciate the amount of labor and ingenuity that went into Mendel’s experiments, proceed through one of Mendel’s dihybrid crosses.

Question : What will be the offspring of a dihybrid cross?

Background : Consider that you have access to a large garden in which you can cultivate thousands of pea plants. There are several true-breeding plants with the following pairs of traits: tall plants with inflated pods, and dwarf plants with constricted pods. Before the plants have matured, you remove the pollen-producing organs from the tall/inflated plants in your crosses to prevent self-fertilization. When the plants mature, they are manually crossed by transferring pollen from the dwarf/constricted plants to the stigmata of the tall/inflated plants.

Hypothesis : Both trait pairs will sort independently according to Mendelian principles. When the true-breeding parents are crossed, all of the F1 offspring are tall and have inflated pods, which indicates that the tall (T ) and inflated (I) traits are dominant over the dwarf (t) and constricted (i) traits, respectively. A self-cross of the F1 heterozygotes results in 2,000 F2 progeny.

Test the hypothesis : You cross the dwarf and tall plants and then self-cross the offspring. For best results, this is repeated with hundreds or even thousands of pea plants. What special precautions should be taken in the crosses and in growing the plants?

If these traits sort independently, the ratios of tall:dwarf and inflated:constricted will each be 3:1. Each member of the F1 generation therefore has a genotype of TtIi . Figure 18.1 0 shows a cross between two TtIi individuals. There are 16 possible offspring genotypes. The offspring proportions: tall/inflated:tall/constricted:dwarf/inflated:dwarf/constricted show a 9:3:3:1 ratio. Notice from the grid that when considering the tall/dwarf and inflated/constricted trait pairs in isolation, they are each inherited in 3:1 ratios.

Analyze your data: You observe the following plant phenotypes in the F2 generation: 2706 tall/inflated, 930 tall/constricted, 888 dwarf/inflated, and 300 dwarf/constricted. Reduce these findings to a ratio and determine if they are consistent with Mendelian principles.

Form a conclusion: Were the results close to the expected 9:3:3:1 phenotypic ratio? Do the results support the prediction? What might be observed if far fewer plants were used, given that alleles segregate randomly into gametes? Try to imagine growing that many pea plants, and consider the potential for experimental error. For instance, what would happen if it was extremely windy one day?

18.3 | Exceptions to Mendel’s Principles of Inheritance

• Identify non-Mendelian inheritance patterns such as incomplete dominance, codominance, and sex linkage.
• Describe genetic linkage.
• Describe how chromosome maps are created.
• Explain the phenotypic outcomes of epistatic effects between genes.

Although Mendel’s principles still apply to some situations, many situations exist in which they do not apply. These “exceptions” to Mendelian genetics are discussed below.

18.3.1 Alternatives to Dominance and Recessiveness

Since Mendel’s experiments with pea plants, other researchers have found that the principle of dominance does not always hold true. Instead, several different patterns of inheritance have been found to exist.

Incomplete Dominance

Mendel’s results, that traits are inherited as dominant and recessive pairs, contradicted the view at that time that offspring exhibited a blend of their parents’ traits. However, the heterozygote phenotype occasionally does appear to be intermediate between the two parents. For example, in the snapdragon, Antirrhinum majus ( Figure 18. 11 ), a cross between a homozygous parent with white flowers ( CWCW ) and a homozygous parent with red flowers ( CRCR ) will produce offspring with pink flowers ( CRCW ). (Note that different genotypic abbreviations are used for Mendelian extensions to distinguish these patterns from simple dominance and recessiveness.) This pattern of inheritance is described as incomplete dominance , denoting the expression of two contrasting alleles such that the individual displays an intermediate phenotype. The allele for red flowers is incompletely dominant over the allele for white flowers. However, the results of a heterozygote self-cross can still be predicted, just as with Mendelian dominant and recessive crosses. In this case, the genotypic ratio would be 1 CRCR :2 CRCW :1 CWCW , and the phenotypic ratio would be 1:2:1 for red:pink:white.

Codominance

A variation on incomplete dominance is codominance , in which both alleles for the same characteristic are simultaneously expressed in the heterozygote. An example of codominance is the MN blood groups of humans. The M and N alleles are expressed in the form of an M or N antigen present on the surface of red blood cells. Homozygotes ( LMLM and LNLN ) express either the M or the N allele, and heterozygotes ( LMLN ) express both alleles equally. In a self-cross between heterozygotes expressing a codominant trait, the three possible offspring genotypes are phenotypically distinct. However, the 1:2:1 genotypic ratio characteristic of a Mendelian monohybrid cross still applies.

Multiple Alleles

Mendel implied that only two alleles, one dominant and one recessive, could exist for a given gene. We now know that this is an oversimplification. Although individual humans (and all diploid organisms) can only have two alleles for a given gene, multiple alleles may exist at the population level such that many combinations of two alleles are observed. Note that when many alleles exist for the same gene, the convention is to denote the most common phenotype or genotype among wild animals as the wild type (often abbreviated “+”); this is considered the standard or norm. All other phenotypes or genotypes are considered variants of this standard, meaning that they deviate from the wild type. The variant may be recessive or dominant to the wild-type allele.

An example of multiple alleles is coat color in rabbits ( Figure 18. 12 ). Here, four alleles exist for the c gene. The wild-type version, C+C+ , is expressed as brown fur. The chinchilla phenotype, cchcch , is expressed as black-tipped white fur. The Himalayan phenotype, chch , has black fur on the extremities and white fur elsewhere. Finally, the albino, or “colorless” phenotype, cc , is expressed as white fur. In cases of multiple alleles, dominance hierarchies can exist. In this case, the wild- type allele is dominant over all the others, chinchilla is incompletely dominant over Himalayan and albino, and Himalayan is dominant over albino. This hierarchy, or allelic series, was revealed by observing the phenotypes of each possible heterozygote offspring.

An example of multiple allelism in humans pertains to ABO blood type. A person’s blood type (e.g., type A or type O) is caused by different combinations of three alleles: IA, IB, and IO. A person with type A blood could have either IAIA or IAIO genotype. A person with type B blood could have IBIB or IBIO genotype. A person with type O blood must have the IOIO genotype. Note that type AB blood is an example of codominance (IAIB).

The complete dominance of a wild-type phenotype over all other mutants often occurs as an effect of “dosage” of a specific gene product, such that the wild-type allele supplies the correct amount of gene product whereas the mutant alleles cannot. For rabbit fur color, the wild-type allele may supply a given dosage of fur pigment, whereas the mutants supply a lesser dosage or none at all.

Multiple Alleles Confer Drug Resistance in the Malaria Parasite

Malaria is a parasitic disease that is transmitted to humans by infected female  Anopheles gambiae mosquitos ( Figure 18.13a ). It is characterized by cyclic high fevers, chills, flu-like symptoms, and severe anemia. Plasmodium falciparum is the most deadly causative agent of malaria ( Figure 18.13b ). When promptly and correctly treated,  P. falciparum  malaria has a mortality rate of 0.1%. However, in some parts of the world, the parasite has evolved resistance to commonly used malaria treatments, so the most effective malarial treatments can vary by geographic region.

In Southeast Asia, Africa, and South America,  P. falciparum has developed resistance to the anti-malarial drugs chloroquine, mefloquine, and sulfadoxine-pyrimethamine.  P. falciparum , which is haploid during the life stage in which it infects humans, has evolved multiple drug-resistant mutant alleles of the  dhps gene. Varying degrees of sulfadoxine resistance are associated with each of these alleles. Being haploid,  P. falciparum needs only one drug-resistant allele to express this trait.

Environmental Effects

Interestingly, the Himalayan phenotype in rabbits is the result of an allele that produces a temperature-sensitive gene product that only produces pigment in the cooler extremities of the rabbit’s body. In this case, the protein product of the gene does not fold correctly at high temperatures. A similar gene gives Siamese cats their distinctive coloration.

Temperature-sensitive proteins are also at work in arctic foxes and rabbits, which are white in the winter and darker colored during the summer. In these cases, the protein product of the gene does not fold correctly at colder temperatures. The mutation that caused this coloration was advantageous to these species, so they persisted in the populations.

18.3.2 X-Linked Traits are an Exception to the Principle of Segregation

In humans, as well as in many other animals and some plants, the sex of the individual is determined by sex chromosomes. The sex chromosomes are one pair of non-homologous chromosomes. Until now, we have only considered inheritance patterns among non-sex chromosomes, or autosomes. In addition to 22 homologous pairs of autosomes, human females have a homologous pair of X chromosomes, whereas human males have an XY chromosome pair. Although the Y chromosome contains a small region of similarity to the X chromosome so that they can pair during meiosis, the Y chromosome is much shorter and contains many fewer genes. When a gene is present on the X chromosome, it is said to be X-linked .

Eye color in Drosophila was one of the first X-linked traits to be identified. Like humans, Drosophila males are XY and females are XX. In flies, the wild-type eye color is red (X W ) which is dominant to white eye color (X w ) ( Figure 18.1 4) . Females can be X W X W , X W X w  or X w X w . However, Drosophila males lack a second allele copy on the Y chromosome, so their genotype can only be X W Y or X w Y. Males are said to be hemizygous , because they have only one allele for any X- linked characteristic. Hemizygosity makes the descriptions of dominance and recessiveness irrelevant for XY males.

In an X-linked cross, the genotypes of F1 and F2 offspring depend on whether the recessive trait was expressed by the male or the female in the P generation. When the P male expresses the white-eye phenotype and the female is homozygous red-eyed, all members of the F1 generation exhibit red eyes ( Figure 18.1 5 ). The F1 females are heterozygous (X W X w ), and the males are all X W Y, since they received their X chromosome from the homozygous dominant P female and their Y chromosome from the P male. A cross between a X W X w female and an X W Y male would produce only red-eyed females and both red- and white-eyed males. A cross between a homozygous white-eyed female and a male with red eyes would produce only heterozygous red-eyed females and only white-eyed males.

What ratio of offspring would result from a cross between a white-eyed male and a female that is heterozygous for red eye color?

In some groups of organisms with sex chromosomes, the gender with the non-homologous sex chromosomes is the female rather than the male. This is the case for all birds. In this case, sex-linked traits will be more likely to appear in the female, in which they are hemizygous.

Human Sex-linked Disorders

Sex-linkage studies in Morgan’s laboratory provided the fundamentals for understanding X-linked recessive disorders in humans, which included red-green color blindness, Types A and B hemophilia, and muscular dystrophy. Because human males need to inherit only one recessive mutant X allele to be affected, X-linked disorders are disproportionately observed in males. Females must inherit recessive X-linked alleles from both of their parents in order to express the trait. When they inherit one recessive X-linked mutant allele and one dominant X-linked wild-type allele, they are carriers of the trait and are typically unaffected. Carrier females can manifest mild forms of the trait due to the inactivation of the dominant allele located on one of the X chromosomes. However, female carriers can contribute the trait to their sons, resulting in the son exhibiting the trait, or they can contribute the recessive allele to their daughters, resulting in the daughters being carriers of the trait ( Figure 18.1 6 ). Although some Y-linked recessive disorders exist, typically they are associated with infertility in males and are therefore not transmitted to subsequent generations.

18.3.3 Lethal Alleles are Apparent Exceptions to the Principle of Segregation

A large proportion of genes in an individual’s genome are essential for survival. Occasionally, a nonfunctional allele for an essential gene can arise by mutation and be transmitted in a population through heterozygous carriers. The wild-type allele functions at a capacity sufficient to sustain life and is therefore considered to be dominant over the nonfunctional allele. If two heterozygous parents mate, one quarter of their offspring will be homozygous recessive. Because the gene is essential, these individuals will die. This will cause the genotypic ratio among surviving offspring to be 2:1 rather than 3:1. This inheritance pattern is referred to as recessive lethal .

The dominant lethal inheritance pattern is one in which an allele is lethal both in the homozygote and the heterozygote. Dominant lethal alleles are very rare because, as you might expect, the allele only lasts one generation and is not transmitted. However, dominant lethal alleles might not be expressed until adulthood. The allele may be unknowingly passed on, resulting in a delayed death in both generations. An example of this in humans is Huntington disease, in which the nervous system gradually wastes away ( Figure 18.1 7 ). People who are heterozygous for the dominant Huntington allele ( Hh ) will inevitably develop the fatal disease. However, the onset of Huntington disease may not occur until age 40, at which point the afflicted persons may have already passed the allele to 50 percent of their offspring.

18.3.4 Linked Genes Violate the Principle of Independent Assortment

Although all of Mendel’s pea characteristics behaved according to the principle of independent assortment, we now know that some allele combinations are not inherited independently of each other. Genes that are located on different chromosomes will always sort independently. However, each chromosome contains hundreds or thousands of genes, organized linearly on chromosomes like beads on a string. Genes that are on the same chromosome are linked and are therefore likely to be inherited together. When homologs separate during meiosis I, entire chromosomes segregate into separate daughter cells, carrying all of their linked genes with them.

However, because of crossover, it is possible for two genes on the same chromosome to behave independently, or as if they are not linked. To understand this, let’s consider the biological basis of gene linkage and recombination.

Homologous chromosomes possess the same genes in the same order. However, since each homolog came from a different parent, the alleles may differ on homologous chromosome pairs. Prior to meiosis I, homologous chromosomes replicate and synapse so that genes on the homologs align with each other. At this stage, segments of homologous chromosomes cross over and exchange segments of genetic material ( Figure 18.1 8 ). Because the genes are aligned, the gene order is not altered. Instead, the result of recombination is that maternal and paternal alleles are combined onto the same chromosome. Across a given chromosome, several recombination events may occur, causing extensive shuffling of alleles.

When two genes are located in close proximity on the same chromosome, their alleles are more likely to be transmitted through meiosis together. To exemplify this, imagine a dihybrid cross involving flower color and plant height in which the genes are next to each other on the chromosome. If the homologous chromosome from one parent has alleles for tall plants and red flowers, and the homolog from the other parent has alleles for short plants and yellow flowers, then when the gametes are formed, the tall and red alleles will go together into a gamete and the short and yellow alleles will go into other gametes. These are called the parental genotypes because they have been inherited intact from the parents of the individual producing gametes. Since the genes were close together on the same chromosomes, the chance of a crossover event happening between them is slim. Therefore, there will be no gametes with tall and yellow alleles and no gametes with short and red alleles. If you create the Punnett square with these gametes, you will see that the classical Mendelian prediction of a 9:3:3:1 outcome of a dihybrid cross would not apply

As the distance between two genes increases, the probability of crossovers between them increases, and the genes behave more as if they are on separate chromosomes. The further apart two linked genes are on a chromosome, the more progeny with nonparental genotypes will appear.

Genetic Linkage and Distances

Geneticists have used the proportion of nonparental gametes as a measure of how far apart genes are on a chromosome. Using this information, they have constructed elaborate maps of genes on chromosomes. Briefly, the more crossover that occurs between two linked genes, the further apart they are on the chromosome. The frequency of crossover is measured by counting the number of offspring that have nonparental genotypes. By using recombination frequency to predict genetic distance, the relative order of genes on chromosome 2 could be inferred.

18.3.5 Epistasis is an Exception to the Principle of Independent Assortment

Mendel’s studies in pea plants implied that every characteristic was distinctly and completely controlled by a single gene. In fact, single observable characteristics are almost always under the influence of multiple genes (each with two or more alleles) acting in unison. For example, at least eight genes contribute to eye color in humans.

Genes may function in complementary or synergistic fashions, such that two or more genes need to be expressed simultaneously to affect a phenotype. Genes may also oppose each other. In epistasis , the interaction between genes is antagonistic, such that one gene masks or interferes with the expression of another. Often the biochemical basis of epistasis is a gene pathway in which the expression of one gene is dependent on the function of a gene that precedes or follows it in the pathway.

An example of epistasis is pigmentation in mice. The wild-type coat color, agouti ( AA ), is dominant to solid-colored fur ( aa ). However, a separate gene ( C ) is necessary for pigment production. A mouse with a recessive c allele at this locus is unable to produce pigment and is albino regardless of the allele present at locus A . Therefore, the genotypes AAcc , Aacc , and aacc all produce an albino phenotype. A cross between heterozygotes for both genes ( AaCc x AaCc ) would generate offspring with a phenotypic ratio of 9 agouti:3 solid color:4 albino ( Figure 18.19 ). In this case, the C gene is epistatic to the A gene.

Epistasis can also occur when a dominant allele masks expression at a separate gene. Fruit color in summer squash is expressed in this way. Homozygous recessive expression of the W gene ( ww ) coupled with homozygous dominant or heterozygous expression of the Y gene ( YY or Yy ) generates yellow fruit, and the wwyy genotype produces green fruit. However, if a dominant copy of the W gene is present in the homozygous or heterozygous form, the summer squash will produce white fruit regardless of the Y alleles. A cross between white heterozygotes for both genes ( WwYy × WwYy ) would produce offspring with a phenotypic ratio of 12 white:3 yellow:1 green.

Finally, epistasis can be reciprocal such that either gene, when present in the dominant (or recessive) form, expresses the same phenotype. In the shepherd’s purse plant ( Capsella bursa-pastoris ), the characteristic of seed shape is controlled by two genes in a dominant epistatic relationship. When the genes A and B are both homozygous recessive ( aabb ), the seeds are ovoid. If the dominant allele for either of these genes is present, the result is triangular seeds. That is, every possible genotype other than aabb results in triangular seeds, and a cross between heterozygotes for both genes ( AaBb x AaBb ) would yield offspring with a phenotypic ratio of 15 triangular:1 ovoid.

As you work through genetics problems, keep in mind that any single characteristic that results in a phenotypic ratio that totals 16 is typical of a two-gene interaction. Recall the phenotypic inheritance pattern for Mendel’s dihybrid cross, which considered two non-interacting genes—9:3:3:1. Similarly, we would expect interacting gene pairs to also exhibit ratios expressed as 16 parts. Note that we are assuming the interacting genes are not linked; they are still assorting independently into gametes.

• Johann Gregor Mendel, Versuche über Pflanzenhybriden Verhandlungen des naturforschenden Vereines in Brünn, Bd. IV für das Jahr, 1865 Abhandlungen, 3–47. [for English translation see http://www.mendelweb.org/Mendel.plain.html] ↵

Introduction to Molecular and Cell Biology Copyright © 2020 by Katherine R. Mattaini is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Natural GM: how plants and animals steal genes from other species to accelerate evolution

Natural Environment Research Council Independent Research Fellow, University of Sheffield

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Little did biologist Gregor Mendel know that his experiments with sweet peas in a monastery garden in Brno, Czech Republic, would lay the foundations for our understanding of modern genetics and inheritance. His work in the 19th century helped scientists to establish that parents pass their genetic information onto their offspring, and in turn, they pass it on to theirs.

Indeed, this premise forms the basis of much of our understanding of evolution. But we now know that this process is not sacrosanct and some of our most widely grown crops may be fiddling the system by supplementing their genetic information with stolen genetic secrets. Our new study, published in New Phytologist , shows that this does in fact happen in grasses.

Grasses aren’t the only culprits, however. Bacteria are the master criminals in this regard. They are able to freely absorb genetic information from their environment. This process is termed lateral or horizontal gene transfer, and is thought to play an important role in the spread of traits such as antibiotic resistance.

Although scientists originally thought this process was restricted to bacteria, it has since been documented in a broad range of animals and plants. Examples include aphids that can synthesise a red fungal pigment to avoid predation, mushrooms that have shared the genetic instructions to assemble psychoactive compounds, and whiteflies that have turned their host plants’ defences against them .

Mysterious gene transfer

Grasses are the most ecologically and economically important group of plants. Grasslands cover between 20% and 40% of the world’s landmass , and several of the most widely grown global crops are grasses, including rice, maize, wheat and sugar cane. Our new study is the first to show that lateral gene transfer is widespread in this important plant group, and it occurs in wild and cultivated species alike.

Our discovery is based on genetic detective work, helping us trace the origin of each gene in the genomes of 17 grass species from around the world. As expected, an overwhelming majority of genes had the same evolutionary history as that of the species they were found in – indicating they were passed down through the generations from parent to offspring. However, we found over a hundred examples where the evolutionary history of the species and genes did not tell the same story.

The results showed that these genes had a past life in another distantly related grass species before being transferred into the recipient’s genome. We know that species boundaries are porous in nature, and that hybrid can occur as a result of reproduction between closely related organisms. Hybridisation and lateral gene transfer ultimately have similar effects generating novel combinations of genes that may or may not be advantageous.

However, lateral gene transfer is not a reproductive process and therefore has the potential to connect deeper branches within the tree of life, facilitating the movement of genetic material across much broader evolutionary distances. The genes transferred between grass species have functions relating to energy production, stress tolerance and disease resistance, potentially giving them an evolutionary advantage by allowing them to grow bigger, taller and stronger.

Foreign DNA was detected in the genomes of 13 of the 17 grasses sampled, including crops such as maize, millet and wheat. The million-dollar question is, how are these genes moving between species? In truth, we don’t know and we may never know for certain as there are several potential mechanisms and more than one may be involved.

After all, evolution is studying events that happened thousands and even millions of years ago. But there is a significant statistical increase in the number of transferred genes present today in grass species with rhizomes – modified roots that allow plants to propagate themselves asexually (a process in which part of a plant can be used to generate a new plant). The transfer of DNA into the rhizome could be facilitated via direct contact between species underground, possible through root fusion. Interestingly, scientists have recently observed DNA moving between tobacco plants that have been grafted together, further supporting this hypothesis.

Any foreign DNA transferred into the rhizome would then be replicated in all the cells in the daughter clone that arises from this tissue as the plant reproduces asexually. This foreign DNA would subsequently make its way into the germline (cells that pass on their genetic material to offspring) and future generations when the daughter clone flowers and produces seed.

The results of this study show that grasses have been genetically engineering themselves. Whether this is ammunition for the pro- or anti-GM lobby depends on your existing preconceptions in this debate.

It could be argued that if grasses are already doing this naturally, then why shouldn’t we? Conversely, this research shows that genes can freely move between grass species regardless of how closely related they are. Therefore, any gene inserted into a modified grass crop may eventually escape into wild species generating so-called superweeds.

Ultimately, if we can determine how lateral gene transfer is happening in grasses it may allow us to harness the process so we can naturally modify crops and make them more resistant to the effects of climate change.

• Genetic modification
• Gene transfer

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• Review Article
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• Published: 01 June 2021

Molecular basis of heterosis and related breeding strategies reveal its importance in vegetable breeding

• Daoliang Yu 1 ,
• Xingfang Gu 1 ,
• Shengping Zhang 1 ,
• Shaoyun Dong 1 ,
• Han Miao 1 ,
• Kiros Gebretsadik   ORCID: orcid.org/0000-0002-0540-0180 1 , 2 &
• Kailiang Bo   ORCID: orcid.org/0000-0001-8841-5195 1  

Horticulture Research volume  8 , Article number:  120 ( 2021 ) Cite this article

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• Gene regulation
• Plant breeding

Heterosis has historically been exploited in plants; however, its underlying genetic mechanisms and molecular basis remain elusive. In recent years, due to advances in molecular biotechnology at the genome, transcriptome, proteome, and epigenome levels, the study of heterosis in vegetables has made significant progress. Here, we present an extensive literature review on the genetic and epigenetic regulation of heterosis in vegetables. We summarize six hypotheses to explain the mechanism by which genes regulate heterosis, improve upon a possible model of heterosis that is triggered by epigenetics, and analyze previous studies on quantitative trait locus effects and gene actions related to heterosis based on analyses of differential gene expression in vegetables. We also discuss the contributions of yield-related traits, including flower, fruit, and plant architecture traits, during heterosis development in vegetables (e.g., cabbage, cucumber, and tomato). More importantly, we propose a comprehensive breeding strategy based on heterosis studies in vegetables and crop plants. The description of the strategy details how to obtain F 1 hybrids that exhibit heterosis based on heterosis prediction, how to obtain elite lines based on molecular biotechnology, and how to maintain heterosis by diploid seed breeding and the selection of hybrid simulation lines that are suitable for heterosis research and utilization in vegetables. Finally, we briefly provide suggestions and perspectives on the role of heterosis in the future of vegetable breeding.

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Introduction

Heterosis occurs in a variety of species and has been observed and recorded in China since ancient times. For example, Jia Sixie described in “The Manual of Important Arts for the People” that interbreeding between horses and donkeys produced stronger mules, and the famous agricultural work “Tian Gong Kai Wu” also recorded crossbreeding techniques for silkworms. Heterosis has also been extensively studied in other countries. In 1763, the German scholar Koelreuter 1 was the first to present concrete evidence that the growth of hybrid tobacco is superior to that of its parents. By comparing the height of hybrid and self-crossing offspring in maize, Darwin 2 found that the average height of hybrid offspring was higher than that of self-crossing offspring. Beal 3 found that the yield of maize hybrid offspring was greater than that of both parents. Shull 4 , 5 observed heterosis in maize hybrid offspring and first proposed the concept of heterosis; he then formally named this phenomenon “heterosis.” Heterosis was first applied to genetic breeding in maize, and many excellent maize hybrids have been produced since the 1930s. Since 2011, the yield of maize increased by at least eightfold in America, due mostly to the cultivation of hybrids 6 .

As heterosis has been applied in cereal crop production, crossbreeding in vegetables has also rapidly progressed. Under natural planting conditions, 40–80% of seeds produced are usually hybrids due to fertilization competition between self-pollination and pollen from other plants 7 . Although the traits of randomly generated hybrid seeds are not organized at first, F 1 hybrids exhibit higher yield, better adaptability, and higher stress resistance than pure line seeds under optimum production and fertilization protection management conditions. Therefore, farmers have paid much attention to the cultivation of hybrid seeds 8 . The first hybrid of eggplant ( Solanum melongena ) was released in 1924 9 . Subsequently, hybrids of other vegetables, such as watermelon ( Citrullus lanatus L.), cucumber ( Cucumis sativus L.), radish ( Raphanus sativus L.), tomato ( Solanum lycopersicum L.), and cabbage ( Brassica oleracea L.), were developed over the next 20 years 7 . The number of hybrid vegetable varieties is rapidly increasing, at a rate of 8–10% each year, while nonhybrid vegetable varieties are gradually being eliminated 10 .

The application of heterosis to vegetable cultivation was first proposed by Hayes and Jones 11 using cucumbers. However, because of the high cost of producing hybrid seeds, hybrid cucumber seeds were not used until the 1930s 7 . Similarly, self-pollination and the occasional presence of indehiscent anthers in eggplant 12 and styles that are shorter than anthers in tomato 13 have resulted in a high degree of self-pollination, which in turn has limited hybrid utilization. Pearson (1933) and Jones and Clarke (1943) used the mechanisms of self-incompatibility in cabbage and cytoplasmic male sterility in onion, respectively, to produce pure line and hybrid seeds on a large scale 8 . To avoid undesirable selfing, various genetic and nongenetic mechanisms, including genic male sterility, cytoplasmic male sterility, self-incompatibility, gynoecious lines, auxotrophy, and the use of sex regulators and chemical hybridizing agents, have been applied to facilitate hybrid seed production in vegetables 8 , 14 . The various traits that exhibit remarkable heterosis in F 1 hybrids, including yield, earliness, growth vigor, and stress tolerance 15 , 16 , 17 , 18 , have become a major area of research on vegetables. In an experiment with hybrid eggplant conducted by Balwani et al. 19 and Makani et al. 20 heterosis in the optimal F 1 hybrid resulted in yield increases of 125.78% and 88.88%, respectively. A more productive eggplant hybrid will effectively decrease the time to first harvest 18 . Transgressive phenotypes have also been observed in other Solanaceae 21 , 22 , Cruciferae 23 , 24 , and Cucurbitaceae vegetables 25 , 26 .

Although heterosis in vegetables has historically been used in research and crossbreeding experiments, its genetic mechanism remains elusive. Different genetic models for heterosis have been described in various reviews 27 , 28 , 29 , 30 , 31 . However, it is apparent that the classical genetic hypothesis of heterosis cannot explain all mechanisms of heterosis. Therefore, genetic models of heterosis have been included in this review. In addition to genetic models, we also present a schematic diagram depicting the involvement of epigenetics in heterosis. Simultaneously, we discuss studies on heterosis at the molecular level based on QTL effects and differential gene expression analyses. We also describe the effects of QTL on heterosis in crop plants based on Shang et al. 32 to guide future research studies on the genetic mechanisms of heterosis. We summarize recent findings on the interactions of QTL sites with regard to heterosis and discuss the contribution of various QTL effects to heterosis. Differential expression analysis of genes related to heterosis can also provide a different perspective on heterosis 31 . In addition, we present morphological improvement as another measure to increase yield and an important component of breeding 7 and describe how to combine heterosis utilization and morphological improvement.

To date, studies on heterosis in vegetables mainly involve obtaining F 1 hybrids through crossbreeding. The utilization of cucumber hybrids proposed by Hayes and Jones 11 was likely the first instance of effective vegetable breeding that exploits heterosis. Kumar et al. 30 introduced methods of predicting heterosis in eggplant hybrids, such as genetic distance prediction and combining ability tests, and proposed the application of a sterile line system as well as transgenic and gene editing techniques in eggplant breeding. Herath et al. 33 summarized the QTL mapping of yield-related traits in chili, introduced the use of heterosis breeding to improve the economic and agronomic traits of chili, and suggested the use of genomic technology and sterile line materials in chili breeding. Mallikarjunarao et al. 34 reviewed the progress of various balsam pear (bitter gourd) hybridization tests and indicated that heterosis does occur in the yield of balsam pear hybrids. However, studies on the genetic mechanisms of heterosis in vegetables are limited, which hinders the application of heterosis in vegetable breeding. Therefore, in this review, we describe the progress of research on the genetic mechanisms of heterosis, analyze the use of hybrid production systems and molecular biology technology in vegetable production, and propose a breeding strategy that can predict, obtain, and maintain heterosis. This review will provide a reference for the utilization of heterosis in vegetable breeding.

Study on the genetic mechanisms of heterosis

Genetic regulation of heterosis.

Heterosis is a complex biogenetic phenomenon caused by the combination of many factors that is manifested in the performance of hybrid offspring. The classical hypotheses for the genetic mechanisms of heterosis include the dominance and overdominance hypotheses, which are based on allelic interactions, and epistasis, which is based on nonallelic interactions.

Davenport 35 first proposed the dominance hypothesis (Fig. 1A ), and Bruce 36 and Jones 37 developed it further. In the dominant hypothesis, favorable genes controlling growth and development are dominant, and unfavorable genes are recessive. In the hybrid generation, the alleles from the two parents are complementary, and the unfavorable recessive genes are suppressed by the favorable dominant genes; therefore, the hybrid generation exhibits heterobeltiosis.

Suppose that the biomass is the sum of the genetic effects (A, B, C) and that the biomass of an organism is represented by the circular area. A Dominance effect: the dominant allele ( A ) inhibits the recessive allele (a); ( B ) overdominance effect: a single heterozygous allele (B/B − ) promotes the development of heterosis; ( C ) Epistasis effect: nonallelic (A 1 /B 1 ) interactions in the parents promote the development of heterosis; ( D ) active gene effect: genes from parents ( C ) promote heterosis when heterozygous and produce genome imprinting when homozygous, which inhibits the occurrence of heterosis; ( E ) gene network system: genes from parents (A, B, C) are combined into a coordinated gene network system that enables F 1 to develop heterosis; ( F ) single-cross hybrids P 1 (AB) and P 2 (CD) produced from four homozygous inbred tetraploids (with genotypes A, B, C, and D) are crossed to produce F 1 (ABCD), a double-cross tetraploid hybrid

The overdominance hypothesis (Fig. 1B ) was originally proposed by Shull 4 and East 38 as the opposite of the dominance hypothesis. This hypothesis denies that there is dominant-recessive relationship between alleles and suggests that the main cause of heterosis is the interaction of heterogeneous alleles from parents. Heterozygous alleles interact more strongly than homozygous alleles; thus, the hybrids exhibit heterobeltiosis. Using the isozyme technique, Dranginis 39 found that the enzymes in heterozygotes exhibit many unique conformations of hybrid enzymes. For example, the regulatory proteins of heterozygotes often present as polymers that regulate genes, and different heterozygous and homozygous proteins consistently show different activity characteristics. In addition, the anthocyanin content heterobeltiosis that occurs due to the heterozygosity of a single locus ( pl ) in maize 40 and the yield heterosis induced by the heterozygosity of a single locus ( sft ) in tomato 15 also provide experimental evidence for the overdominance hypothesis. However, the interaction of closely linked alleles can also result in an overdominance effect that is known as pseudo-overdominance 41 .

The dominance and overdominance hypotheses for the heterosis phenomenon both suggest that heterosis is caused by individual allele loci. However, several reports have shown that plant traits such as yield and growth vigor are complex quantitative traits 42 . Wright 43 visualized the network structure of population genotypes, i.e., multiple loci control the variations in most traits; in such networks, the replacement of anu gene may affect multiple traits. Based on this perspective, Sheridan 44 proposed the concept of epistasis. He believed that heterosis may arise from interactions between nonalleles. In genetics, the phenomenon in which the genetic effect of a nonallele deviates from its additive effect is called epistasis (Fig. 1C ). The significant special combining ability (SCA) effects in the hybridization experiment of Sao and Mehta indicated that epistasis plays a predominant role in the genetic control of eggplant heterosis 45 . Using a genetic map that covered the whole rice ( Oryza sativa ) genome, QTL mapping for yield-related traits was conducted in 250 F 2:3 lines. The results showed that the correlation between marker heterozygosity and yield-related traits was low and that the interaction between most genes could not be detected on the basis of single-gene loci; the interactions were classified as dominance by dominance, additive by dominance, and additive by additive 46 . Therefore, Yu et al. 46 also believed that epistasis is an important genetic basis for the development of heterosis.

Other ideas in addition to the classical hypotheses have been proposed. Zhong 47 proposed the active gene effect hypothesis (Fig. 1D ) by comparing the relationship between genomic imprinting and heterosis; this hypothesis suggests that heterosis is caused by additive effects between the active genes. When alleles are homozygous, only one of them is active. When genes are heterozygous, genomic imprinting does not occur, and all genes are active, showing all effects. The interaction between active genes increases the overall effect of gene expression; as a result, the hybrid exhibits heterosis. For example, in maize, the red1 ( r1 ) gene, when inherited from both parents, causes different colors in corn kernels 48 . Genomic imprinting affects the differential expression of genes by affecting DNA methylation and histone modification 49 . Bao 50 suggested that individuals have a specific set of genetic information that controls their growth. Genetic information is expressed as different coding genes in organisms; these genes form an orderly network of expression, and the activities of each gene are related to each other. An alteration in a single gene may cause changes in the entire network. The network of F 1 hybrids is a new gene network system that is formed from the two different gene networks of the parents. If the interactions between alleles bring the whole genetic network system to an optimal state, the F 1 hybrid exhibits heterosis; otherwise, it remains typical (Fig. 1E ). In addition, the effects caused by genomic imprinting or active gene effects may be components of genomic dosage effects 51 ; the other part of genomic dosage effects usually caused by polyploidy, which is a specific phenomenon in polyploid plants called progressive heterosis (Fig. 1F ) 52 , 53 . The genomic dosage effects produced by allopolyploids are usually stronger than those produced by homologous polyploids 38 , 51 , 54 , 55 . The formation of polyploids is accompanied by extensive genetic and epigenetic changes 56 , which may provide the molecular basis for the development of heterosis.

Epigenetics is involved in the development of heterosis

Although many hypotheses have been proposed to explain the mechanisms of plant heterosis at the genetic level, studies have shown that the genetic mechanisms of heterosis cannot be fully explained by one or even several hypotheses at the genetic level. Through the intensive study of epigenetics, epigenetic factors such as DNA methylation, small RNAs, and histone modifications have been found to be involved in the development of heterosis in plants 57 , 58 , 59 , 60 , 61 , 62 .

Epigenetic modifications play an important role in the formation of plant phenotypes by regulating gene transcription and gene expression 63 , 64 , 65 . Alleles of known phenotypes have been studied more extensively in the context of DNA methylation than in the context of other epigenetic modifications 63 . RNA-directed de novo methylation (RdDM) is one of the pathways that triggers DNA methylation by 24 nt-siRNA, which is regulated by two key genes, namely, NRPD1 and NRPE1 66 (Fig. 2A, B ). A silent epigenetic variant caused by differentially methylated regions (DMRs) in the promoter, sulfurea ( sulf/+ ), can result in homozygous lethal tomato plants that exhibit only chlorotic leaf sectors 64 , 65 . This may occur due to the random combination of genetic information from the parents of the F 1 hybrids because their genotypes are more prone to heterozygosity at the DNA methylation level; this is in line with the findings of Shen et al. 59 . The gene effect caused by such heterozygosity may enable F 1 hybrids to avoid producing common phenotypes or hybrid weakness, thus achieving heterobeltiosis. Using experiments involving heterograft eggplants, Cerruti et al. 62 found that scion vigor is related to DNA methylation and that the reduction in methylation in the CHH context promotes scion vigor. Tomato grafting experiments revealed that RdDM can cause a heritable enhancement-through-grafting phenotype 67 , 68 .

A DNA methylation: De novo methylation was catalyzed by DRM2, a homologous enzyme of DNMT3. In maintenance methylation, CG is catalyzed by MET1, a homologous enzyme of DNMT1; CHG is catalyzed by CMT3; and CHH is still catalyzed by DRM2. B Small RNA: Includes the miRNA produced by premiRNA and the siRNA produced by dsRNA. In general, 24 nt-siRNA mediates de novo DNA methylation catalyzed by the AGO4 protein. C Histone modifications: The modifications of histone amino acid residue includes acetylation, phosphorylation, methylation, and ubiquitination processes. Epigenetic modifications are produced by the parents. New epigenetic modifications may occur in F 1 hybrids. D Epigenetic modification status of the parents and F 1 hybrid: the increase and decrease in or recombination of epigenetic modifications induces the F 1 hybrid to exhibit heterosis

Because de novo DNA methylation is mediated by siRNAs (Fig. 2B ), siRNAs may also be involved in the regulation of heterosis. The level of siRNAs decreased in different genome regions between parents and hybrids, but this phenomenon was limited to 24 nt-siRNAs; in contrast, the levels of siRNAs of other sizes did not decrease 67 . Noncoding small RNAs can be used as signaling molecules in plants 67 . Shivaprasad et al. 61 observed that miR395 is differentially expressed, mediates transgressive phenotypes in the hybrid progeny of tomato and is associated with suppression of the corresponding target genes, which indicates that the combination of parental genetic information can cause differences in miR395 abundance in the progeny. Simultaneously, 21–24 nt small RNAs can move through the intercellular filaments and phloem of the graft site 69 , and 24 nt sRNAs can guide genomic DNA methylation in recipient cells 70 ; this information provides a theoretical basis for guiding grafting. In addition, sRNAs in plants usually play a major role in inducing gene expression silencing and gene posttranscriptional silencing 71 , 72 . This may be due to the downregulation of sRNA levels in hybrids, which lifts the silencing of some favorable genes and thus allows hybrids to exhibit heterobeltiosis 71 , 72 .

Different modifications, such as acetylation, phosphorylation, methylation, and ubiquitination, occur at the amino terminus of histones (Fig. 2C ). These histone modifications can affect the binding of related proteins to chromatin and thereby affect the transcriptional activity of genes. At the same time, the combination of modifications of the amino terminus of histones expands the genetic information for and changes the phenotype of an individual 73 . Histone modifications are related to the stability of heterosis. Studies have shown that histone deacetylases cause the nonadditive expression of some genes in hybrids 58 . In addition, histone acetylation and methylation are related to the activation of regulatory (circadian-regulated) genes in F 1 hybrids 73 . The biological clock controls the physiological activities of plants, including the synthesis of physiological and biochemical substances. Therefore, histone modifications can influence plant biomass heterosis.

The recombination of genetic information from parents may lead to new combinations of epigenetic modifications in the F 1 generation (Fig. 2D ). Epigenetic modifications essentially affect the expression of genes, causing them to be overexpressed or silenced. Therefore, epigenetic modifications may indirectly influence the development of heterosis in F 1 by affecting the expression pattern of genes.

Study on heterosis at the molecular level

Progress in heterosis research based on qtl analysis.

The genome contains all the genetic information of a species and determines whether an individual gene is expressed as well as its degree of expression. Heterosis is usually indicated if the hybrid generation is superior to the parents in terms of quantitative traits. Thus, it is essential to conduct a genetic analysis of heterosis from the perspective of the whole genome. With the rapid development of genome sequencing technology, it has become possible to identify gene loci related to heterosis by genome-wide association studies 74 , which lay a foundation for the study of individual phenotypic differences. This review summarizes the QTL effects on heterosis based on 35 studies that mainly addressed 6 crops and vegetables, i.e., rice ( Oryza sativa ), maize ( Zea mays ), cotton ( Gossypium hirsutum ), oilseed rape ( Brassica campestris ), sorghum ( Sorghum vulgare ), and tomato ( Solanum lycopersicum ) (Table S1 ). Among the six types of QTL effects, dominance and epistasis had equal proportions (19%, 23%, Fig. 3 ). Interestingly, the overdominance effect accounted for the largest proportion of all the effects (42%, Fig. 3 ). This means that although there are many gene loci in the plant genome, these interacted to produce different, complex, hard-to-imitate effects and resulted in heterosis; among these effects, overdominance effects occurred consistently and contributed significantly to heterosis. In addition, the overdominance effect can be conveniently used for artificial breeding, which has been well demonstrated in tomato 15 . However, efficiently and accurately locating the gene loci that impart the overdominance effect is necessary to make use of this effect. Heterosis may be the result of many traits. In addition, the results of QTL mapping differ among species and even within different groups of the same species 75 , 76 , 77 . Therefore, it is necessary to select a suitable genetic population based on the genetic background of the plants exhibiting heterosis.

Statistical analysis of the effect of quantitative trait loci on crop heterosis. A In the statistical analysis of the effect of quantitative trait loci on crop heterosis, the species and frequency of each species were studied; ( B ) in the statistical analysis of the effect of quantitative trait loci on crop heterosis, the quantitative trait locus effect on each species and the proportion of each type of effect were analyzed

Advances in gene action related to heterosis based on differential expression analysis of genes

The genome controls the formation of a biological phenotype by regulating the differential expression of genes 78 , 79 . Molecular-based expression analyses, such as allele-specific expression, DNA microarray, expression quantitative trait loci, RNA-seq, quantitative SNP-based Sequenom technology, and allele-specific RT-PCR, have made it possible to detect differential gene expression.

Yield and biomass heterosis in F 1 hybrids may occur due to the altered expression patterns of genes that control biological functions such as carbon fixation, glucose metabolism, and circadian rhythm 80 . Gene Ontology (GO) analysis of pakchoi line parents and hybrids indicated that most of the differentially expressed genes between parents and hybrids enriched the photosynthetic pathway and that the enhancement of the photosynthetic capacity of the hybrids was related mainly to an increase in the number of thylakoids 17 . In addition, the increase in the number of thylakoids also promoted the enhancement of the carbon fixation capacity in the hybrids 17 ; this is similar to the finding that differentially expressed genes that significantly enrich the optical signaling pathway occur between F 1 and their parents in broccoli 24 . The same results were also found in other plants 79 , 81 . Transcriptome and differential gene expression analyses revealed that the modes of action of heterosis genes were mainly additive (F 1  = MPV), overdominance (F 1  > HPV), and underdominance (F 1  < LPV) 82 (Fig. 4 ). When the expression value of a differentially expressed gene in the hybrid line was higher or lower than that of the parent, the gene action patterns were classified as high-parent dominance (F 1  ≈ HPV) and low-parent dominance (F 1  ≈ LPV), respectively 82 (Fig. 4 ). Li et al. 24 reported that most genes exhibited additive expression patterns in hybrid broccoli and that nonadditive action was involved mainly in light and hormone signal pathways related to heterosis; a similar finding was reported in Chinese cabbage ( Brassica campestris ssp. pekinensis cv. “ spring flavor ”) 23 . These gene expression patterns may have occurred due to selective inhibition or activation by the epigenetic modification of hybrid F 1 genes 83 , 84 ; the genes from inactive inbred lines can be activated by genes or regulatory factors of active inbred lines 85 , 86 . Epigenetic modifications and the interactions of heterogeneous factors occur in only a few genes, and the genome that produces differential expression in F 1 hybrids and parents accounts for only a small part of the total genome 87 . Moreover, Springer and Stupar 88 have shown that additive gene expression accounts for the majority of gene expression, while nonadditive gene expression is responsible for a small proportion of gene expression. These findings suggest that nonadditive expression of this fraction facilitates the development of heterosis.

Midparent value [MPV = (HPV + LPV)/2]; High-parent value (HPV); low-parent value (LPV)

Traits contributing to yield heterosis in vegetables

Traits related to yield heterosis.

Hybrids that exhibit heterosis show significant heterobeltiosis in yield, which is a complex trait that is usually measured by weight. To clearly study the mechanisms of yield increase in hybrids, it is essential to divide yield into other, simpler traits. This review describes the traits that contribute to vegetable yields. Fruits are the source of the yield of most plants; the yield contributing traits related to fruits usually include the fruit number, fruit size and fruit weight; earliness is usually also taken into account. Cabbage is a typical leafy, head-forming vegetable in Cruciferae, so its main yield contributing traits are head weight and head size (Fig. 5A, C ). Similar to that of cabbage, the yield of radish is determined by its taproot. For leafy vegetables that do not form heads, the main yield heading traits are the number and size of the leaves. Unlike cruciferous vegetables, Cucurbitaceae and Solanaceae vegetables are produce multiple harvests and multiple fruits per plant (Fig. 5B, D ), so the average single fruit weight and fruit yield per plant should be taken into account. In addition, Solanaceae vegetable flowers consist mostly of compound inflorescences 89 , so the numbers of flowers per cluster and fruits per cluster contribute greatly to production. Cucurbitaceae are single-inflorescence vegetables; only the fruits on the main vine are harvested in production, and the first nodal position of female flowers and sex ratio (M/F) affect the days to first harvest and the number of fruits per plant, respectively. Regardless of the trait considered, the total yield can be affected only by changes in yield-related traits. Therefore, it is necessary to analyze the mechanisms that regulate yield-related traits.

Contributing traits of yield heterosis in cucumber, cabbage and tomato. A Traits contributing to yield heterosis in cucumber, cabbage, and tomato: cucumber yield contributing traits include the number of fruits, days to first female flowering, days to first harvest, first nodal position of female flower, sex ratio (M/F), fruit length, fruit diameter, and fruit weight; cabbage yield contributing traits include fruit length, fruit diameter, and fruit weight; tomato yield contributing traits include number of fruits, days to first female flowering, days to first harvest, number of flowers/fruits per cluster, fruit length, fruit diameter, and fruit weight. B Cucumber: cucumber model in production, gynoecious line with a small number of branches. C Cabbage: an aerial and cross-sectional model of cabbage consisting of leaves and heads. D Tomato: a tomato with single inflorescences and indeterminate growth is crossbred with a tomato with compound inflorescences and determinate growth to produce the hybrid F 1 with earlier fruiting, more compound inflorescences, and determinate growth

Relationship between yield heterosis and plant architecture

Since the “green revolution”, interest in breeding for specific plant architecture has significantly increased, and the idea of combining heterosis breeding with plant architecture breeding has been proposed 90 . Donald 91 conducted research on half-dwarf plant architecture, which gradually turned into the concept of the ideotype. Donald introduced the ideotype concept, which refers to the plant architecture form that results in the minimum competitive intensity in population breeding. Although this definition is no longer used, the concept of an ideal plant architecture has played a major role in promoting plant breeding for high yields. Research on ideotypes first made progress in rice. It is worth mentioning that a key gene regulating ideotype, IPA1 , was proven by Huang et al. 75 to influence genes that are important in heterosis by using the indica-japonica hybrid rice group. Studies of heterozygosity and ideotype were also combined effectively in tomato. The self-pruning ( sp ) gene promotes indeterminate growth in tomato, while the sft gene changes indeterminate growth into determinate growth by inhibiting the sp gene 92 . The sft gene results in the development of heterosis in tomatoes through the heterozygosity of a single gene 15 and induces changes in plant architecture on the ground, causing tomato to produce compound inflorescences rather than single inflorescences 93 . The earliness of F 1 was also higher than that of its parent (Fig. 5D ), which increased tomato yield. Other vegetables in addition to tomato may also have ideotypes, and the key genes controlling plant architecture may also be important genes that are involved in the development of heterosis. Therefore, it is particularly important to study the genetic mechanisms of heterosis. By identifying the important genes involved in heterosis, the key genes that control plant ideotypes can be characterized.

Advances in heterosis utilization and biotechnology in vegetables

Breeding for heterosis has been extensively studied in plants, and research on the heterobeltiosis of hybrid offspring in vegetables has focused mainly on yield 94 and disease resistance 29 . Wellington 95 and Tschermak 96 showed that tomato hybrids exhibit heterosis in early maturity and during yield production. Krieger et al. 15 cloned the single-gene sft that affects the female flower fertility rate in tomato by infiltrating the IL and TC populations. When the sft gene exhibited heterozygosity, the tomato yield exhibited heterosis. According to this study, tomato plants that showed yield heterosis also showed resistance to both biological and abiotic stresses. The heterozygous state of the Tm and Tm22 genes contributes to tobacco mosaic virus resistance 97 , 98 and high-temperature stress tolerance 99 , 100 . Naresh et al. 101 suggested that heterosis is the result of nonadditive gene effects and that it also plays an important role in improving Cercospora leaf spot resistance in eggplant in the field. Similar to studies on other vegetables, studies on heterosis in Cucurbitaceae vegetables have also focused mainly on yield and disease resistance. Pandey et al. 102 used 77 cucumber hybrid generations and their parents to study the yield heterosis and contributing traits of different cucumber hybrid varieties and found that DC–1 × B–159 and VRC–11–2 × Bihar–10 were the best hybrid combinations for yield and prematurity. Using 48 F 1 hybrids and their parents, the gene effects caused by diseases and insect pests under natural conditions 29 were investigated. The results indicated that nonadditive gene effects had a significant regulatory effect on other traits in cucumber (except morbidity caused by Drosophila), demonstrating the importance of heterosis in cucumber breeding for disease resistance.

Different molecular markers, such as simple sequence repeats (SSRs), inter-simple sequence repeats (ISSRs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs), and sequence-related amplified polymorphisms (SRAPS), have provided the molecular basis for the construction of genetic maps and the mapping of important trait genes (Table 1 ). Whole-genome sequencing has been conducted for a variety of vegetables (Table 1 ), which has provided a basis for whole-genome strategies. Whole-genome approaches can help obtain complete sequences of germplasm resources, increase the coverage of molecular markers, and increase the accuracy of genetic maps 103 . Molecular markers are often used for the determination of genetic distance and the classification of heterotic groups. To elucidate the breeding processes and to improve the efficiency of breeding techniques in cabbage, heterotic cabbages are usually divided into two groups: The round head type and the flat head type. Xing et al. 104 further divided 21 flat cabbage inbred lines into three heterotic groups and divided 42 round cabbage inbred lines into five heterotic groups in order to provide a more definite direction for the preparation of hybrid combinations of cabbage. The method of dividing heterotic groups by molecular markers and genetic distance is widely used in vegetable breeding (Table 1 ).

Chen 83 proposed that determining how to obtain hybrid seeds is the key to the utilization of heterosis. The purpose of obtaining hybrid seeds is to make heterosis in the offspring permanent. The sporophyte of cruciferous vegetables is a self-incompatible system 105 that can prevent self-pollination and produce normal seeds through cross-pollination. Hence, this system is convenient for the generation of hybrid seeds. In cabbage 106 , 107 and Chinese cabbage 108 , hybrids are usually obtained using self-incompatible and male-sterile lines. To produce hybrid tomato seeds, pollen-abortive type and functionally sterile lines are often used 109 , 110 , 111 . Cytoplasmic male sterility occurs in eggplant 112 , 113 and pepper 114 , 115 . Gynoecious lines tend to exist in Cucurbitaceae 116 . A new male-sterile system in tomato was developed by Du et al. 117 . Plant growth regulators such as ethylene, auxins, and brassinosteroids 118 , 119 can increase the number of female flowers in Cucurbitaceae; this effect and male sterility are both convenient for hybrid seed production.

Strategies for heterosis breeding in vegetables (with tomato as an example)

Obtaining f 1 hybrids that exhibit heterosis based on heterosis prediction.

It is not advisable to conduct extensive hybridization tests to obtain hybrid F 1 lines that exhibit heterosis, as this approach requires considerable resources and time and produces unreliable results 13 . Melchinger and Gumber 120 proposed that heterotic groups should be used as the basis for crossbreeding. The heterotic group is the population that is classified according to breeding requirements, with abundant genetic variation and high combining ability. Chen et al. 121 carried out a genome-wide association study (GWAS) on the yield traits, general combining ability (GCA), and SCA of rice. The study provided strong evidence for the use of combining ability to classify heterotic groups and provided a reference for studies on combining ability in vegetables (Fig. 6 ). Other studies have also shown that combining ability, genetic distance, and molecular markers can provide the basis for evaluating parental inbred lines and predicting F 1 hybrid heterosis in vegetables 122 , 123 , 124 , 125 .

There are two strategies for obtaining heterotic lines in crop breeding. The first is the use of crossbreeding or molecular biotechnology. Genealogical analysis, molecular markers, combining ability, and genetic distance can usually predict heterosis development, so they are often used to classify heterotic groups. The inbred lines from different heterotic groups can be crossed with each other to obtain elite lines that exhibit heterosis. The second strategy is to use modern molecular biotechnology. Elite lines were obtained based on GWAS and linkage analysis, mapping and cloning genes related to heterosis, gene editing, and gene transformation

The GCA characterizes the average performance of a set of hybrid combinations and is mainly the consequence of additive gene effects and additive × additive interactions; SCA evaluates the average performance of certain hybrid combinations compared to the parental lines and is the result of dominance, epistatic deviation and genotype × environmental interactions 126 . Parents with a high GCA effect have higher adaptability and fewer environmental effects 127 . Parents with superior traits do not always pass on their traits to offspring 126 ; hence, the evaluation of combining ability is more reliable than the performance of the lines per se. Many types of combining ability tests can be used to identify superior parental lines for developing heterotic hybrids, including line × tester analysis, topcross tests, single-cross tests, poly-cross tests, and diallel mating 128 . Singh et al. 129 conducted a complete diallel cross test on seven diverse bitter gourd lines and found that combinations with high × high GCA usually produced high SCA effects and could therefore be considered for use in developing superior variants through the pedigree method. High/low × low GCA combinations can also achieve high but unstable SCA effects that are suitable for heterosis breeding and are in line with the results of Kenga et al. 130 in sweet sorghum and Franco et al. 131 in common bean.

In addition to combining ability, heterotic groups are often classified by genealogical information 132 . For parents with known genealogical relationships, heterosis in hybrids can usually be predicted according to these genealogical relationships. Genetic distance is a quantitative description of the genetic differences that provide the genetic basis for the development of heterosis in offspring 133 , 134 . Parental lines with a longer genetic distance are more likely to produce hybrids with strong predominance 135 , 136 . Molecular markers can also be used to directly or indirectly classify heterotic groups by assessing their genetic distance 125 , 137 , 138 . RAPD and AFLP have been successfully used to detect the genetic distance between tested lines, and the yield of carrots was found to be significantly correlated with genetic distance 125 . Genetic distance has also been applied to predict hybrid pepper fruit diameter 139 and hybrid melon ( Cucumis melo L.) fruit shape diameter 140 . The scientific classification of heterotic groups improves the efficiency of selecting hybrid combinations of superior parents and utilizing heterosis (Fig. 6 ).

In addition, some omics approaches, such as genomics, transcriptomics, and metabolomics, have become tools for predicting hybrid yield in rice 141 . Xu et al. 141 analyzed metabolomic and genomic data from 21,445 hybrids developed by 210 recombinant inbred lines and found that metabolomic data were more effective than genomic data in predicting hybrid yield. Research on the prediction of heterosis in vegetables with omics data has not been published. However, the genome or epigenome is the most fundamental source of the plant phenotype, and the transcriptome, proteome, and metabolism are the direct sources of plant phenotypes. Therefore, omics data could represent a more accurate way to predict vegetable hybrid heterosis, and studies of crop hybrid yields can provide a reference for predicting heterosis in vegetables.

Obtaining elite lines based on molecular biotechnology

GWAS is a method used to identify the gene loci that control certain traits in a population by combining phenotypes with genotypes. GWAS is often used to identify certain traits, such as green flesh color or thermotolerance, in cucumber 142 , 143 but can also be used to analyze complex traits, such as yield and biomass 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 . In addition, whole-genome sequencing of various vegetables provides a basis for GWAS (Table 1 ). Due to the unique phenotype of heterosis and its genetic background sources, a genetic population can be composed of different populations or ecotype hybrid populations. A segregated F 2 population that was produced by a strongly predominant F 1 population is regarded as the best population for studying heterosis 27 . Such an F 2 population not only has a reasonable proportion of lines with heterozygous genotypes and homozygous genotypes but also has allele combinations that are distributed evenly at each site 27 .

DeVicente and Tanksley 157 randomly paired an RIL population obtained by strong F 1 self-crossing to produce a new population. This population not only preserves the genotype of the RIL population but also reproduces the F 2 population; thus, it is called an IF 2 population. At present, IF 2 populations have been established in rice 158 , 159 , 160 , 161 , maize 150 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , cotton 170 , and other crops. In addition, there are also diverse F 1 156 , IL 171 , 172 , 173 , 174 , 175 , BILF 1 176 , 177 , and SSSL 178 populations that can be used to study heterosis. Except for two studies on tomato, there are few relevant studies on heterosis in vegetables using such populations that would provide a reference for conducting heterosis-related studies in other vegetables.

Using genome editing techniques to knockout adverse genes or overexpress favorable genes can transform ordinary lines into strong predominance lines. For example, biomass, plant height, and leaf photosynthetic pigment contents increased in rice expressing maize GLK genes compared with those in wild-type rice; 179 such results may cause researchers to think about studying mutual heterosis promotion among different vegetables. Dominance and overdominance effects account for a large proportion of the effects that produce heterosis and are easy to mimic (Fig. 3B ). Understanding the mechanisms of heterosis helps breeders to improve current varieties and generate novel cultivars 27 (Fig. 6 ).

Maintaining heterosis

The hybridization of the selfing line of two heterotic groups can generate hybrid offspring that exhibit heterosis. Through hybrid seed production, self-incompatibility and male-sterile line technology can be used to maintain the hybrid vigor of the hybrid F 1 line. Some of the characteristics of the vegetables themselves, such as the gynoecious characteristic of Cucurbitaceae 116 and asexual reproduction in potato ( Solanum tuberosum L.) 180 , are convenient for hybrid seed production or heterosis maintenance. In addition, some plant hormones or chemical reagents can also be used for plant sex regulation 14 . However, exogenous regulation is often not completely effective 14 , which may affect the purity of hybrid seeds. Therefore, it is necessary to study hybrid systems of vegetables for hybrid seed production.

Du et al. 117 used gene editing technology (Cas9) to knock out the male-specific gene SlSTR1 in tomato to obtain a sterile line and generated a maintainer line by transferring a fertility-restoration gene to the sterile line; it was easy to distinguish whether offspring of crosses between the maintainer and male-sterile lines were male-fertile maintainer plants because a seedling-color gene was linked to the fertility-restoration gene. This system combined tomato sterile lines and gene editing technology and represents a highly practical potential approach to hybrid seed production in tomatoes. Moreover, it may serve as an important reference for the use of gene editing technology for hybrid seed production in other vegetables.

Khanday et al. 181 and Wang et al. 182 found that genome editing can cause mitosis to replace meiosis in rice such that diploid clonal seeds have the original F 1 gene heterozygosity and maintain F 1 traits (Fig. 6 ). Unlike with knocking out the infertility gene using gene editing technology, with this method, fertilization and cell division are necessary for hybridization. Some vegetables do not have sterile line material. Therefore, this method, in which plant fertilization involves only mitosis and not meiosis, will be more widely applicable.

In addition, by repeatedly screening the F 2 lines that were close to the F 1 phenotype, Wang et al. 85 obtained pure F 5 /F 6 lines that were close to the F 1 phenotype; these were called hybrid simulation lines, indicating that the phenotype of the F 1 hybrids was fixed in this line. This method has also been used to maintain F 1 heterosis in other vegetables, such as tomatoes 183 and peas ( Pisum sativum L.) 184 . Therefore, the heterosis of hybrid F 1 vegetables produced by hybridization or molecular biotechnology can be maintained by diploid seed breeding and selection for hybrid simulation lines in the future (Fig. 6 ).

Conclusions and future perspectives

Research on vegetable heterosis has focused mainly on its applications in heterosis breeding. Studies on its genetic mechanism are limited, which hinders its utilization. Extensive progress has been made in the study of heterosis in cereal crops such as rice and maize. In vegetables, both hybrid production systems (male sterility lines, self-incompatibility lines, and gynoecious lines) and molecular biological techniques (gene editing, transgenosis, and asexual reproduction) have been used. Therefore, the methods and strategies proposed by this paper for studying the genetic mechanisms of heterosis can be applied to vegetable breeding. In the near future, we will identify certain heterosis-related gene loci in vegetables to understand the molecular genetics and mechanism of heterosis formation in vegetables and to make new breakthroughs in improving the yield, quality, and safety of vegetables. This review emphasizes the following points: (1) The application of heterosis in vegetable crops allows improvements in yield and quality and enhances plant resistance to biological and environmental stresses. (2) In the future, more attention should be paid to the study of the genetic mechanisms of vegetable heterosis to identify the important genes involved in the development of heterosis and to understand the regulation and activity modes of the key genes affecting vegetable heterosis. (3) By fully referencing and adapting the strategies used in cereal crop heterosis studies, exogenous genes can be applied to produce the same function in different species 179 . Therefore, transgenic and genomic editing technologies can significantly improve the efficiency of research on heterosis gene identification in vegetables. (4) Although a certain basic molecular knowledge of vegetable heterosis has been obtained, applying the knowledge acquired from cereal crops to vegetables will improve vegetable production and quality. It will also be useful to compare sterile line seed production with optimized transgenic systems to achieve more breakthroughs in vegetable production. (5) The study of heterosis can promote the study of ideal plant architecture in vegetable breeding. A breeding strategy that combines heterosis with the ideal plant architecture can achieve substantial gains in vegetable yield and quality. (6) Maintaining heterosis is the core factor of the extensive use of heterosis and has been reflected mainly in F 1 hybrid seed production. With the development of gene editing technology, sterile line gene editing systems, MiMe (Cas9) systems and even new biotechnology approaches will have opportunities to be widely applied; this will be of great significance for hybrid seed production. (7) Progressive heterosis caused by the dosage effect in polyploid hybrids is also an important component of the genetic mechanisms of heterosis, and these phenomena have been observed in different plants 55 , 185 . Polyploid systems allow experiments to be performed that are impossible in diploid systems; hence, polyploid crossbreeding may lead to different plant performance results than diploid breeding. However, polyploids have highly heterozygous genomes and complex genetic structures, and we may not be able to evaluate their phenotypes and genetic structures using diploid criteria. This topic deserves future investigation.

Koelreuter, J. In Methods of Plant Breeding (eds. Hayes, H. K. & Immer, F. R. & Smith, B. C.) (Mcgraw Hill Book Co. Inc., 1763).

Darwin, C. The Effects of Cross and Self Fertilisation in the Vegetable Kingdom (D. Appleton, 1885).

Beal, W. Re. Michigan Board Agric. P 287-288. Cited in Hallauer, AR and J. B. Miranda (1988). Quantitative genetics in maize breeding. (IOWA State Univ. Press Ames, 1880).

Shull, G. H. The composition of a field of maize. J. Hered . 4 , 296–301 (1908).

Shull, G. H. Duplicate genes for capsule-form in Bursa bursa-pastoris. Z. Indukt. Abstamm. Vererbungsl . 12 , 97–149 (1914).

Google Scholar  

Tian, F. et al. Genome-wide association study of leaf architecture in the maize nested association mapping population. Nat. Genet. 43 , 159–162 (2011).

Article   CAS   PubMed   Google Scholar  

Liedl, B. E. & Anderson, N. O. Reproductive barriers: identification, uses and circumvention. Plant Breed. Rev. 11 , 11–154 (1993).

Kumar, S. & Singh, P. Mechanisms for hybrid development in vegetables. J. N. Seeds 6 , 381–407 (2005).

Article   Google Scholar  

Nishi, S. F 1 seed production in Japan. Proc. XVIII Int. Hort. Cong . 3 , 231–257 (1967).

da Silva Dias, J. C. Guiding strategies for breeding vegetable cultivars. Agric. Sci. 5 , 9 (2014).

Hayes, H. K. & Jones, D. First generation crosses in cucumbers. Conn. Storrs. Agric. Exp. Stn. Res. Rpt. 40 , 319–322 (1917).

Daunay, M. C. Eggplant in Vegetables II 163–220 (Springer, 2008).

Singh, P. K., Dasgupta, S. K. & Tripathi, S. K. Hybrid Vegetable Development (CRC Press, 2005).

Colombo, N. & Galmarini, C. R. The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review. Plant Breed. 136 , 287–299 (2017).

Krieger, U., Lippman, Z. B. & Zamir, D. The flowering gene SINGLE FLOWER TRUSS drives heterosis for yield in tomato. Nat. Genet. 42 , 459 (2010).

Zhang, W. L. et al. Cloning and sequence analysis of CsMYB108 gene in cucumber ( Cucumis sativa L.). Mol. Plant Breed. 18 , 4555–4561 (2020).

Liu, T. et al. Enhanced photosynthetic activity in pak choi hybrids is associated with increased grana thylakoids in chloroplasts. Plant J. 103 , 2211–2224 (2020).

Kakizaki, Y. Hybrid vigor in egg-plants and its practical utilization. Genetics 16 , 1 (1931).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Balwani, A., Patel, J., Acharya, R., Gohil, D. & Dhruve, J. Heterosis for fruit yield and its component traits in brinjal ( Solanum melongena L.). J. Pharmacogn. Phytochem. 6 , 187–190 (2017).

CAS   Google Scholar  

Makani, A., Patel, A., Bhatt, M. & Patel, P. Heterosis for yield and its contributing attributes in brinjal ( Solanum melongena L.). bioscan 8 , 1369–1371 (2013).

Tamta, S. & Singh, J. Heterosis in tomato for growth and yield traits. Int. J. Veg. Sci. 24 , 169–179 (2018).

Spaldon, S., Hussain, S., Jabeen, N. & Lay, P. Heterosis studies for earliness, fruit yield and yield attributing traits in chilli ( Capsicum Annum L.). Bioscan 10 , 813–818 (2015).

Kong, X. et al. Transcriptome analysis of biological pathways associated with heterosis in Chinese cabbage. Genomics 112 , 4732–4741 (2020).

Li, H. et al. Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli ( Brassica oleracea L. var. italic ). BMC Plant Biol. 18 , 1–17 (2018).

Article   CAS   Google Scholar  

Sharma, M., Singh, Y., Singh, S. K. & Dhangrah, V. Exploitation of gynoecious lines in cucumber ( Cucumis Sativus L.) for heterosis breeding. Int. J. Bio-Resour. Stress Manag. 7 , 184–190 (2016).

El-Adl, A., Abd El-Hadi, A., Fathy, H. M. & Abdein, M. Heterosis, Heritability and Combining Abilities for some Earliness Traits in Squash ( Cucurbita pepo , L.). Alex. Sci. Exch. J. 35 , 203–214 (2014).

Liu, J., Li, M., Zhang, Q., Wei, X. & Huang, X. Exploring the molecular basis of heterosis for plant breeding. J. Integr. Plant Biol. 62 , 287–298 (2020).

Article   PubMed   Google Scholar  

Qin, J. et al. Identification and characterization of a repertoire of genes differentially expressed in developing top ear shoots between a superior hybrid and its parental inbreds in Zea mays L. Mol. Genet. Genom. 288 , 691–705 (2013).

Kumar, R., Kumar, S., Kumar, D. & Kansal, S. Heterosis, combining ability and gene action studies for insect-pest and disease resistance in cucumber. Electron. J. Plant Breed. 9 , 815–828 (2018).

Kumar, A., Sharma, V., Jain, B. T. & Kaushik, P. Heterosis breeding in eggplant ( Solanum melongena L.): Gains and provocations. Plants 9 , 403 (2020).

Article   CAS   PubMed Central   Google Scholar  

Fujimoto, R. et al. Recent research on the mechanism of heterosis is important for crop and vegetable breeding systems. Breed. Sci . 68 , 145–158 (2018).

Shang, L. G., Gao, Z. Y. & Qian, Q. Progress in Understanding the Genetic Basis of Heterosis in Crops. Chin. Bull. Bot. 52 , 10–18 (2017).

Herath, H. N., Rafii, M., Ismail, S., Nakasha, J. J. & Ramlee, S. Improvement of important economic traits in chilli through heterosis breeding: a review. J. Hortic. Sci. Biotechnol. 96 , 1–10 (2020).

Mallikarjunarao, K., Badu, M., Bandi, H. R. K. & Tripathy, B. Heterosis breeding in Bitter Gourd ( Momordica charantia L.): a review. J. Pharmacogn. Phytochem. 9 , 308–313 (2020).

Davenport, C. B. Degeneration, albinism and inbreeding. Science 28 , 454–455 (1908).

Bruce, A. The Mendelian theory of heredity and the augmentation of vigor. Science 32 , 627–628 (1910).

Jones, D. F. Dominance of linked factors as a means of accounting for heterosis. Genetics 2 , 466 (1917).

East, E. M. Heterosis. Genetics 21 , 375 (1936).

Dranginis, A. Binding of yeast al and α2 as a heterodimer to the operator DNA of a haploid-specific gene. Nature 347 , 682–685 (1990).

Hollick, J. B. & Chandler, V. L. Epigenetic allelic states of a maize transcriptional regulatory locus exhibit overdominant gene action. Genetics 150 , 891–897 (1998).

Stuber, C. W., Lincoln, S. E., Wolff, D., Helentjaris, T. & Lander, E. Identification of genetic factors contributing to heterosis in a hybrid from two elite maize inbred lines using molecular markers. Genetics 132 , 823–839 (1992).

Schnable, P. S. & Springer, N. M. Progress toward understanding heterosis in crop plants. Annu. Rev. Plant Biol . 64 , 71–88 (2013).

Wright, S. Evolution and the Genetics of Populations, volume 1: Genetic and Biometric Foundations (University of Chicago press, 1984).

Sheridan, A. Cross breeding and heterosis Animal Breeding. Anim. Breed. Abst. 49 , 131–144 (1981).

Sao, A. & Mehta, N. Heterosis in relation to combining ability for yield and quality attributes in brinjal ( Solanum melongena L.). Electron. J. Plant Breed. 1 , 783–788 (2010).

Yu, S. et al. Importance of epistasis as the genetic basis of heterosis in an elite rice hybrid. P. Natl Acad. Sci. USA. 94 , 9226–9231 (1997).

Zhong, J. C. Active gene effect hypothesis. J. Southwest Nationalities Nat. Sci. Ed. 20 , 2 (1994).

Kermicle, J. Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission. Genetics 66 , 69 (1970).

Zhang, M. et al. Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize. Genome Res. 24 , 167–176 (2014).

Bao, W. K. Opportunity and risk–considerations for 40 years of breeding research. Plant J. 4 , 4–5 (1990).

Goff, S. A. A unifying theory for general multigenic heterosis: energy efficiency, protein metabolism, and implications for molecular breeding. N. Phytol. 189 , 923–937 (2011).

Yao, H., Gray, A. D., Auger, D. L. & Birchler, J. A. Genomic dosage effects on heterosis in triploid maize. Proc. Natl Acad. Sci. USA. 110 , 2665–2669 (2013).

Fu, D. et al. Utilization of crop heterosis: a review. Euphytica 197 , 161–173 (2014).

Chen, Z. J. Molecular mechanisms of polyploidy and hybrid vigor. Trends Plant Sci. 15 , 57–71 (2010).

Birchler, J. A., Yao, H., Chudalayandi, S., Vaiman, D. & Veitia, R. A. Heterosis. Plant Cell 22 , 2105–2112 (2010).

Liu, C., Wang, M., Wang, L., Guo, Q. & Liang, G. Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids. Genome 61 , 437–447 (2018).

Zhao, X., Chai, Y. & Liu, B. Epigenetic inheritance and variation of DNA methylation level and pattern in maize intra-specific hybrids. Plant Sci. (Amst., Neth.) 172 , 930–938 (2007).

He, G. et al. Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrids. Plant Cell 22 , 17–33 (2010).

Shen, H. et al. Genome-wide analysis of DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids. Plant Cell 24 , 875–892 (2012).

Lauss, K. et al. Parental DNA methylation states are associated with heterosis in epigenetic hybrids. Plant Physiol. 176 , 1627–1645 (2018).

Shivaprasad, P. V., Dunn, R. M., Santos, B. A., Bassett, A. & Baulcombe, D. C. Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs. EMBO J. 31 , 257–266 (2012).

Cerruti, E. et al. Epigenetic bases of grafting-induced vigour in eggplant. bioRxiv https://doi.org/10.1101/831719 (2019).

Kenchanmane, R. S. K. & Niederhuth, C. E. Epigenetic diversity and application to breeding. Adv. Bot. Res. 88 , 49–86 (2018).

Gouil, Q., Novák, O. & Baulcombe, D. C. SLTAB2 is the paramutated SULFUREA locus in tomato. J. Exp. Bot. 67 , 2655–2664 (2016).

Hagemann, R. Somatische Konversion (Paramutation) am sulfurea Locus von Lycopersicon esculentum Mill. Theor. Appl. Genet. 39 , 295–305 (1969).

Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11 , 204–220 (2010).

Kundariya, H. et al. MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants. Nat. Commun. 11 , 1–14 (2020).

Barber, W. T. et al. Repeat associated small RNAs vary among parents and following hybridization in maize. Proc. Natl Acad. Sci. USA. 109 , 10444–10449 (2012).

Nogueira, F. T. Tomato epigenetics: deciphering the “beyond” genetic information in a vegetable fleshy-fruited crop in Epigenetics in Plants of Agronomic Importance: Fundamentals and Applications 247–265 (Springer, 2019).

Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328 , 872–875 (2010).

Lippman, Z. et al. Role of transposable elements in heterochromatin and epigenetic control. Nature 430 , 471–476 (2004).

Vance, V. & Vaucheret, H. RNA silencing in plants-defense and counterdefense. Science 292 , 2277–2280 (2001).

Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293 , 1074–1080 (2001).

Huang, X. & Han, B. Natural variations and genome-wide association studies in crop plants. Annu. Rev. Plant Biol. 65 , 531–551 (2014).

Huang, X. et al. Genomic architecture of heterosis for yield traits in rice. Nature 537 , 629–633 (2016).

Lippman, Z. B. & Zamir, D. Heterosis: revisiting the magic. Trends Genet. 23 , 60–66 (2007).

Xiao, J., Li, J., Yuan, L. & Tanksley, S. D. Dominance is the major genetic basis of heterosis in rice as revealed by QTL analysis using molecular markers. Genetics 140 , 745–754 (1995).

Groszmann, M. et al. Hormone-regulated defense and stress response networks contribute to heterosis in Arabidopsis F1 hybrids. Proc. Natl Acad. Sci. USA. 112 , E6397–E6406 (2015).

Song, G. S. et al. Comparative transcriptional profiling and preliminary study on heterosis mechanism of super-hybrid rice. Mol. Plant 3 , 1012–1025 (2010).

Chen, Z. J. Genomic and epigenetic insights into the molecular bases of heterosis. Nat. Rev. Genet. 14 , 471–482 (2013).

Ni, Z. et al. Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids. Nature 457 , 327–331 (2009).

Baranwal, V. K., Mikkilineni, V., Zehr, U. B., Tyagi, A. K. & Kapoor, S. Heterosis: emerging ideas about hybrid vigour. J. Exp. Bot. 63 , 6309–6314 (2012).

Chen, S. R. Vegetable Breeding (Agricultural Press, 1980).

Preuss, S. B. et al. Multimegabase silencing in nucleolar dominance involves siRNA-directed DNA methylation and specific methylcytosine-binding proteins. Mol. Cell 32 , 673–684 (2008).

Wang, L. et al. Hybrid mimics and hybrid vigor in Arabidopsis. Proc. Natl Acad. Sci. USA. 112 , E4959–E4967 (2015).

Paschold, A. et al. Complementation contributes to transcriptome complexity in maize ( Zea mays L.) hybrids relative to their inbred parents. Genome Res. 22 , 2445–2454 (2012).

Li, D. et al. Integrated analysis of phenome, genome, and transcriptome of hybrid rice uncovered multiple heterosis-related loci for yield increase. Proc. Natl Acad. Sci. USA. 113 , E6026–E6035 (2016).

Springer, N. M. & Stupar, R. M. Allelic variation and heterosis in maize: how do two halves make more than a whole? Genome Res. 17 , 264–275 (2007).

Barboza, G. E. et al. Solanaceae in Flowering plants. Eudicots 295–357 (Springer, 2016).

Khush, G. S. Rice germplasm enhancement at IRRI. Phillipp. J. Crop Sci. 25 , 45–51 (2000).

Donald, C. T. The breeding of crop ideotypes. Euphytica 17 , 385–403 (1968).

Yeager, A. Determinate growth in the tomato. J. Hered. 18 , 263–265 (1927).

Jiang, K., Liberatore, K. L., Park, S. J., Alvarez, J. P. & Lippman, Z. B. Tomato yield heterosis is triggered by a dosage sensitivity of the florigen pathway that fine-tunes shoot architecture. PLoS Genet. 9 , e1004043 (2013).

Article   PubMed   PubMed Central   Google Scholar  

Khan, A. & Jindal, S. Exploiting yield potential in tomato ( Solanum lycopersicum L.) through heterosis breeding. Plant Gene Trait 7 , 1–7 (2016).

Wellington, R. Influence of Crossing in Increasing the Yield of the Tomato (New York Agricultural Experiment Station, 1912).

Tschermak, E. V. Steigerung der Ertragsfähigkeit der Tomaten durch Bastardierung in der F 1 Generation. Nachr. Dtseh. Landwirtsch. Ges. Öst . 51 (1918).

Laterrot, H. Résistance de la tomate au virus de la mosaïque du tabac. Difficultés rencontrées pour la sélection de variétés résistantes. Ann. Am.élior Plantes 23 , 287–316 (1973).

Soost, R. Tobacco mosaic resistance. TGC Rep. 9 , 46 (1959).

Lapushner, D. & Frankel, R. Rationale and practice of tomato F 1 hybrid breeding and seed production. Monographic di Genetica Agraria IV. Roma 259–273 (1979).

Philouze, J. Les hybrides de la tomate: leur intérêt, les techniques d’hybridation, l’utilisation de la stérilité mâle. PHM Revue Hortieole. 164 , 11–18 (1976).

Naresh, B. V., Dubey, A., Tiwari, P. & Dabbas, M. Line x Tester analysis for yield components and cercospora leaf spot resistance in brinjal ( Solanum melongena L.). Electron. J. Plant Breed. 5 , 230–235 (2014).

Pandey, S., Singh, B., Singh, M. & Rai, M. Heterosis in cucumber ( Cucumis sativus L.). Veg. Sci. 32 , 143–145 (2005).

Xu, Y. et al. Whole-genome strategies for marker-assisted plant breeding. Mol. Breed. 29 , 833–854 (2012).

Xing, L. et al. Heterotic group classification of 63 inbred lines and hybrid purity identification by using SSR markers in winter cabbage ( Brassica oleracea L. var. capitata ). Hortic. Plant J. 4 , 158–164 (2018).

Muthuselvi, R. & Praneetha, S. Molecular basis of self-incompatibility in vegetable crops. Int. J. Chem. Stud. 7 , 506–510 (2019).

Earle, E., Stephenson, C., Walters, T. & Dickson, M. Cold-tolerant Ogura CMS brassica vegetables for horticultural use. Cruciferae Newsletter 16 , 80–81 (1994).

Pelletier, G. et al. Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet. MGG 191 , 244–250 (1983).

Xiao, Z. et al. Overcoming cabbage crossing incompatibility by the development and application of self-compatibility-QTL-specific markers and genome-wide background analysis. Front. Plant Sci. 10 , 189 (2019).

Atanassova, B. & Georgiev, H. Using genic male sterility in improving hybrid seed production in tomato ( Lycopersicon esculentum Mill.). Acta Hortic 579 , 185–188 (2000).

Dhall, R. Status of male sterility in vegetables for hybrid development. A review. Adv. Hortic. Sci. 24 , 263–279 (2010).

Georgiev, H. Heterosis in tomato breeding in Genetic improvement of tomato 83–98 (Springer, 1991).

Mizanur, M., Khan, R. & Isshiki, S. Cytoplasmic male sterility in eggplant. Hortic. J . 85 , 1–7 (2016).

Krommydas, K. S. et al. Development and fertility restoration of CMS eggplant lines carrying the cytoplasm of Solanum violaceum. J. Agric. Sci. 8 , 10–26 (2016).

Lin, S. W. et al. Restorer breeding in sweet pepper: introgressing Rf allele from hot pepper through marker-assisted backcrossing. Sci. Hortic. 197 , 170–175 (2015).

Liu, C., Ma, N., Wang, P. Y., Fu, N. & Shen, H. L. Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper ( Capsicum annuum L.). PLoS ONE 8 , e65209 (2013).

Kumar, S. Male sterility in vegetables. Olericulture Fundam. Veg. Prod. 1 , 431–439 (2013).

Du, M. et al. A biotechnology-based male-sterility system for hybrid seed production in tomato. Plant J . 102 , 1090–1100 (2020).

Papadopoulou, E. & Grumet, R. Brassinosteriod-induced femaleness in cucumber and relationship to ethylene production. HortScience 40 , 1763–1767 (2005).

Shannon, S. & Guardia, deLa M. D. Sex expression and the production of ethylene induced by auxin in the cucumber ( Cucumis sativum L.). Nature 223 , 186–186 (1969).

Melchinger, A. E. & Gumber, R. K. Overview of heterosis and heterotic groups in agronomic crops. Concepts Breed. Heterosis Crop Plants 25 , 29–44 (1998).

Chen, J. et al. Genome‐wide association analyses reveal the genetic basis of combining ability in rice. Plant Biotechnol. J. 17 , 2211–2222 (2019).

Mather, K. The balance of polygenic combinations. J. Genet. 43 , 309–336 (1942).

Griffing, B. Concept of general and specific combining ability in relation to diallel crossing systems. Aust. J. Biol. Sci. 9 , 463–493 (1956).

Toker, C. et al. Association between heterosis and genetic distance based on morphological traits and SSR markers in Cicer species. In Pre Breeding–fishing in the Gene Pool» . Abstract of Oral Presentation And Posters of the European Plant Genetic Resources Conference 85 (2013).

Jagosz, B. The relationship between heterosis and genetic distances based on RAPD and AFLP markers in carrot. Plant Breed. 130 , 574–579 (2011).

Sharma, B. B., Sharma, V. K., Dhakar, M. K. & Punetha, S. Combining ability and gene action studies for horticultural traits in garden pea: A review. Afr. J. Agric. Res. 8 , 4718–4725 (2013).

Fasahat, P., Rajabi, A., Rad, J. & Derera, J. Principles and utilization of combining ability in plant breeding. Biom. Biostat. Int J. 4 , 1–24 (2016).

Rajendrakumar, P., Hariprasanna, K. & Seetharama, N. Prediction of heterosis in crop plants—status and prospects. J. Exp. Agric. Int. 9 , 1–16 (2015).

Singh, A., Pan, R. & Bhavana, P. Heterosis and combining ability analysis in bittergourd ( Momordica charantia L.). Bioscan 8 , 1533–1536 (2013).

Kenga, R., Alabi, S. & Gupta, S. Combining ability studies in tropical sorghum ( Sorghum bicolor (L.) Moench ). Field Crops Res. 88 , 251–260 (2004).

Franco, M. C. et al. Combining ability for nodulation in common bean ( Phaseolus vulgaris L.) genotypes from Andean and Middle American gene pools. Euphytica 118 , 265–270 (2001).

Gerdes, J. T. & Tracy, W. F. Pedigree diversity within the Lancaster surecrop heterotic group of maize. Crop Sci. 33 , 334–337 (1993).

Arunachalam, V. & Bandyopadhyay, A. Limits to genetic divergence for occurrence of heterosis Experimental evidence from crop plants. Indian J. Genet. Plant Breed. (India) 44 , 548–554 (1984).

Zhong-hu, H. An investigation of the relationship between the F 1 potential and the measures of genetic distance among wheat lines. Euphytica 58 , 165–170 (1991).

Kaeppler, S. Heterosis: many genes, many mechanisms—end the search for an undiscovered unifying theory. Int. Sch. Res. Not. 2012 , 1–12 (2012).

Poehlman, J. & Sleper, D. Breeding Field Crops (Iowa State University Press, 1995).

Tomkowiak, A., Bocianowski, J., Kwiatek, M. & Kowalczewski, P. Ł. Dependence of the heterosis effect on genetic distance, determined using various molecular markers. Open Life Sci. 15 , 1–11 (2020).

Xiao, J., Li, J., Yuan, L., McCouch, S. & Tanksley, S. Genetic diversity and its relationship to hybrid performance and heterosis in rice as revealed by PCR-based markers. Theor. Appl. Genet. 92 , 637–643 (1996).

Geleta, L., Labuschagne, M. & Viljoen, C. Relationship between heterosis and genetic distance based on morphological traits and AFLP markers in pepper. Plant Breed. 123 , 467–473 (2004).

José, M. A., Iban, E., Silvia, A. & Pere, A. Inheritance mode of fruit traits in melon: Heterosis for fruit shape and its correlation with genetic distance. Euphytica 144 , 31–38 (2005).

Xu, S., Xu, Y., Gong, L. & Zhang, Q. Metabolomic prediction of yield in hybrid rice. Plant J. 88 , 219–227 (2016).

Bo, K. et al. QTL mapping and genome-wide association study reveal two novel loci associated with green flesh color in cucumber. BMC Plant Biol. 19 , 1–13 (2019).

WEI, S. et al. Evaluation and Genome-wide Association Study (GWAS) of seedling thermotolerance in cucumber core germplasm. J. Plant Genet. Resour. 23 , 1223–1231 (2019).

Huang, X. et al. Genomic analysis of hybrid rice varieties reveals numerous superior alleles that contribute to heterosis. Nat. Commun. 6 , 1–9 (2015).

Frascaroli, E. et al. Classical genetic and quantitative trait loci analyses of heterosis in a maize hybrid between two elite inbred lines. Genetics 176 , 625–644 (2007).

Guo, T. et al. Genetic basis of grain yield heterosis in an “immortalized F 2” maize population. Theor. Appl. Genet. 127 , 2149–2158 (2014).

Alpert, K., Grandillo, S. & Tanksley, S. fw 2.2: a major QTL controlling fruit weight is common to both red-and green-fruited tomato species. Theor. Appl. Genet. 91 , 994–1000 (1995).

Zhuang, J., Fan, Y., Wu, J., Xia, Y. & Zheng, K. Identification of over-dominance QTL in hybrid rice combinations. Hereditas 22 , 205–208 (2000).

Mei, H. et al. Gene actions of QTLs affecting several agronomic traits resolved in a recombinant inbred rice population and two backcross populations. Theor. Appl. Genet. 110 , 649–659 (2005).

Li, L. et al. Dominance, overdominance and epistasis condition the heterosis in two heterotic rice hybrids. Genetics 180 , 1725–1742 (2008).

Jiang, G., Zeng, J. & He, Y. Analysis of quantitative trait loci affecting chlorophyll content of rice leaves in a double haploid population and two backcross populations. Gene 536 , 287–295 (2014).

Wei, X. et al. Heterotic loci for various morphological traits of maize detected using a single segment substitution lines test-cross population. Mol. Breed. 35 , 94 (2015).

Li, H. et al. Quantitative trait locus analysis of heterosis for plant height and ear height in an elite maize hybrid zhengdan 958 by design III. BMC Genom. 18 , 36 (2017).

Guo, X. et al. Mapping heterotic loci for yield and agronomic traits using chromosome segment introgression lines in cotton. J. Integr. Plant Biol. 55 , 759–774 (2013).

Tian, S. et al. Overdominance is the major genetic basis of lint yield heterosis in interspecific hybrids between G. hirsutum and G. barbadense. Heredity 123 , 384–394 (2019).

Yang, M. et al. Genomic architecture of biomass heterosis in Arabidopsis. Proc. Natl Acad. Sci. USA. 114 , 8101–8106 (2017).

DeVicente, M. & Tanksley, S. QTL analysis of transgressive segregation in an interspecific tomato cross. Genetics 134 , 585–596 (1993).

Zhou, G. et al. Genetic composition of yield heterosis in an elite rice hybrid. Proc. Natl Acad. Sci. USA. 109 , 15847–15852 (2012).

Radoev, M., Becker, H. C. & Ecke, W. J. G. Genetic analysis of heterosis for yield and yield components in rapeseed ( Brassica napus L.) by quantitative trait locus mapping. Genetics 179 , 1547–1558 (2008).

Li, C. et al. Genetic basis of heterosis for yield and yield components explored by QTL mapping across four genetic populations in upland cotton. BMC Genom. 19 , 910 (2018).

Luo, X. et al. Additive and over‐dominant effects resulting from epistatic loci are the primary genetic basis of heterosis in rice. J. Integr. Plant Biol. 51 , 393–408 (2009).

Tang, J. et al. Dissection of the genetic basis of heterosis in an elite maize hybrid by QTL mapping in an immortalized F 2 population. Theor. Appl. Genet. 120 , 333–340 (2010).

Liu, H. et al. Genome‐wide identification and analysis of heterotic loci in three maize hybrids. Plant Biotechnol. J. 18 , 185–194 (2020).

Ma, L., Wang, Y., Ijaz, B. & Hua, J. Cumulative and different genetic effects contributed to yield heterosis using maternal and paternal backcross populations in Upland cotton. Sci. Rep. UK. 9 , 1–11 (2019).

Li, Z.-K. et al. Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. I. Biomass. Grain Yield. 158 , 1737–1753 (2001).

Luo, L. et al. Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. II. Grain Yield Compon. 158 , 1755–1771 (2001).

Zhu, D., Zhou, G., Xu, C. & Zhang, Q. Genetic components of heterosis for seedling traits in an elite rice hybrid analyzed using an immortalized F 2 population. J. Genet. Genom. 43 , 87–97 (2016).

Song, F. et al. Heterosis for plant height and ear position in maize revealed by quantitative trait loci analysis with triple testcross design. Acta Agronomica Sin. 37 , 1186–1195 (2011).

Liang, Q., Shang, L., Wang, Y. & Hua, J. Partial dominance, overdominance and epistasis as the genetic basis of heterosis in upland cotton ( Gossypium hirsutum L.). PLoS ONE 10 (2015).

Liu, R., Wang, B., Guo, W., Wang, L. & Zhang, T. Differential gene expression and associated QTL mapping for cotton yield based on a cDNA-AFLP transcriptome map in an immortalized F 2. Theor. Appl. Genet. 123 , 439–454 (2011).

Semel, Y. et al. Overdominant quantitative trait loci for yield and fitness in tomato. P. Natl Acad. Sci. USA. 103 , 12981–12986 (2006).

Larièpe, A. et al. The genetic basis of heterosis: multiparental quantitative trait loci mapping reveals contrasted levels of apparent overdominance among traits of agronomical interest in maize ( Zea mays L.). Genetics 190 , 795–811 (2012).

Article   PubMed   PubMed Central   CAS   Google Scholar  

Li, Q. et al. The identification of Cucumis sativus Glabrous 1 ( CsGL1 ) required for the formation of trichomes uncovers a novel function for the homeodomain-leucine zipper I gene. J. Exp. Bot. 66 , 2515–2526 (2015).

Hua, J. et al. Single-locus heterotic effects and dominance by dominance interactions can adequately explain the genetic basis of heterosis in an elite rice hybrid. P. Natl Acad. Sci. USA. 100 , 2574–2579 (2003).

Zhang, L. et al. A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice. Nat. Commun. 8 , 1–10 (2017).

Yu, Y. et al. Genome sequence and QTL analyses using backcross recombinant inbred lines (BILs) and BILF 1 lines uncover multiple heterosis-related loci. Int. J. Mol. Sci. 21 , 780 (2020).

Liu, Y. et al. Identification of quantitative trait loci for kernel-related traits and the heterosis for these traits in maize ( Zea mays L.). Mol. Genet. Genom. 295 , 121–133 (2020).

Xu, M. et al. Heterotic loci analysis for root traits of maize seedlings using an SSSL test population under different nitrogen conditions. Mol. Breed. 40 , 1–12 (2020).

Li, X. et al. Maize GOLDEN2-LIKE genes enhance biomass and grain yields in rice by improving photosynthesis and reducing photoinhibition. Commun. Biol. 3 , 1–12 (2020).

Mohapatra, P. P. & Batra, V. Tissue culture of potato ( Solanum tuberosum L.): A review. Int. J. Curr. Microbiol. Appl. Sci. 6 , 489–495 (2017).

Khanday, I., Skinner, D., Yang, B., Mercier, R. & Sundaresan, V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565 , 91–95 (2019).

Wang, C. et al. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nat. Biotechnol. 37 , 283–286 (2019).

Williams, W. The isolation of ‘pure lines’ from F 1 hybrids of tomato, and the problem of heterosis in inbreeding crop species. J. Agric. Sci. 53 , 347–353 (1959).

Sarawat, P., Stoddard, F. & Marshall, D. Derivation of superior F 5 lines from heterotic hybrids in pea. Euphytica 73 , 265–272 (1993).

Manrique-Carpintero, N. C. et al. Genome reduction in tetraploid potato reveals genetic load, haplotype variation, and loci associated with agronomic traits. Front. plant Sci. 9 , 944 (2018).

Wang, X. et al. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 43 , 1035 (2011).

Li, X. et al. Establishment of Agrobacterium-mediated genetic transformation and application of CRISPR/Cas9 gene-editing system to Chinese cabbage ( Brassica rapa L. ssp. pekinensis ). (2020).

Liu, S. et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 5 , 3930 (2014).

Ma, C. et al. Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Hortic. Plant J. 5 , 164–169 (2019).

Kitashiba, H. et al. Draft sequences of the radish ( Raphanus sativus L.) genome. DNA Res. 21 , 481–490 (2014).

Kim, K. et al. Quantitative Trait Loci (QTLs) Associated with Microspore Culture in Raphanus sativus L.(Radish). Genes 11 , 337 (2020).

Consortium, T. G. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485 , 635 (2012).

Lee, M. H. et al. Temporal and spatial expression analysis of shoot-regeneration regulatory genes during the adventitious shoot formation in hypocotyl and cotyledon explants of tomato (cv. Micro-Tom). Int. J. Mol. Sci. 21 , 5309 (2020).

Consortium, P. G. S. Genome sequence and analysis of the tuber crop potato. Nature 475 , 189 (2011).

Zhang, C., Wang, D., Yang, Y. & Chen, Q. Construction of Potato DM1-3-516-R44 (DM) transgenic system based on agrobacterium transformation. (2020).

Kim, S. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat. Genet. 46 , 270–278 (2014).

Qin, C. et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc. Natl Acad. Sci. USA. 111 , 5135–5140 (2014).

Chee, M. J. Y., Lycett, G. W. & Chin, C. F. Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L. Electron. J. Biotechnol. 34 , 51–58 (2018).

Hirakawa, H. et al. Draft genome sequence of eggplant ( Solanum melongena L.): the representative solanum species indigenous to the old world. DNA Res. 21 , 649–660 (2014).

Khan, H. An Efficient Plant Regeneration System Via Leaf Derived Callus of Solanum melongena L in Propagation and Genetic Manipulation of Plants 93-100 (Springer, 2021).

Huang, S. et al. The genome of the cucumber, Cucumis sativus L. Nat. Genet. 41 , 1275–1281 (2009).

Du, C., Fan, H., Liu, C. & Si, Y. Improved the Agrobacterium tumefaciens-mediated transformation of cucumber by a modified the using of antibiotics and acetosyringone. (2020).

Garcia-Mas, J. et al. The genome of melon ( Cucumis melo L.). Proc. Natl Acad. Sci. USA. 109 , 11872–11877 (2012).

Chenarani, Z., Shokouhifar, F. & Mamarabadi, M. Study the Effects of Explants and Hormonal Levels onDirect Regeneration in Melon ( Cucumis melo L., cv. Khatooni ). J. Hortic. Sci. 32 , 13–22 (2018).

Guo, B. et al. Comparative proteomic analysis of embryos between a maize hybrid and its parental lines during early stages of seed germination. PLoS ONE 8 , e65867 (2013).

Vasudevan, V., Siva, R., Krishnan, V. & Manickavasagam, M. Polyamines, sonication and vacuum infiltration enhances the Agrobacterium-mediated transformation in watermelon ( Citrullus lanatus Thunb.). S. Afr. J. Bot. 128 , 333–338 (2020).

Montero‐Pau, J. et al. De novo assembly of the zucchini genome reveals a whole‐genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 16 , 1161–1171 (2018).

Article   PubMed   CAS   Google Scholar  

Sun, H. et al. Karyotype stability and unbiased fractionation in the paleo-allotetraploid Cucurbita genomes. Mol. Plant 10 , 1293–1306 (2017).

Guo, J., Li, Y., He, C., Yan, Y. & Yu, X. Establishing a High-efficiency Regeneration System in Pumpkin (Cucurbita moschata). Chin. Bull. Bot. 54 , 539 (2019).

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Acknowledgements

This work was supported by the National Key Research and Development Program of China [2016YFD0101705], the Earmarked Fund for Modern Agro–industry Technology Research System [CARS–25] and the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, China.

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Daoliang Yu, Xingfang Gu, Shengping Zhang, Shaoyun Dong, Han Miao, Kiros Gebretsadik & Kailiang Bo

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D.Y. and K.B. conducted the literature review and wrote the paper. X.G., S.Z., S.D., H.M. and K.G. helped review the crop heterosis data. K.B. conceived of and supervised the study. All authors reviewed and approved the final submission.

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Yu, D., Gu, X., Zhang, S. et al. Molecular basis of heterosis and related breeding strategies reveal its importance in vegetable breeding. Hortic Res 8 , 120 (2021). https://doi.org/10.1038/s41438-021-00552-9

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DOI : https://doi.org/10.1038/s41438-021-00552-9

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Unit 7: Inheritance and variation

About this unit.

How do the instructions in genes create the unique traits that we see in every living thing? Learn about how genes work, how they get passed from parent to offspring, and why most living things are genetically unique.

Chromosomes

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4.14: Experiments and Hypotheses

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Now we’ll focus on the methods of scientific inquiry. Science often involves making observations and developing hypotheses. Experiments and further observations are often used to test the hypotheses.

A scientific experiment is a carefully organized procedure in which the scientist intervenes in a system to change something, then observes the result of the change. Scientific inquiry often involves doing experiments, though not always. For example, a scientist studying the mating behaviors of ladybugs might begin with detailed observations of ladybugs mating in their natural habitats. While this research may not be experimental, it is scientific: it involves careful and verifiable observation of the natural world. The same scientist might then treat some of the ladybugs with a hormone hypothesized to trigger mating and observe whether these ladybugs mated sooner or more often than untreated ones. This would qualify as an experiment because the scientist is now making a change in the system and observing the effects.

Forming a Hypothesis

When conducting scientific experiments, researchers develop hypotheses to guide experimental design. A hypothesis is a suggested explanation that is both testable and falsifiable. You must be able to test your hypothesis, and it must be possible to prove your hypothesis true or false.

For example, Michael observes that maple trees lose their leaves in the fall. He might then propose a possible explanation for this observation: “cold weather causes maple trees to lose their leaves in the fall.” This statement is testable. He could grow maple trees in a warm enclosed environment such as a greenhouse and see if their leaves still dropped in the fall. The hypothesis is also falsifiable. If the leaves still dropped in the warm environment, then clearly temperature was not the main factor in causing maple leaves to drop in autumn.

In the Try It below, you can practice recognizing scientific hypotheses. As you consider each statement, try to think as a scientist would: can I test this hypothesis with observations or experiments? Is the statement falsifiable? If the answer to either of these questions is “no,” the statement is not a valid scientific hypothesis.

Practice Questions

Determine whether each following statement is a scientific hypothesis.

• No. This statement is not testable or falsifiable.
• No. This statement is not testable.
• No. This statement is not falsifiable.
• Yes. This statement is testable and falsifiable.

[reveal-answer q=”429550″] Show Answers [/reveal-answer] [hidden-answer a=”429550″]

• d: Yes. This statement is testable and falsifiable. This could be tested with a number of different kinds of observations and experiments, and it is possible to gather evidence that indicates that air pollution is not linked with asthma.
• a: No. This statement is not testable or falsifiable. “Bad thoughts and behaviors” are excessively vague and subjective variables that would be impossible to measure or agree upon in a reliable way. The statement might be “falsifiable” if you came up with a counterexample: a “wicked” place that was not punished by a natural disaster. But some would question whether the people in that place were really wicked, and others would continue to predict that a natural disaster was bound to strike that place at some point. There is no reason to suspect that people’s immoral behavior affects the weather unless you bring up the intervention of a supernatural being, making this idea even harder to test.

[/hidden-answer]

Testing a Vaccine

Let’s examine the scientific process by discussing an actual scientific experiment conducted by researchers at the University of Washington. These researchers investigated whether a vaccine may reduce the incidence of the human papillomavirus (HPV). The experimental process and results were published in an article titled, “ A controlled trial of a human papillomavirus type 16 vaccine .”

Preliminary observations made by the researchers who conducted the HPV experiment are listed below:

• Human papillomavirus (HPV) is the most common sexually transmitted virus in the United States.
• There are about 40 different types of HPV. A significant number of people that have HPV are unaware of it because many of these viruses cause no symptoms.
• Some types of HPV can cause cervical cancer.
• About 4,000 women a year die of cervical cancer in the United States.

Practice Question

Researchers have developed a potential vaccine against HPV and want to test it. What is the first testable hypothesis that the researchers should study?

• HPV causes cervical cancer.
• People should not have unprotected sex with many partners.
• People who get the vaccine will not get HPV.
• The HPV vaccine will protect people against cancer.

[reveal-answer q=”20917″] Show Answer [/reveal-answer] [hidden-answer a=”20917″]Hypothesis A is not the best choice because this information is already known from previous studies. Hypothesis B is not testable because scientific hypotheses are not value statements; they do not include judgments like “should,” “better than,” etc. Scientific evidence certainly might support this value judgment, but a hypothesis would take a different form: “Having unprotected sex with many partners increases a person’s risk for cervical cancer.” Before the researchers can test if the vaccine protects against cancer (hypothesis D), they want to test if it protects against the virus. This statement will make an excellent hypothesis for the next study. The researchers should first test hypothesis C—whether or not the new vaccine can prevent HPV.[/hidden-answer]

Experimental Design

You’ve successfully identified a hypothesis for the University of Washington’s study on HPV: People who get the HPV vaccine will not get HPV.

The next step is to design an experiment that will test this hypothesis. There are several important factors to consider when designing a scientific experiment. First, scientific experiments must have an experimental group. This is the group that receives the experimental treatment necessary to address the hypothesis.

The experimental group receives the vaccine, but how can we know if the vaccine made a difference? Many things may change HPV infection rates in a group of people over time. To clearly show that the vaccine was effective in helping the experimental group, we need to include in our study an otherwise similar control group that does not get the treatment. We can then compare the two groups and determine if the vaccine made a difference. The control group shows us what happens in the absence of the factor under study.

However, the control group cannot get “nothing.” Instead, the control group often receives a placebo. A placebo is a procedure that has no expected therapeutic effect—such as giving a person a sugar pill or a shot containing only plain saline solution with no drug. Scientific studies have shown that the “placebo effect” can alter experimental results because when individuals are told that they are or are not being treated, this knowledge can alter their actions or their emotions, which can then alter the results of the experiment.

Moreover, if the doctor knows which group a patient is in, this can also influence the results of the experiment. Without saying so directly, the doctor may show—through body language or other subtle cues—his or her views about whether the patient is likely to get well. These errors can then alter the patient’s experience and change the results of the experiment. Therefore, many clinical studies are “double blind.” In these studies, neither the doctor nor the patient knows which group the patient is in until all experimental results have been collected.

Both placebo treatments and double-blind procedures are designed to prevent bias. Bias is any systematic error that makes a particular experimental outcome more or less likely. Errors can happen in any experiment: people make mistakes in measurement, instruments fail, computer glitches can alter data. But most such errors are random and don’t favor one outcome over another. Patients’ belief in a treatment can make it more likely to appear to “work.” Placebos and double-blind procedures are used to level the playing field so that both groups of study subjects are treated equally and share similar beliefs about their treatment.

The scientists who are researching the effectiveness of the HPV vaccine will test their hypothesis by separating 2,392 young women into two groups: the control group and the experimental group. Answer the following questions about these two groups.

• This group is given a placebo.
• This group is deliberately infected with HPV.
• This group is given nothing.
• This group is given the HPV vaccine.

[reveal-answer q=”918962″] Show Answers [/reveal-answer] [hidden-answer a=”918962″]

• a: This group is given a placebo. A placebo will be a shot, just like the HPV vaccine, but it will have no active ingredient. It may change peoples’ thinking or behavior to have such a shot given to them, but it will not stimulate the immune systems of the subjects in the same way as predicted for the vaccine itself.
• d: This group is given the HPV vaccine. The experimental group will receive the HPV vaccine and researchers will then be able to see if it works, when compared to the control group.

Experimental Variables

A variable is a characteristic of a subject (in this case, of a person in the study) that can vary over time or among individuals. Sometimes a variable takes the form of a category, such as male or female; often a variable can be measured precisely, such as body height. Ideally, only one variable is different between the control group and the experimental group in a scientific experiment. Otherwise, the researchers will not be able to determine which variable caused any differences seen in the results. For example, imagine that the people in the control group were, on average, much more sexually active than the people in the experimental group. If, at the end of the experiment, the control group had a higher rate of HPV infection, could you confidently determine why? Maybe the experimental subjects were protected by the vaccine, but maybe they were protected by their low level of sexual contact.

To avoid this situation, experimenters make sure that their subject groups are as similar as possible in all variables except for the variable that is being tested in the experiment. This variable, or factor, will be deliberately changed in the experimental group. The one variable that is different between the two groups is called the independent variable. An independent variable is known or hypothesized to cause some outcome. Imagine an educational researcher investigating the effectiveness of a new teaching strategy in a classroom. The experimental group receives the new teaching strategy, while the control group receives the traditional strategy. It is the teaching strategy that is the independent variable in this scenario. In an experiment, the independent variable is the variable that the scientist deliberately changes or imposes on the subjects.

Dependent variables are known or hypothesized consequences; they are the effects that result from changes or differences in an independent variable. In an experiment, the dependent variables are those that the scientist measures before, during, and particularly at the end of the experiment to see if they have changed as expected. The dependent variable must be stated so that it is clear how it will be observed or measured. Rather than comparing “learning” among students (which is a vague and difficult to measure concept), an educational researcher might choose to compare test scores, which are very specific and easy to measure.

In any real-world example, many, many variables MIGHT affect the outcome of an experiment, yet only one or a few independent variables can be tested. Other variables must be kept as similar as possible between the study groups and are called control variables . For our educational research example, if the control group consisted only of people between the ages of 18 and 20 and the experimental group contained people between the ages of 30 and 35, we would not know if it was the teaching strategy or the students’ ages that played a larger role in the results. To avoid this problem, a good study will be set up so that each group contains students with a similar age profile. In a well-designed educational research study, student age will be a controlled variable, along with other possibly important factors like gender, past educational achievement, and pre-existing knowledge of the subject area.

What is the independent variable in this experiment?

• Sex (all of the subjects will be female)
• Presence or absence of the HPV vaccine
• Presence or absence of HPV (the virus)

[reveal-answer q=”68680″]Show Answer[/reveal-answer] [hidden-answer a=”68680″]Answer b. Presence or absence of the HPV vaccine. This is the variable that is different between the control and the experimental groups. All the subjects in this study are female, so this variable is the same in all groups. In a well-designed study, the two groups will be of similar age. The presence or absence of the virus is what the researchers will measure at the end of the experiment. Ideally the two groups will both be HPV-free at the start of the experiment.

List three control variables other than age.

[practice-area rows=”3″][/practice-area] [reveal-answer q=”903121″]Show Answer[/reveal-answer] [hidden-answer a=”903121″]Some possible control variables would be: general health of the women, sexual activity, lifestyle, diet, socioeconomic status, etc.

What is the dependent variable in this experiment?

• Sex (male or female)
• Rates of HPV infection
• Age (years)

[reveal-answer q=”907103″]Show Answer[/reveal-answer] [hidden-answer a=”907103″]Answer b. Rates of HPV infection. The researchers will measure how many individuals got infected with HPV after a given period of time.[/hidden-answer]

Contributors and Attributions

• Revision and adaptation. Authored by : Shelli Carter and Lumen Learning. Provided by : Lumen Learning. License : CC BY-NC-SA: Attribution-NonCommercial-ShareAlike
• Scientific Inquiry. Provided by : Open Learning Initiative. Located at : https://oli.cmu.edu/jcourse/workbook/activity/page?context=434a5c2680020ca6017c03488572e0f8 . Project : Introduction to Biology (Open + Free). License : CC BY-NC-SA: Attribution-NonCommercial-ShareAlike

Module 12: Trait Inheritance

Genetics and the environment, discuss the role environment plays on phenotypes.

In recent years, scientists have begun to research how our environment can impact our phenotypes. In this outcome, we’ll learn about a few different ways our environment can impact us.

Learning Objectives

• Explain how epistasis impacts trait expression
• Describe polygenic inheritance and how to recognize it
• Describe continuous variation and how to recognize it
• Identify gene-environment interaction and how this impacts trait expression
• Explain pleiotropy and its impact on traits in a population

Mendel’s studies in pea plants implied that the sum of an individual’s phenotype was controlled by genes (or as he called them, unit factors), such that every characteristic was distinctly and completely controlled by a single gene. In fact, single observable characteristics are almost always under the influence of multiple genes (each with two or more alleles) acting in unison. For example, at least eight genes contribute to eye color in humans.

In some cases, several genes can contribute to aspects of a common phenotype without their gene products ever directly interacting. In the case of organ development, for instance, genes may be expressed sequentially, with each gene adding to the complexity and specificity of the organ. Genes may function in complementary or synergistic fashions, such that two or more genes need to be expressed simultaneously to affect a phenotype. Genes may also oppose each other, with one gene modifying the expression of another.

In epistasis, the interaction between genes is antagonistic, such that one gene masks or interferes with the expression of another. “Epistasis” is a word composed of Greek roots that mean “standing upon.” The alleles that are being masked or silenced are said to be hypostatic to the epistatic alleles that are doing the masking. Often the biochemical basis of epistasis is a gene pathway in which the expression of one gene is dependent on the function of a gene that precedes or follows it in the pathway.

An example of epistasis is pigmentation in mice. The wild-type coat color, agouti ( AA ), is dominant to solid-colored fur ( aa ). However, a separate gene ( C ) is necessary for pigment production. A mouse with a recessive c allele at this locus is unable to produce pigment and is albino regardless of the allele present at locus A (Figure 1). Therefore, the genotypes AAcc , Aacc , and aacc all produce the same albino phenotype. A cross between heterozygotes for both genes ( AaCc x AaCc ) would generate offspring with a phenotypic ratio of 9 agouti:3 solid color:4 albino (Figure 1). In this case, the C gene is epistatic to the A gene.

Figure 1. In mice, the mottled agouti coat color ( A ) is dominant to a solid coloration, such as black or gray. A gene at a separate locus ( C ) is responsible for pigment production. The recessive c allele does not produce pigment, and a mouse with the homozygous recessive cc enotype is albino regardless of the allele present at the A locus. Thus, the C gene is epistatic to the A gene.

Epistasis can also occur when a dominant allele masks expression at a separate gene. Fruit color in summer squash is expressed in this way. Homozygous recessive expression of the  W gene ( ww ) coupled with homozygous dominant or heterozygous expression of the Y gene ( YY or Yy ) generates yellow fruit, and the wwyy genotype produces green fruit. However, if a dominant copy of the W gene is present in the homozygous or heterozygous form, the summer squash will produce white fruit regardless of the Y alleles. A cross between white heterozygotes for both genes ( WwYy × WwYy ) would produce offspring with a phenotypic ratio of 12 white:3 yellow:1 green.

Finally, epistasis can be reciprocal such that either gene, when present in the dominant (or recessive) form, expresses the same phenotype. In the shepherd’s purse plant ( Capsella bursa-pastoris ), the characteristic of seed shape is controlled by two genes in a dominant epistatic relationship. When the genes A and B are both homozygous recessive ( aabb ), the seeds are ovoid. If the dominant allele for either of these genes is present, the result is triangular seeds. That is, every possible genotype other than aabb results in triangular seeds, and a cross between heterozygotes for both genes ( AaBb x AaBb ) would yield offspring with a phenotypic ratio of 15 triangular:1 ovoid.

As you work through genetics problems, keep in mind that any single characteristic that results in a phenotypic ratio that totals 16 is typical of a two-gene interaction. Recall the phenotypic inheritance pattern for Mendel’s dihybrid cross, which considered two non-interacting genes—9:3:3:1. Similarly, we would expect interacting gene pairs to also exhibit ratios expressed as 16 parts. Note that we are assuming the interacting genes are not linked; they are still assorting independently into gametes.

Whether or not they are sorting independently, genes may interact at the level of gene products such that the expression of an allele for one gene masks or modifies the expression of an allele for a different gene. This is called epistasis.

Polygenic Inheritance and Environmental Effects

How is height inherited.

Many heritable human characteristics don’t seem to follow Mendelian rules in their inheritance patterns. For example, consider human height. Unlike a simple Mendelian characteristic, human height displays:

• Continuous variation. Unlike Mendel’s pea plants, humans don’t come in two clear-cut “tall” and “short” varieties. In fact, they don’t even come in four heights, or eight, or sixteen. Instead, it’s possible to get humans of many different heights, and height can vary in increments of inches or fractions of inches. As an example, consider the bell curve-shaped graph in Figure 2, which shows the heights of a group of male high school seniors.
• A complex inheritance pattern. If you’ve paid attention to the heights of your friends and family, you may have noticed that many different patterns of inheritance are possible. Tall parents can have a short child, short parents can have a tall child, and two parents of different heights may or may not have a child of intermediate height. In addition, siblings with the same two parents may have a range of heights, ones that don’t fall into clear, distinct categories. Simple models involving one or two genes can’t accurately predict all of these inheritance patterns.

Figure 2. Heights of male high school seniors. Image modified from “ Continuous variation: Quantitative traits ,” by J. W. Kimball (CC BY 3.0).

How, then, is height inherited? Height and other similar features are controlled not just by one gene, but rather, by multiple (often many) genes that each make a small contribution to the overall outcome. This inheritance pattern is called polygenic inheritance ( poly – = many). For instance, a recent study found over 400 genes linked to variation in height [1] . When there are large numbers of genes involved, it becomes hard to distinguish the effect of each individual gene, and even harder to see that gene variants (alleles) are inherited according to Mendelian rules. In a further complication, height doesn’t just depend on genetics: it also depends a lot on environmental factors, such as a child’s overall health and the type of nutrition he or she receives while growing up.

In this article, we’ll look in more detail at how complex human traits such as height are inherited, as well as how factors like genetic background and environment can influence the phenotype (observable features) produced by a particular genotype (set of gene variants, or alleles).

Polygenic Inheritance

Some human characteristics, such as height, eye color, and hair color, don’t come in just a few distinct forms. Instead, they vary in small gradations, forming a spectrum or continuum of possible phenotypes. Features like these are called quantitative characters, and they’re typically controlled by multiple genes (often, many genes), each of which contributes to the overall phenotype. For example, although there are two major eye color genes, there are at least 14 additional genes that play roles in determining a person’s exact eye color [2] .

Looking at a real example of a human polygenic trait would get complicated, largely because we’d have to keep track of tens, or even hundreds, of different allele pairs. However, we can use an example involving the color of wheat kernels to see how Mendelian inheritance of multiple genes (plus a little incomplete dominance of alleles) can produce a broad spectrum of phenotypes [3] . In this example, there are three genes that make reddish pigment in wheat kernels, which we’ll call A , B , and C . Each comes in two alleles, one of which makes a unit of pigment (the capital-letter allele) and one of which does not make any pigment (the lowercase allele). Thus, the aa genotype would contribute zero units of pigment, the Aa genotype would contribute one unit, and the AA genotype would contribute two—basically, a form of incomplete dominance.

Now, let’s imagine that two plants heterozygous for all three genes ( AaBbCc ) were crossed to one another (or, equivalently, allowed to self-fertilize). Each of the parent plants would have three units of pigment, or pinkish kernels. Their offspring, however, could display seven different categories of phenotypes, ranging from zero units of pigment ( aabbcc ) and pure white kernels to six units of pigment ( AABBCC ) and dark red kernels, with the intermediate phenotypes being most common.

This example illustrates how we can get a spectrum of slightly different phenotypes (something approaching continuous variation) with just three genes whose alleles display incomplete dominance. It’s not hard to imagine that, as we increased the number of genes involved, we’d be able to get even finer variations in color, or in another trait such as height. Real polygenic traits aren’t usually quite this clean and simple. (For instance, genes may make unequal contributions to the phenotype, alleles may or may not display incomplete dominance, and there may be non-additive interactions between genes.) However, the basic idea—that multiple genes obeying Mendelian rules can produce a spectrum of finely differing phenotypes—holds true for human traits such as skin and eye color.

PRactice Questions

We’ve learned about polygenic inheritance and continuous variation. Just what is the difference between these two types of inheritance?

Effect of the Environment

Characteristics that are influenced by environmental as well as genetic factors are called multifactorial . The idea of “nature versus nurture” — in other words, the relative influence of genetics versus environmental factors — has been and still is debated. Just looking at the genes of a given organism will not determine how that organism will develop and act. Even identical twins will show different characteristics, depending on the environment in which they live. Everyone is a product of their environment as well as their genetics.

Even when influenced by the environment, phenotypes have a normal range of expression. For instance, human height varies based on nutrition and genetics, but not many people are shorter than 4½ feet or taller than 7 feet. The range of phenotypic possibilities is called the norm of reaction . Hydrangeas, for example, may be blue, pink, or purple, but they are never naturally orange. Hydrangeas are blue in acidic soil with available aluminum, and they are pink in alkaline soil without available aluminum.

Some human characteristics have a narrow norm of reaction, such as blood type. Others have a wide norm of reaction, such as the number of blood cells in humans, which varies depending on factors that include physical fitness, presence or history of infections, and even the altitude at which a person lives.

Figure 4. Taking a newborn blood sample for PKU testing. By Staff Sgt Eric T. Sheler, U.S. Air Force (Phenylketonuria testing) Public Domain

The environment also affects human genes. Serotonin, a neurotransmitter that acts inside brain cells, lowers anxiety and depression during traumatic times. Mutations in the serotonin transporter gene may cause a reduced ability to cope with stress. That does not mean that the person is always depressed, but if the environment produces stress, the person may become depressed more easily than a person with unmutated serotonin transporter genes.

You already learned about PKU, a pleiotropic disorder caused by defects in a single gene coding for an enzyme that converts the amino acid phenylalanine to tyrosine. Newborns are tested for this defect very early in life (Figure 4), so that if the results are positive, they can be given a diet limiting phenylalanine ingestion. That way, the toxic buildup is prevented and the children can develop normally. PKU is an example in which environmental factors can modify gene expression.

Practice Question

Two identical twins (female) live in different parts of the country. One is very committed to a healthy lifestyle: not smoking, exercising regularly, eating a diet rich in fresh produce, and avoiding red meats and processed foods. The other is not as careful: she smokes, is overweight, and often eats fast and processed foods. They are aware that several women in their family have had breast cancer, and decide to consult a doctor about their odds of developing the disease. Which of the following statements by the doctor sounds most correct?

• As identical twins, you are genetically the same, so your chances of developing breast cancer are identical.
• The twin with the healthy lifestyle should not be terribly concerned, while the one with the unhealthy lifestyle is at a higher risk.
• Breast cancer has a genetic component, and the twins have identical genes, so they have the same genetic risk. However, environmental factors such as smoking, obesity, and consumption of red meat have been shown to increase the risk of cancer. While both twins should monitor themselves closely, the twin who smokes and is overweight may want to consider a healthier lifestyle to decrease her risk of breast cancer.

While genes and genetic causes play a large role in health and phenotypes, the environment also plays an important role. Understanding this can enable the treatment of some disorders, such as the case with PKU in which limiting the intake of phenylalanine can prevent toxic build up of this amino acid. Often the norm of reaction is set by genetic factors but ultimately determined by environmental exposures.

Pleiotropy and Human Disorders

Based on Mendel’s experiments, you might imagine that all genes control a single characteristic, are present in two copies, and affect some harmless aspect of an organism’s appearance (such as color, height, or shape). Although those predictions are accurate in many cases, there are also some important exceptions. For instance, how can we explain observations like the following?

• The genetic disorder Marfan syndrome is caused by a mutation in one gene, yet it affects many aspects of growth and development, including height, vision, and heart function.

To understand observations like these, we need to look more deeply at what genes are. Rather than abstract “heritable factors,” genes are stretches of DNA found on chromosomes, and most of them encode (specify the sequence of) proteins that do a certain job in the cell or body. In this article, we’ll look in more detail at genes affecting multiple characteristics (pleiotropy).

When we discussed Mendel’s experiments with purple-flowered and white-flowered plants, we didn’t mention any other phenotypes associated with the two flower colors. However, Mendel noticed that the flower colors were always correlated with two other features: the color of the seed coat (covering of the seed) and the color of the axils (junctions where the leaves met the main stem) [4] . In plants with white flowers, the seed coats and axils were colorless, while in plants with purple flowers, the seed coats were brown-gray and the axils were reddish. Thus, rather than affecting just one characteristic, the flower color gene actually affected three.

Figure 5. Based on similar diagram by Ingrid Lobo

Genes like this, which affect multiple, seemingly unrelated aspects of an organism’s phenotype, are said to be pleiotropic ( pleio – = many, – tropic = effects) [5] . We now know that Mendel’s flower color gene encodes a regulator protein that activates pigment biosynthesis, and that it works in several different parts of the pea plant (flowers, seed coat, and leaf axils). Thus, the seemingly unrelated phenotypes can all be traced back to a defect in a single gene with several jobs.

Importantly, alleles of pleiotropic genes are transmitted in the same way as alleles of genes that affect single traits. Although the phenotype has multiple elements, these elements are specified as a package, and the dominant and recessive versions of the package would appear in the progeny of a monohybrid cross in a ratio of 3:1.

Pleiotropy in Human Genetic Disorders

Genes affected in human genetic disorders are often pleiotropic. For example, people with the hereditary disorder Marfan syndrome may have a constellation of seemingly unrelated symptoms [6] :

• Unusually tall height
• Thin fingers and toes
• Dislocation of the lens of the eye
• Heart problems (in which the aorta, the large blood vessel carrying blood away from the heart, bulges or ruptures).

These symptoms don’t appear directly related to one another, but as it turns out, they can all be traced back to the mutation of a single gene. This gene encodes a protein that assembles into chains, making elastic fibrils that give strength and flexibility to the body’s connective tissues [7] . Disease-causing mutations in the Marfan syndrome reduce the amount of functional protein produced, resulting in fewer fibrils. The eye and the aorta normally contain many fibrils that help maintain structure, explaining why these two organs are strongly affected in Marfan syndrome [8] . In addition, the fibrils serve as “storage shelves” for growth factors. When there are fewer of them in Marfan syndrome, the growth factors cannot be shelved and thus cause excess growth (leading to the characteristic tall, thin Marfan build) [9] .

Check Your Understanding

Answer the question(s) below to see how well you understand the topics covered in the previous section. This short quiz does  not  count toward your grade in the class, and you can retake it an unlimited number of times.

Use this quiz to check your understanding and decide whether to (1) study the previous section further or (2) move on to the next section.

• Wood, A. R., Esko, T., Yang, J., Vedantam, S., Pers, T. H., Gustafsson, S., ... Frayling, T. M. (2014). Defining the role of common variation in the genomic and biological architecture of adult human height. Nature Genetics , 46 , 1173–1186. http://dx.doi.org/10.1038/ng.3097 . ↵
• White, D. and Rabago-Smith, M. (2011). Genotype-phenotype associations and human eye color. Journal of Human Genetics , 56 , 5–7. http://dx.doi.org/10.1038/jhg.2010.126 . ↵
• Kimball, J. W. (2011, March 8). Continuous variation: Quantitative traits. Retrieved from  http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/Q/QTL.html . ↵
• Lobo, I. (2008). Pleiotropy: One gene can affect multiple traits. Nature Education , 1 (1), 10. Retrieved from http://www.nature.com/scitable/topicpage/pleiotropy-one-gene-can-affect-multiple-traits-569 . ↵
• Ibid . ↵
• Marfan syndrome. (2012). In Genetics home reference . Retrieved from  http://ghr.nlm.nih.gov/condition/marfan-syndrome . ↵
• FBN1. (2015). In Genetics home reference . Retrieved from http://ghr.nlm.nih.gov/gene/FBN1 . ↵
• Marfan syndrome. (2015, November 3). Retrieved November 21, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Marfan_syndrome . ↵
• Introduction to Genetics and the Environment. Authored by : Shelli Carter and Lumen Learning. Provided by : Lumen Learning. License : CC BY: Attribution
• Biology. Provided by : OpenStax CNX. Located at : http://cnx.org/contents/[email protected] . License : CC BY: Attribution . License Terms : Download for free at http://cnx.org/contents/[email protected]
• Polygenic inheritance and environmental effects. Provided by : Khan Academy. Located at : https://www.khanacademy.org/science/biology/classical-genetics/variations-on-mendelian-genetics/a/polygenic-inheritance-and-environmental-effects . License : CC BY-NC-SA: Attribution-NonCommercial-ShareAlike
• Application Spotlight: Effect of the Environment. Provided by : Open Learning Initiative. Located at : https://oli.cmu.edu/jcourse/workbook/activity/page?context=434a5f4180020ca600915a031e68a874 . Project : Introduction to Biology (Open + Free). License : CC BY-NC-SA: Attribution-NonCommercial-ShareAlike
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4 Genes and Cells

• The Fundamental Biological Unit of All Living Organisms Is the Cell

To a surprising degree, all cells are similar in design and function, whether in human beings, in plants, or as simple single-celled organisms such as bacteria. One major difference, however, is the presence or absence of a distinct compartment, the nucleus, for the genome. Cells with a nucleus, called eukaryotes, are found in advanced single-celled organisms and multicellular organisms. Those without nuclei, called prokaryotes are the simplest single-celled organisms, the bacteria.

The importance of the cell as a biological unit is made clear when we consider the life cycle of advanced multicellular organisms such as human beings. The cycle begins with the fusion of egg and sperm, themselves single cells of specialized types, to form the one-celled embryo. At the earliest stage of our life cycle, therefore, we exist as a single cell. This cell divides into two cells, each of those into two more, and so on, to give rise to the adult organism, which may contain as many as 1 million billion cells (see Chapter 5 ). Every one of these cells is autonomous in some functions and dependent or interdependent in others.

The entire developmental process is regulated by the myriad interactions within and among individual cells. These interactions regulate the capacity of cells to multiply, to differentiate into the hundreds of different cell types that make up our bodies, and to organize themselves into tissues, organs, and finally the human body itself, according to a specific, well-defined architectural plan.

This cycle of activity characterizes normal healthy individuals, but the process can go awry, leading to abnormal states characterized by disease in humans, animals, and plants. Since both normal and abnormal progression of the life cycle is governed in a fundamental way by cells, it is logical that to understand and exert control over these phenomena and, in a real sense, over our own lives and biological environment, it is necessary to learn all we can about the cell and how it conducts its activities.

• Research Strategies

Since all organisms are related through evolution, we can use information from simple organisms to determine protein and gene functions in higher and more complicated organisms. Furthermore, new technologies allow us to test these inferences by expressing the genes of more complicated species, such as humans, in the cells of simple organisms such as yeast or bacteria.

The identification of the genes and the proteins involved in various cellular functions is only part of the story. A deep understanding of the roles played by individual proteins requires detailed characterization of their mechanisms of action. Ultimately this requires knowledge of the three-dimensional structure of each protein and of the rates of its reaction with other molecules. This characterization requires the development of test-tube (in vitro) assays of function. Genetics can contribute to this effort by providing components from mutant cells that can be used to identify essential components and to test the molecular roles inferred from the physiology of live cells. Likewise, components identified as part of in vitro mechanisms can be used to isolate the corresponding genes and produce defective mutants to test in the live cell the functions inferred in vitro.

Many Cellular Functions Are Carried Out By Macromolecular Assemblies That Can Be Isolated and Reconstituted from Their Constituent Molecules

Some biological functions such as the fermentation of sugar to alcohol are carded out by individual proteins, alone or in a series of catalytic reactions, but many others including photosynthesis and the beating of sperm tails require complex assemblies consisting of many different types of proteins and other molecules such as lipids and carbohydrates. These subcellular molecular machines include the organelles that can be seen by light microscopy as well as many smaller structures.

Perhaps the most successful research strategy in cell biology for the past 30 years has been to purify these molecular machines so that their functions can be studied outside the living cell. Fortunately, the organelles and many smaller macromolecular complexes are so well constructed that they emerge from the trauma of separation still able to perform complex, highly integrated functions such as contraction, transport of molecules across membranes, photosynthesis, the production of adenosine triphosphate (ATP), protein synthesis, and gene transcription. This strategy has yielded a biochemical inventory and, in addition, a rather detailed functional characterization of each organelle showing that functional specialization and division of biochemical and biophysical labor prevail at the subcellular level.

At the same time, studies of reconstituted cell fractions have demonstrated that most macromolecular machines are formed by the serf-assembly of their component molecules. More recently, this approach has been used to demonstrate that cell organelles can interact in vitro to reconstitute a specific intracellular process. Whole organelles such as the nucleus can now be reversibly disassembled and reassembled in the test tube. A striking feature of the current state of research in molecular and cell biology is the remarkable degree to which the effectiveness of the reductionist strategy has been confirmed by experiments.

In Each Field of Cell Biology, Particularly Favorable Cells Have Been Identified for Experimental Work

To a great extent, progress in molecular and cellular biology depends on finding cell types that are suitable for experiments. Fortunately, because of the parsimony of nature, mechanisms for determining the most important cellular processes are shared by cells all along the phylogenetic tree. For example, the force-producing protein myosin from muscle can bind functionally to actin filaments from all eukaryotic organisms. Similarly, protein synthesis can be reconstituted in vitro from different components from plant and animal cells.

Fruit flies, sea urchins, and chickens have all been excellent sources of model systems for analyzing cell behavior during embryonic development. Amphibian eggs, because of their large size, are ideal for the production of foreign proteins by microinjection of messenger RNAs (mRNAs). Highly motile amoebas that can be grown in large quantities have yielded much of the basic information about cytoplasmic contractile proteins. Chlamydomonas , an alga with two flagella and well-characterized genetics, has provided much of what we know about the molecular biology of flagella and cilia.

The occurrence in yeast of three specific cell types, each of which plays a distinctive role in the cell's life cycle, makes the organism suitable for investigations in several important areas of cell biology, including genetic programming for cell differentiation. Yeasts are also especially valuable for combined biochemical and genetic studies of control of the cell cycle, exocytosis (secretion), endocytosis (uptake), and biogenesis of such cell organelles as mitochondria.

• The Nucleus

The Nucleus is Both the Warehouse and the Factory for Most of the Cell's Genetic Material and Activity

The nucleus has three main functions: the storage, replication, and expression of the genes. The nucleus in most human cells is a sphere roughly 10 micrometers in diameter that contains the almost 2 meters of DNA that makes up the human genome.

The DNA contains the code for all cellular proteins and RNAs, as illustrated in the pathway represented by the ''central dogma":

This information is processed with exquisite precision. In most cells only a tiny fraction of the total number of genes are actually expressed at any given time (or ever). Furthermore, the patterns of gene expression change in precisely programmed ways during development. Thus, cells in kidney and brain express independent repertoires of genes even though they originate from a single fertilized ovum and have identical copies of the genetic material. The cells of a developing embryo act like tiny computers that accumulate and remember information concerning their past and present locations relative to other cells and express genes appropriate to this information. The study of the control of gene expression is an important focus of modern cell biology research. How do the cells of complex multicellular organisms turn on and off their genes? And how is the position of a cell "read out" by the cell to control specific genes? These are difficult but central questions to cell biology that are being actively pursued in many laboratories.

DNA is complexed with proteins to form chromosomes. All human body cells (somatic cells) contain 46 chromosomes. The germ cells (sperm in the male and eggs in the female) that contribute to the embryos of the next generation contain only half that number—23. In addition, the nucleus contains an abundance of proteins and RNAs, representing various structural elements and the enzymes and products of replication, transcription, and RNA processing. How all of these components are organized within the nucleus is unknown, and research on the three-dimensional structure of chromosomes and other nuclear components is needed to better understand how genes function and are controlled.

Nuclear Envelope

All molecular traffic between the nucleus and cytoplasm is by way of the nuclear envelope.

Replication of chromosomes and synthesis of most mRNA molecules is restricted to the nucleus, whereas translation of messenger RNA into protein occurs only in the cytoplasm. Separation of chromosomes from the cytoplasmic space is thought to ensure proper regulation of nucleic acid and protein synthesis in the cell. Except at the time of mitosis, when the nuclear envelope breaks down, all molecular traffic between the nucleus and cytoplasm is by way of this envelope, which consists of two parallel membranes joined at regions called nuclear pores.

Nuclear pores consist of a precise geometrical arrangement of structural elements. The transport of large molecules across the pores and the direction in which individual molecules are transported (cytoplasm to nucleus or vice versa) seems to be selective. Selective proteins are actively moved from their site of synthesis in the cytoplasm into the nucleus by means of an active transport system in the nuclear pores. Selective RNAs are pumped out of the nucleus through the same pores. For the transported proteins, the specificity is known to reside in a short sequence of their amino acids that seems to be recognized by a component of the nuclear pore. A number of pore complex proteins have been identified. However, the organization of these proteins in the pore complex and their role in regulating traffic in and out of the nucleus are largely unknown. Future analyses of the structure, molecular composition of the pore complex, and the genes encoding its proteins will undoubtedly reveal how this gatekeeper of the nucleus works.

Attached to the inner surface of the nuclear envelope with its pores and pore complexes is a fibrous network known as the nuclear lamina. The major proteins of the lamina, called lamins, have been identified and shown to be related to the intermediate filaments found in the cytoplasm. (See the section on cytoskeleton in this chapter.) The nuclear envelope seems to be a major site for anchoring chromosomes and may also facilitate the packing of DNA in the nucleus. The reversible assembly of the lamina may help control the breakdown and reformation of the nuclear envelope during mitosis.

Chromosomes

Chromosomes are the structural units that contain the dna.

Each chromosome consists of one extraordinarily long DNA molecule complexed with a multitude of proteins. An orderly condensation of long DNA molecules into much smaller chromosomes is mediated by nuclear proteins. The first level of folding involves a set of five proteins called histories. DNA interacts with histones to form a regular beadlike structure, the nucleosome. Approximately 160 nucleotide pairs of DNA are wrapped around a core formed from two copies each of four histone molecules (the histone octamer). This basic structure is repeated over and over to give a beads-on-a-string appearance when chromosome fibers are viewed in the electron microscope.

DNA complexed with histones is generally referred to as chromatin. Chromatin fibers consisting of nucleosome beads are folded into still more complex structures known as superbeads or supercoils, depending upon one's view of their organization. For technical reasons this higher-order folding of chromatin has been hard to study, and we are only beginning to understand how chromatin folding may be related to important processes such as the turning on and off of genes.

Beyond superbeads or supercoils, one major level of chromosome folding can be recognized, the so-called loops. Loops have been studied in the lampbrush chromosomes of oocytes for more than 100 years; the giant lampbrush chromosomes consist of several hundred individually recognizable loops of chromatin extending laterally from the main axis. These lampbrush loops are regions of unusually intense RNA synthesis that provide a unique opportunity to visualize active gene transcription. When RNA synthesis is completed, the loops contract back into the main axis of the chromosomes. The loops are thought to retain their individuality even during mitosis, the time of maximal chromosome condensation. Loops of chromatin similar to those of the lampbrush chromosomes can be seen when normal mitotic chromosomes are chemically treated to loosen their structure.

We know very little about the composition (or intranuclear location) of the macromolecular complexes that anchor the DNA into loop-domains inside nuclei. Fortunately, however, the genome does not remain in the dispersed state characteristic of physiological activity during the entire cell cycle. At cell division (mitosis), the nuclear envelope disassembles and the genome condenses into discrete chromosomes. These mitotic chromosomes provide an opportunity to study the organization of the DNA loop-domains in the absence of the many soluble components that participate in transcription and replication. Methods are currently being developed to identify proteins that regulate mitotic chromosome architecture. Recently one putative component of the loop-domain anchor complex was identified as the enzyme DNA topoisomerase II, which had previously been identified as able to knot and unknot DNA molecules in vitro. Genetic analysis shows that this activity is required at mitosis if the two sets of intertwined DNA molecules are to be successfully partitioned to daughter cells. Future studies should identify other components of the anchor complex and eventually enable us to determine what role they may play in the regulation of a gene's activity.

The domain idea has matured sufficiently during the past decade that the genome can now be conceived of as being constrained into loops whose average size is roughly 100,000 base pairs. In theory, the loop-domain model permits the coordinate control of complex arrays of genes, since regions that may be very distant in the DNA sequence may actually be physically adjacent (at the base of a loop) in the nucleus. The nucleolus is an example of the clustering of dispersed DNA regions into a single-functional domain where the genes (often found on different chromosomes) encoding ribosomal RNAs associate physically and are expressed together.

It is a striking coincidence that DNA replicates in multiple independent blocks, which again are about 100,000 base pairs long. Thus, loop-domains (or clusters of loop-domains) may constitute the control units for both transcription and replication. This model predicts the existence of a new set of nuclear components that control DNA function—the structural components that anchor the loop-domains; at present, however, these ideas have yet to be demonstrated.

Centromeres and Kinetochores Play a Key Role in the Migration of Chromosomes During Mitosis

During the separation of chromosomes at mitosis, microtubules of the mitotic spindle attach to a specific site on each chromosome known as the centromere or kinetochore. (As now used, centromere refers to the DNA sequences at this site and kinetochore to a rather complex structure of unknown composition visible by electron microscopy.) The centromere is fundamentally important because movement of the chromosome at mitosis and meiosis into daughter cells depends on this region; chromosomes without a centromere fail to move normally and are eventually lost from one of the daughter cells. Centromeric DNA has been cloned and sequenced from the yeast Saccharomyces . Remarkably, the DNA region necessary for accurate segregation of a chromosome is no more than a few hundred base pairs long in this organism. The centromeres of higher eukaryotes are larger and their characterization will be difficult, but should prove of great interest for comparison with the presumably simpler condition in yeast. The use of antibodies produced by patients with an autoimmune disease has made it possible to identify and clone the DNA sequence for a centromeric protein. Characterization of this human protein and others that mediate association of the centromere with the spindle microtubules should give insight into critical questions of chromosome movement during mitosis and meiosis.

Telomeres Maintain the Structural Integrity of Chromosomes

The ends of eukaryotic chromosomes, the telomeres, are special in several ways. Telomeres stabilize chromosomes and prevent their fusion with other broken or natural ends. In addition, their structure allows replication without loss of DNA. The ends represent a vanishingly small amount of the total DNA in a typical chromosome. For this reason telomeric sequences were first recognized in certain ciliates, which have thousands of extremely small chromosomes and hence thousands of telomeres. Telomeres have subsequently been identified in yeast and several other organisms, including humans. In all cases they consist of hundreds of nucleotides of simple repeated DNA (such as CCCCAA and CCCCAAAA repeats) associated with unique proteins. Telomere sequences are added to the ends of chromosomes at the time of chromosome replication by a special enzyme or enzymes without the need for a DNA template, and in this way they form a protective cap at each end of a chromosome.

Artificial Chromosomes Are Valuable Research Tools

The identification of centromeres and telomeres as well as sequences that initiate DNA replication now permits the synthesis of artificial "minichromosomes" by genetic engineering techniques. Such minichromosomes have already been introduced into yeast cells, where they function normally during both mitosis and meiosis. Work on artificial chromosomes in yeast will undoubtedly lead to increased knowledge about chromosome structure and mechanics; eventually similar studies will be possible in higher eukaryotes, simplifying the introduction of specific genes or gene combinations into experimental organisms. One important use for the artificial yeast chromosomes is in the cloning of very large fragments of DNA (as many as a million nucleotide pairs).

The Nucleolus

The nucleolus is the site in the nucleus for the transcription of ribosomal rna.

The nucleolus is the major structural differentiation seen in nondividing nuclei. It is formed from a specific chromosomal locus, the nucleolar organizer, which contains the genes coding for ribosomal RNA (rRNA). When rRNA is synthesized, it first accumulates in the nucleolus in association with a large number of ribosomal proteins. Eventually the rRNA and proteins are transported to the cytoplasm, where they constitute the mature ribosomes. Although the major features of rRNA synthesis and its relation to the nucleolus were worked out more than 15 years ago, the nucleolus continues to be of interest as a model for RNA transcription and processing. In particular, we are just beginning to learn about the ribosomal proteins and the ways they interact with rRNA to form the ribosomes. Ribosomes themselves are crucially important to cell function, since they are the machines that catalyze all protein synthesis.

• Genes and Gene Action

The Primary Questions That Have Been Asked for the Past 100 Years by Geneticists Are Still Inspiring Research Innovation

Geneticists have always wanted to know how traits are passed from one generation to the next. We now seek to answer these questions at higher levels of resolution. For example, we can ask, What is the structure of genes? How do they replicate? How are they organized on chromosomes? How do they mutate, recombine, and repair themselves? What controls the timing of gene expression and repression? What mechanisms control tissue-specific and cell type-specific gene expression? The answers that we anticipate are at the level of nucleotide sequences, the three-dimensional structure of chromatin, the mechanism of action of enzyme complexes and specific DNA binding proteins. As answers to these questions emerge, we can use the resulting picture of how genes act to ask even more sophisticated questions about their products and functions.

Genetic Analysis

The combination of classical genetics and biochemistry has resulted in an explosion of genetic understanding.

The field of genetics has made dramatic advances within the past 25 years largely because biologists learned that genes are made out of DNA. In classical genetics, the arrangement of genes on the chromosomes was inferred by analysis of crosses between organisms. In modern genetics the order of genes can in principle be determined directly by sequencing the DNA molecules. The rapidity and certainty with which genes can be sequenced by biochemical procedures, even in organisms having no convenient sexual cycle or, as in humans, having a long cycle, have come to give the illusion that classical genetics has been subsumed by biochemistry. The most elegant insights, however, have emerged in the collaboration between classical genetics and biochemistry, which has created the field of molecular genetics. In this collaboration, the goals of genetic research have not changed. Geneticists still seek to learn the set of instructions that specify the architectural plans encoded in the DNA for building a functional organism.

Mutations in Essential Metabolic Pathways Have Contributed to Our Understanding of Both Genetics and Biochemistry

Classically, genetic analysis starts with mutations. These are alterations in a single gene that result in changes in an organism's appearance or biochemistry (phenotype). For example, normal yeast cells can grow on a simple medium with no amino acid supplements. Mutations can be found that lead to the requirement that an amino acid, say histidine, which the cells would normally produce itself, be supplied in the growth medium. Mutations of this type are extremely useful because the cells can be maintained and genetic crosses can be made on complete medium, yet the mutation can be detected at any time by plating a sample of cells onto medium that lacks the required nutrient. Such conditional and selectable mutants have been the basis for learning the fundamentals of molecular genetics, the physical nature of the gene, and the structure of chromosomes. These mutations have also been important to our current understanding of biochemistry, The analysis of many mutants, each blocked in the same biochemical pathway, but at different steps, can lead to an understanding of how complex molecules could be formed by sequential chemical reactions.

When coupled with genetic crosses, mutant analysis becomes a valuable tool for dissecting some of the most complex biological functions by correlating genotype (the nucleotide sequence) with phenotype (how the organism looks). A decade of intensive research correlated gene structure and function with biochemical activities of simple organisms. In some cases a single base-pair change in the DNA can be correlated with the loss of the catalytic function of an enzyme in a biochemical pathway. The metabolite required in the diet of the mutant organism reveals the identity of the defective biochemical pathway. The loss of enzyme function easily explains the phenotype of the mutant organism—the failure to grow without a metabolite in the medium—since the mutant cannot carry out one of the steps in the biosynthesis of the metabolite. Thus, classical genetic analysis allowed the elucidation of biochemical pathways because the essential steps in the pathways were easy to eliminate: The genes were discovered through mutations with a metabolite-requiring phenotype, the functions were inferred by inspection of the phenotype, and the proteins were identified by application of chemical and biochemical analysis of the mutants in comparison with the normal.

The Analysis of Mutations in Other Types of Genes Requires a Different Approach

Although these approaches permitted an understanding of metabolic pathways in simple organisms, they are difficult to apply to the direct analysis of cellular structure and to questions involving the complex interactions that determine protein structure and function in higher organisms. The major difficulty lies in our inability to connect genetics and biochemistry at higher levels of complexity. Very often the function that we wish to study is part of a subcellular structure and cannot simply be added back from the outside to correct the effect of the mutation. Furthermore, a component of the cell's architecture may be essential for viability, and then a mutation in this function would be lethal. Undaunted, geneticists still made considerable progress through the mutation analysis approach by using conditional lethal mutations (for example, mutations that permit growth at one temperature, but not at another). Despite many imaginative efforts, it became clear that major cellular processes could not be analyzed simply by examining phenotypes in the old ways that had elegantly sufficed for determining basic biochemical pathways.

To associate a gene with its function in one of these complex cellular processes required development and exploitation of new experimental strategies that can permit the enumeration of genes controlling the synthesis and function of the cell's architecture. The new methods enable scientists to carry out a new chain of discovery that allows them to associate the gene with a cellular function, with a gene product (usually a protein), and ultimately with a mechanism by which the gene product executes the function.

Even with the latest in technologies, one still faces problems in associating genes with products and the products with essential functions related to cellular structure, progress of the cell division cycle, or other functions carried out by macromolecular assemblies. The geneticists can identify candidate genes by observing mutant properties that suggest failure in cell architecture or cell cycle. Biochemists and cell biologists, on the other hand, can find proteins (such as actin and tubulin) that are abundant in structures implicated in basic cell function. The challenge to the molecular geneticist is to find ways to bring these lines of endeavor together so that the geneticists' genes can be associated with the biochemists' proteins and the cell biologists' structure and function.

A Gene Isolation Experiment Usually Begins with the Construction of a Gene Library

The gene library is a collection of DNA fragments carrying every gene from the organism, each recombined with a carrier DNA molecule called a vector. The vector contains genes that permit replication and transmission of the vector and any DNA joined to it when the DNA is transformed into the appropriate cloning host (usually a bacterial or yeast cell). The library is made by chopping the chromosome into gene-sized pieces of DNA and then joining each piece to its vector. The size of the library depends on the size of the organism's genome.

Isolating a Specific Gene from All the Rest of the Recombinant Molecules Can Occur in Two General Ways

The more classical route begins with mutations, which define a gene and whose properties (phenotype) indicate failure in a particular cellular function. One can readily isolate the gene as a DNA fragment by using DNA transformation with gene libraries to complement a mutation. Restriction fragment length polymorphisms (RFLPs), to be discussed later, can also be used to localize genes or chromosomes. The isolated gene can be analyzed as a physical entity, after which one can find the gene product and determine, in favorable cases, something about the way in which this protein contributes to cellular function. By this route one eventually obtains all the elements: the gene, the function, and the protein product. This route works well with bacteria, yeast, and cultured cells of higher organisms if the gene desired has a strongly selectable phenotype.

The second approach begins with a gene product, perhaps a protein involved in some cellular process. Isolation of the gene for that protein depends on what is already known about that protein and often on the ingenuity of the investigator. If the protein sequence is known, oligonucleotide probes can be chemically synthesized on the basis of information contained in the protein's amino acid sequence. These probes can be used to screen the library by hybridization.

Other techniques involve isolating mRNA from the cell type of interest, transcribing it into DNA, and cloning it into a vector that is designed to direct transcription of that mRNA when it has been transformed into a bacterial host. Colonies of bacteria producing the desired protein can be identified by antibody binding (if an antibody is available) and then grown to yield quantities of the desired cloned genes. These clones can be used to screen a library of genomic DNA sequences to obtain the gene encoding the mRNA.

Once the gene is cloned, much can be learned from its sequence. Much more can be learned if the gene can be put back into the species of origin. In many organisms it is now possible to introduce DNA, usually by injection, that will become stably integrated into chromosomes and frequently be normally expressed. It is often possible to use such transformation to show that a cloned gene will repair a mutation.

Specific features of transformation vary significantly with the organism. In yeast and in the slime mold Dictyostelium , the introduced gene can displace the resident gene by homologous recombination (recombination that requires sequence similarity). This feature makes it possible to analyze the new gene without interference by the preexisting one and also ensures that only one copy of the transformed gene will be added. Homologous gene replacements are not yet routinely possible in other organisms, although techniques are being actively sought. In spite of some limitations, gene transformation has proved to be a powerful research tool that has opened analysis of complex function by direct genetic approaches.

The Genome Refers to the Assemblage of All of the Genes of an Organism

The size of the genome (that is, the total amount of DNA) varies widely among organisms ( Figure 4-1 ). Even though the number of genes in different multicellular animals is probably reasonably constant (totaling perhaps tens of thousands), the total amount of DNA in their genomes varies by a factor of 100 or more. Paradoxically, the size of the genome is not directly correlated with the complexity of the organism. Thus, much of the DNA in organisms with high DNA values (such as mammals, including humans) is noncoding DNA. What is the function of this DNA? Some of it may have no function at all, although we are beginning to learn about functions of DNA segments that do not contain conventional genes.

Genome sizes. Shown are the number of telephone books needed to display the DNA sequence of various genomes, ranging from that of a simple virus to the entire human genome.

One intriguing class of "extra" DNA includes transposons (transposable elements). These are short pieces of DNA that have the ability to move from one place in the genome to another or from one genome to another. The transposon usually contains sequences that specify the machinery required for its own mobility and perhaps one or two other genes that can affect the host. Although the transposons may appear to be innocuous passengers in the genome, they play a significant role in generating evolutionary diversity by introducing chromosomal rearrangements and changes in gene expression. Some of these transposons are the inserted DNA copies of RNA retroviruses.

Another fraction of the extra DNA almost certainly plays a role in maintaining the higher-order structure of chromosomes and ensuring their appropriate segregation and disposition in the nucleus. The role of extra DNA is an aspect of genetics that is only beginning to be explored.

The Physical and Chemical Characterization of Bacteriophages and of Bacterial Genomes Has Revealed a Remarkable Array of Molecular Structures

Bacteriophage genomes (the genomes of viruses that attack bacteria) range from single-stranded linear RNA and DNA molecules to small double-stranded linear and circular DNA molecules. By comparison, the double-stranded linear or circular molecules that compose the entire genomes of various bacteria are enormous. The quest for correlations between the physical map of the genome and the genetic map obtained by recombinational studies led to the complete sequencing of bacteriophage genomes and is currently the driving force behind the attempt to completely sequence bacterial genomes. The first genome to be sequenced, that of bacteriophage &phi;x-174, resulted in the surprising finding that some of the stretches of nucleic acids encode overlapping genes.

Although much has been learned about the DNA of bacteria, less is known about the proteins that are bound to that DNA. Specific proteins have been identified that bind adjacent to and perhaps within the coding region of active genes and are necessary for specific gene transcription and function, but many such proteins are as yet uncharacterized. Existing techniques of genetic, chemical, and physical analyses need to be pushed further, and other techniques will need to be invented to yield a dynamic picture of the changes in the structure of the genome during the cell cycle. Both the overall structure and the disposition of specific proteins and nucleic acids within these structures need to be determined.

DNA Replication

The study of DNA replication promises at long last to yield information important for understanding both normal proliferative responses, such as those basic to the immune response, differentiation, tissue regeneration, and abnormal processes such as tumorigenesis and carcinogenesis.

The Major Components of DNA Replication Have Been Determined

Although the problem of how a DNA sequence is transmitted in dividing cells was solved conceptually by Watson and Crick in 1953, the elucidation of the actual molecular basis of replication has required considerable time and effort. At many points in the history of the development of the field it appeared that replication was ''solved" and that all that was left was to work out the details. For instance, in 1959, the discovery of DNA polymerase I of Escherichia coli led to 10 years of study based on the assumption that replication was catalyzed by this single enzyme. Then, in 1969, it was shown that polymerase I mutants were viable and that DNA polymerase I was, thus, not required for replication. This finding led to the search for and discovery of DNA polymerase III. Polymerase III mutants that were replication-defective identified this polymerase as the authentic replicative enzyme. Additional replication mutants showed that more than DNA polymerase was required. What was needed was a strategy to identify the products of the genes defined by the replication mutants. The complexity of replication forced biochemists to first work out the number of components required in prokaryotes.

By using a variety of DNA viruses as model systems, biochemists have been able to identify six essential classes of proteins in addition to DNA polymerase that are required in virtually all replication systems.

The Details of DNA Replication Can Now Be Investigated

A current model of how the purified proteins interact to carry out movement of the replication fork along both strands of DNA simultaneously is shown in Figure 4-2 . Rather than carry out a set of sequential individual reactions with one class of protein carrying out its function, dissociating from the DNA, and the next protein further processing an intermediate, the proteins form a stable complex that remains associated with the DNA during growth. Fork movement of the chain of newly synthesized DNA may be facilitated by ordered conformational changes in the proteins in the complex by the hydrolysis of bound nucleotide triphosphate molecules. Thus the replication complex resembles a machine, with proteins as the moving parts.

The trombone model for the propagation of a replication fork of DNA. [Adapted from B. M. Alberts, Cold Spring Harbor Syrup. Quant. Biol. 59:1-12 (1984)]

Good evidence exists that these replication complexes are stable; despite the conceptual attractiveness of the machine model, however, we actually know very little about how the proteins move along the DNA. In the future, many experimental disciplines will be brought to bear on this problem. What is the detailed architecture of the complexes? What is the driving force for the set of coordinated protein movements, and what is the inherent timing mechanism of the polymerization cycles? How are error rates minimized? How do accessory proteins increase the time of association of DNA polymerase with the template? How is the size of intermediate fragments determined? Are all of the components of the machines defined or have we missed some by our search methods? The key enzyme remains DNA polymerase, for which an x-ray crystal structure is now available, allowing more precise questions about polymerization mechanisms. Sequencing of cloned polymerase genes and in vitro mutagenesis, based on the crystal structure, offer fundamental new insights into how these enzymes work, especially with respect to mechanisms of fidelity (ability to copy accurately) and mutagenesis. Comparisons between cellular and vital polymerases are defining differences that can be exploited for antiviral therapy by rational drug design.

The original idea that replication was carried out by a single enzyme may seem naive to us now, but it would also be naive to think that DNA replication is the only process mediated by such complex protein machines. Impressive evidence has already accumulated that transcription, translation, RNA processing, and muscle contraction are also organized in this way. Yet, only for bacterial DNA replication is the field so advanced as to have all the components not only defined, but purified in abundance, and in a form allowing reconstitution of the entire process in the test tube. Although the current description of the replication apparatus in itself represents an enormous achievement, the most exciting era is just beginning. The basic principles that emerge from future enzymatic replication studies will have a general biological significance far beyond the specific light they shed on DNA synthesis.

The Regulation of Chromosomal Replication Is Poorly Understood

The regulation of chromosome replication will be emphasized in research in the coming years because of its important implications for cellular and developmental biology. In both prokaryotic and eukaryotic cells, regulation of replication occurs when replication is initiated rather than when the DNA chain is elongated. Therefore, the first step toward understanding regulation is the identification of proteins, genes, and DNA sequences involved in initiation. Apparently only one more protein is necessary for initiation than for elongation in bacteria [although this may not be the case for the replication of simian virus 40 (SV40)]. The extra protein is one that selects the sequence of nucleotides in the origin of replication, binds there, and mediates unwinding of the DNA, creating a nucleoprotein structure that directs binding and further unwinding by the enzyme DNA helicase. Interestingly, only months after this simple mechanism was established for bacteria, an identical process was reconstituted from partially purified human proteins in the eukaryotic DNA virus SV40. This finding suggests that initiation mechanisms may have been conserved over long periods of evolutionary time; although the SV40 system is undoubtedly simpler in detail than eukaryotic chromosome replication, the basic enzymology of replication may confidently be assumed to be the same in prokaryotes and eukaryotes.

Since the systems for the study of initiation of replication are new, much less is known about initiation than elongation. In the future, this recent work on initiation will be extended to address the role of transcription in activating DNA replication. Are there additional proteins? What are the primary, secondary, and tertiary structures of the DNA in specific areas where replication begins? Most importantly, how is the process controlled? The prokaryotic systems will continue to be important for enzymological studies because of the ease of genetic and biochemical analysis and because of their recently proven relevance for higher cells. In addition, new technological advances should permit the testing of principles learned from prokaryotes in the more complicated eukaryotic systems.

A second aspect of the regulation of eukaryotic DNA synthesis needs to be understood. The eukaryotic cell apparently retains the ability to initiate new replication units throughout the S phase, but a region once replicated cannot be replicated again in the same cell cycle. This block to reinitiation is essential to ensure that each region of a chromosome is replicated only once per cell cycle. The next few years will see major efforts to try to understand the molecular basis for this aspect of replication regulation.

Initial Efforts Will Emphasize the Further Development of Eukaryotic Experimental Systems for the Study of Replication

Two of the systems that seem to offer the most promise for study of eukaryotic replication are yeast and amphibian eggs. Yeast has been uniquely successful in contributing to our knowledge of eukaryotic DNA replication in at least two ways: (1) in the isolation of the DNA sequences essential for chromosomal replication and segregation: origins of replication, telomeres and centromeres; and (2) in the isolation of genes and mutants that have helped characterize several of the proteins involved in replication. Recently it has been found that extracts of amphibian eggs ( Xenopus ) initiate replication, with two unexpected features: Replication initiation requires the formation of an intact nuclear envelope around the added DNA; and synthesis is periodic, even though there is no periodic decondensation and condensation of the chromosomes. Intensive study of the proteins that catalyze replication in these eukaryotic systems and others should lead to a complete molecular understanding of the process.

Recombination

Genetic recombination is the rearrangement of dna sequences to create new genetic information.

Genetic recombination is one of the few ways to introduce variability into the genome. Recombination is most important in the generation of diversity among individuals in a population, although it is also a mechanism for generating antibody diversity in vertebrates (see Chapter 7 ). Recombination is also important in pathological processes such as cancer. Many viruses, including the cancer-causing retroviruses, rely on recombination for an essential part of their life cycle: the joining of the viral genome with the host genome. In addition, many parasites rely on recombination to alter the genes that code for components of their surface coats so as to escape the host's immune defenses. A final area of importance for genetic recombination centers on the use of recombination by researchers to artificially introduce gene segments into the genome of cells or whole organisms.

General Homologous Recombination Is the Simplest and Most Studied Category of Recombination

General homologous recombination is the exchange of DNA strands that have a certain degree of complementarity. In homologous recombination the two DNA segments need not be completely identical, but only homologous, to recombine. Studies of homologous recombination in bacteria have identified a key protein, RecA, that catalyzes the exchange of genetic information. Many of the essential features of homologous recombination have been reconstituted with purified RecA protein. These biochemical studies have brought researchers to the point of asking two fundamental questions. First, how does RecA locate the homologous segments of DNA and bring them together to initiate the exchange? Second, how does RecA catalyze the extension of the initial point of DNA exchange so that hundreds of thousands of nucleotides of DNA are recombined? We know that this process requires hydrolysis of the high-energy compound adenosine triphosphate.

In eukaryotic systems, the stage is set for major research advances. Molecular genetic techniques should expedite the identification, isolation, and characterization of the genes that encode the recombination apparatus. Because of the ease of genetic manipulation, research on the lower eukaryotes may lead the way in this effort. One hopes, however, that conservation of gene structure will permit identification of related genes in higher eukaryotes. The biochemical study of meiotic components—the complex that aligns homologous chromosomes, DNA strand transferases that initiate exchange, and DNA resolvases that terminate exchange—is in its infancy, but recent progress in yeast should attract more workers to the field.

In addition to understanding the basic mechanism, we need to learn more about important control features of meiotic recombination. For example, failure of homologues to separate at meiosis is the basis of trisomy diseases like Down's syndrome. A second item for further research is the means by which cells prevent recombination between repeated sequences. Such repetitive DNA is a hallmark of higher eukaryotic cells and presents a potentially devastating opportunity for a homologous recombination system. If left unchecked, recombination between repeated sequences could scramble the genome. We need to know how this is limited and whether breakdowns in this control are responsible for some human disease.

A separate issue in the study of general recombination of eukaryotes concerns somatic cells. When recombination has been studied in living organisms (usually Drosophila and yeast), somatic recombination is less frequent than recombination in germline cells and seems to be primarily involved in DNA repair. Nevertheless, many scientists have demonstrated the efficient recombination of homologous segments of DNA when they are artificially introduced into somatic cells in culture. Although ongoing studies attempt to define the mechanism of this recombination, the most exciting possibility is its potential use to manipulate the genetic content of cultured cells. Research will focus on controlling the frequency and fidelity of recombination so that investigators can introduce genes into cultured cells and replace existing genes with modified ones. This sequence of manipulations is readily accomplished in bacteria, yeast, and slime molds, where the technique has been of tremendous value in the genetic analysis of complex loci. Formidable problems arise in extending this technology to higher organisms became of the complexity of their genomes. However, several initial successes in targeting have opened the way to a thorough attack on the problem.

Site-Specific Recombination Has Been Studied for the Past 20 Years and Many Systems Have Been Studied in Depth

In the 1960s it became clear that efficient recombination could take place between segments of DNA that lacked sufficient homology to undergo general homologous recombination. Two features are required for such nonhomologous recombination—the presence of special DNA sites in at least one partner in the recombination process and the activity of specific enzymes (recombinases) that act at these sites. The rearrangements of the genome promoted by site-specific recombination are mechanically fascinating. One wants to know how small segments of the genome are identified, brought close together, and rearranged with high efficiency and fidelity. Moreover, site-specific recombination is a critical step in many important biological processes. As a result, the study of site-specific recombination has emerged as a major area of concentration of biological research that will continue to grow rapidly in the next 5 to 10 years.

The molecular mechanism of these reactions has been worked out by using the molecules and mutant strains identified by genetic analysis to reconstitute the recombination event in a cell-free (in vitro) system. Site-specific recombination can be divided into broad mechanistic categories according to the degree to which both partners must have specific sites and on the amount of DNA synthesis that accompanies the reaction. In some systems, recombination uses a completely conservative mechanism in which the participating DNAs are simply broken, rearranged, and rejoined. In other systems, complete replicas of the DNA must be synthesized during the recombination event. The integration of the bacterial virus called lambda into the E. coli genome is a reaction with high target specificity and strict conservation of the parental DNA. This complete reaction has been reconstituted in vitro. By combining topological investigations with biochemical and molecular genetic studies, the field is reaching the goal of elaborating plausible models for complex processes such as synapsis, the juxtaposition of DNA segments that is a prerequisite for efficient, precise recombination. In the future, structural studies using x-ray diffraction and nuclear magnetic resonance techniques should be able to add atomic details to the understanding of these processes that is being deduced from molecular biological studies.

In contrast to the conservative reactions (no DNA replication), study of the mechanism of the replicative site-specific recombination (which requires DNA replication) is in its infancy. The transposition of the genome of the bacterial virus mu, is the only example that has been reconstituted in vitro. Several features of the DNA sequence of donors and targets suggest close parallels between mu transposition on the one hand and other rearrangements such as the integration of retroviruses (RNA viruses that replicate by way of a DNA intermediate that is inserted into the host's chromosome) and the recombination of antibody genes. The pioneering work on mu transposition has thus opened the way for rapid progress in the biochemistry of these eukaryotic recombination events.

Another important research area, control of the recombination process, has already been extensively studied in prokaryotic and lower eukaryotic systems. Here, the key event seems to be control of the synthesis of a specific recombinase. In some systems, such as the integration of lambda and sexual differentiation of yeast, such control is elicited through a complex chain of events that couples the synthesis of the recombinase to related events in the life cycle of organisms. In higher eukaryotes, we know almost nothing about the control of recombination. We need to learn whether these events are regulated and, if so, by what mechanism. For the immune system, some experiments suggest that accessibility of the recombining sites rather than the availability of the recombinase may be critical. The finding that several human tumors result from an inappropriate rearrangement of immune elements highlights the importance of understanding the control of site-specific recombination in higher organisms.

DNA Repair and Mutagenesis

Organisms correct mistakes by dna repair mechanisms.

It is critical for organisms to maintain and protect the fidelity of the information encoded in their DNA so they can pass this information on to their descendants and so they can access that information during their own lifetimes. However, the integrity of the DNA itself and the information encoded therein are undergoing constant challenges from agents in the environment. Furthermore, mistakes can be introduced into the information by normal cellular metabolic events such as DNA replication and recombination. The study of mechanisms of DNA repair and mutagenesis is of direct relevance to human health, since mutations in DNA are responsible for both genetically inherited diseases and the somatic cell diseases known as cancer.

The Molecular Mechanisms of DNA Repair and Mutagenesis Need to Be Studied in Prokaryotes, Yeasts, and Mammals

Around 1975 it was generally thought that DNA repair and mutagenesis were understood in bacteria and that this knowledge made it possible to approach these issues in eukaryotic organisms. However, new discoveries of a variety of complex independent enzymatic systems specializing in repair of different kinds of damage over the past 10 years have revealed that these processes are much more complex, even in bacteria, than was assumed in 1975. These new revelations about the complexity of bacterial systems explain the relatively modest progress in dissecting DNA repair in mammalian cells. It is important that major studies of DNA repair and mutagenesis be continued at several levels at once: studies involving at least prokaryotes, yeasts, and mammals. The main reason for lack of progress in mammalian systems has been the lack of genetic tools similar to the ones used in work on prokaryotes.

Opportunities Abound for Studies of DNA Repair and Mutagenesis in Prokaryotes such as E. Coli

Recent studies show that the complexity of the repair systems demand the power of E. coli molecular genetics if we are to obtain a framework within which to understand these processes in all organisms. To begin with, the molecular mechanism by which damaged DNA is processed to give rise to mutated DNA ("error-prone repair") is not understood in any organism, and E. coli is likely to be the first organism in which this fundamental problem will be solved. The existence of regulatory networks of genes induced by DNA damage was discovered in E. coli . Studies of two such gene systems that are induced by DNA damage, the SOS repair system and the adaptive response, have led to the discovery that some proteins (such as the proteins RecA and Ada) have both a regulatory and a mechanistic function in DNA repair. The complexities of the regulation of these systems need further study, as do the functions of all of the induced gene products. Detailed analysis of complex proteins such as these are needed to provide models for systems that lack sophisticated genetics.

Major Opportunities Exist to Study DNA Repair in Eukaryotes

Yeast can be used to explore uniquely eukaryotic aspects of DNA repair, such as the relationship of DNA repair processes to the cell cycle and the effect of chromatin on DNA repair. In yeast, many genes affecting survival or mutagenesis after DNA damage have been identified, and many of these have been cloned. However, very little is known about the biochemistry of DNA repair in yeast. Moreover, although a few yeast DNA repair genes have been shown to be induced by DNA damage, virtually nothing is known about the mechanism of their regulation.

The study of DNA repair in mammals, especially humans, is a challenging problem of great significance, but one that needs to be approached with greater sophistication. Many studies to date have been primarily descriptive and have allowed only low-resolution inferences concerning the mechanism. Another problem has been that humans with inherited diseases caused by mutations in DNA repair, such as xeroderma pigmentosum and ataxia telangiectasia, have complex phenotypes that make analysis of their deficiencies difficult. Furthermore, to date, no one has succeeded in cloning genes that complement the DNA repair deficiencies of these naturally occurring mutants. Problems unique to higher organisms that will need to be addressed include the relative DNA repair capabilities of different types of differentiated cells and the DNA repair capabilities of germline cells versus somatic cells.

Gene Expression

Genes provide the information required for the formation, function, and reproduction of cells and organisms.

The information in the genes specifies the sequences of RNAs and proteins that have the biochemical capacity to synthesize other cellular components (sugars and lipids) as well as the DNA, RNA, and proteins themselves. The process that converts information stored in selected genes into RNA and proteins is called gene expression.

Specific gene expression leads to the selected transcription copies of appropriate genes at particular times in the life cycle of the cell or organism. Although we now understand some of the strategies involved, our knowledge of the control of transcription still contains enormous gaps, for both prokaryotic and eukaryotic organisms. An understanding of the regulation of gene expression lies at the heart of understanding how the genome functions to guide the development, growth, and differentiation processes that generate a complex organism from a single-celled fertilized ovum.

Gene expression must be properly controlled during the life cycle of any organism. For example, hemoglobin is produced in red blood cells but not in other cells. Yet the hemoglobin gene is present in all cells of the body. Similarly in plants, proteins of the photosynthetic apparatus are expressed in specialized leaf cells but not in cells of the root. By what mechanisms are the genes in individual cells differentially expressed?

At least two levels of regulation exist: large-scale activation or inactivation of groups of genes and the precise control of individual genes. Since eukaryotic development is characterized by changes in the expression of complex cohorts of genes, these cohorts may be regulated in parallel. This procedure would be straightforward if the structure of the nucleus could in some way regulate the activity of the DNA. For example, groups of genes that show coordinate expression might be packaged in discrete domains or compartments that could be regulated at one or a few key control sites. However, whereas coordinately regulated genes in prokaryotes are frequently linked and controlled by a single promoter (nucleotide sequence to which RNA polymerase initiates transcription), such linkage is not usually found in eukaryotes. In most instances, members of coordinately controlled gene sets in eukaryotes are broadly scattered over the chromosomes, implying that transcription is controlled by trans -acting regulators, which can affect gene regulation at widely separated chromosome sites.

However, large-scale gene control is found in several notable exceptions to the general picture of eukaryotic gene regulation. These include inactivation of the X chromosome in mammals and dosage compensation of the X chromosome in insects, in which the transcription of genes on an entire chromosome or a large chromosome segment is regulated as a unit. In this latter case, regulation is associated with a general change in chromatin structure; for example, the inactive X in mammals can be distinguished by its pattern of staining. Genes that are moved onto the inactivated X chromosome are also inactivated: This type of gene inactivation does not generally occur when a gene is simply moved near a member of a coordinately controlled gene set.

Formation of Primary RNA Transcripts Depends on Specific DNA Signals

For a gene to be transcribed, it must have certain nucleotide sequences arranged in serial order along the DNA chain. The minimal elements are— starting at one end and reading from left to right—a promoter sequence, which binds an enzyme (RNA polymerase) that facilitates transcription; a coding region that specifies the mRNA transcript; and terminal sequences that terminate and stabilize the transcript. Experiments in bacteria showed that the promoter region also serves as a binding site for specific gene-regulatory proteins that regulate the transcription activity of the gene. New techniques, especially gene cloning and DNA sequencing, have enabled investigators to demonstrate that a similar system of transcriptional regulation exists in higher organisms, including humans. The promoter region of each gene in a higher organism has sites for several different DNA-binding proteins. Many of the specific proteins are now being characterized, and their genes cloned, so that the mechanisms by which they work can be studied in detail at the molecular level.

One class of binding site that need not be located precisely at the promoter region of genes is called an enhancer. Enhancers can function from distances of nucleotide pairs away from the RNA start site they affect. Enhancers were originally given this designation because gene activity was enhanced when the element was present and diminished when it was absent; we now know that an enhancer can also repress transcription, depending on the exact proteins bound.

An important property of many enhancers is their tissue-specific action. Sequences in the promoter region of the human insulin gene, for example, bind proteins found in insulin-producing cells but not in cells that do not produce insulin. Such results indicate that tissue-specific gene expression is at least in part caused by DNA-binding proteins that bind at specific sites next to genes.

The demonstration of enhancers helps explain how cells can coordinately express large sets of genes physically separated on different chromosomes. That is, specific DNA-binding proteins may recognize and bind with a similar nucleotide sequence present in the promoter region or enhancer of a number of different genes. In this way, a particular set of genes typical of a certain cell type would be activated or inhibited in a coordinated, programmatic fashion by the activation of a specific type of DNA-binding protein. Many such DNA-binding proteins exist, and a catalogue of many of them is needed before we can understand how the network of regulatory proteins affects cell differentiation in higher organisms. These networks are fundamental to the processes of normal cell differentiation and tissue morphogenesis. Moreover, the inappropriate expression of trans -regulatory proteins could lead to the abnormal gene expression associated with some forms of cancer.

Primary RNA Transcripts Are Modified by Additions at Their Ends and Deletions of Internal Segments

Modifications of RNA transcripts have an important influence on gene expression. When a transcript is first formed, its free end (the 5' end) is chemically modified, or capped. Such capping is believed to stabilize the transcript. In addition, immediately after completion, most transcripts are modified by having a string of adenylnucleotides added to their other end (the 3' end); this polyadenylated, or poly (A), modification is believed to influence their life expectancy.

Most transcripts are also extensively modified by the Precise deletion of internal segments, a process called mRNA splicing. The splicing mechanism must proceed with high fidelity, since an error of even a single nucleotide could change the reading frame (nucleotide sequence that codes for a specific protein), leading to translation of an altered protein. Variations in splicing may lead to greater flexibility in gene formation and in the expression of genes.

RNA splicing is required because of the existence of introns in eukaryotic genes. Introns are segments of DNA that are not used to specify protein sequences but are interspersed among the coding sequences. Their presence explains why long nuclear RNAs are produced as precursors for shorter cytoplasmic mRNAs. The intron sequences are excised from the precursor by RNA splicing, and the mature mRNA is transported to the cytoplasm. The length of an intron can vary from 60 or 70 to more than 100,000 nucleotides, and a typical vertebrate or plant gene might contain several introns.

Since the coding sequences used for translation of a protein are formed during splicing, variations in the pattern of splicing of a precursor RNA permit one gene to specify the formation of multiple proteins. Not only does such variation in splicing exist, but more importantly, the splicing pattern of a precursor can vary from one cell type to another. This means that splicing is regulated by factors specific to cell type.

Several questions are central to the study of RNA splicing. What specifies the set of intron sequences to be excised by splicing? Small nuclear ribonucleoprotein (snRNP) particles are crucial to sequence recognition during splicing. The splicing of the shortest intron requires the activities of at least four different particles, whereas longer introns may require even more. Changes in such factors could regulate different splicing patterns in different cell types.

Another important question concerns the chemistry of the splicing reactions. What are the mechanisms of the endonucleolytic cleavage and ligation steps, and are these reactions catalyzed by RNA or protein components?

The study of splicing is a new frontier in molecular biology. The most startling finding from this research has been that of the serf-splicing of particular types of RNA sequences. As the name suggests, this splicing reaction occurs in the absence of protein and is catalyzed by conserved sequences within the intron. When analyzed in detail, the RNA sequences within this intron have a catalytic activity similar to that commonly associated with several different types of protein enzymes. Catalytic RNAs are now thought to have been common in primitive biological systems and still to be integral to many contemporary processes (such as protein synthesis). In the past few years, they have been discovered in viruses, bacteria, fungi, protozoa, animals, and plants. It is even possible to imagine that the first cells on earth may have contained only RNA enzymes, and that cells existed before there were proteins.

Precursors to mRNAs are transcribed at many sites in the nucleus, processed by splicing and polyadenylation, and then transported through nuclear pores to the cytoplasm. Nothing is known of the mechanism of transport. These precursors are bound to a highly conserved set of basic proteins. In the cell, these basic proteins are retained in the nucleus and may play an important role in both splicing and polyadenylation. The complex steps of RNA processing offer several points at which gene expression could be modulated, such as by differential splicing to yield different mRNAs. The field of RNA processing is young and has only begun to be explored.

Specific DNA Modification May Influence Gene Expression and Confer Heritable Traits

Specific DNA base pairs are modified most frequently by methylation (the addition of methyl groups to DNA). In prokaryotic organisms it was thought that modification was required only to prevent DNA degradation by restriction enzymes. Recently, however, direct effects of methylation on the regulation of gene expression have been found. In mammalian genomes it has long been thought that methylation might correlate with the heritable control of specific gene expression. A variety of indirect experiments suggests that demethylation can derepress gene activity (activate a gene from a repressed state). In mammalian embryos, the expression of certain genes depends on whether the genes have been transmitted by the egg or by the sperm (see Chapter 5 ). The basis of this ''imprinting" is not known, but data suggest that specific methylation changes that occur during the formation of egg and sperm may be involved.

Although DNA methylation could explain the heritable patterns of gene expression displayed by various cell lineages in multicellular organisms, we do not know how large a role methylation plays, nor do we know what controls the patterns of DNA modification. In some complex multicellular organisms (for example, Drosophila ), the absence of evidence of DNA methylation suggests that it evolved rather late in evolution as a back-up system. Clearly we have much to learn about the role of methylation and other heritable modifications in the control of gene expression.

The Study of the Regulation of Gene Expression Offers Great Potential for New Research Opportunities and Practical Applications

At each level—DNA structure, transcription, RNA processing, mRNA translocation, translation, and the determination of the three-dimensional structure of the protein product and its introduction to the appropriate cellular compartment— new genetic techniques can be used to probe and understand the controlling processes. It is these processes that constitute "gene action" and determine cellular function. We know very little about how RNA polymerase reads the various promoter and enhancer sequences that determine the genes to be transcribed. It is not at all clear that the presence of transcription factors is sufficient to generate a regulatory system that can persist even after cells replicate, yet we know that cell determination results in patterns of gene expression that are inherited by daughter cells. We need to know the details of these and associated processes to understand the mechanisms that control cellular differentiation.

The ability to intervene and affect any of these processes specifically would make it feasible to design a host of new therapeutic drugs. For example, if we understood how genes such as those that control the production of interferon are induced, we might be able to mimic such a process selectively to enhance resistance to viral infections. The same kind of arguments apply to the regulation of growth in cancer cells and of immune responses.

Gene Transfers Provide Many Opportunities to Study in Detail How Genes Function

Using gene transfer technology, one can remove the control regions (enhancer or promoter) of a gene a bit at a time to identify segments that are critical for normal function. One can also switch control regions from one gene to another, so as to cause genes to be expressed under circumstances in which they would normally be silent. Although such studies have been possible for only a short time, they have provided much information (and some surprises) about gene regulation. In many organisms it is now possible to insert the modified gene directly into germline cells so that entire organisms carrying the new DNA are obtained. Studies of transgenic animals are now providing important information on both the mechanisms that regulate tissue-specific gene expression and those that cause cancer (for a discussion of transgenic animals, see Chapter 2 ).

Genome Organization

The organization of complex genomes can be studied by gene mapping.

Every cell of the human body contains an identical set of an estimated 60,000 to 100,000 genes. Most genes are present only once (single-copy genes). Each gene can be assigned to a particular chromosome site. This activity, called gene mapping, is improving our understanding of cells.

Gene mapping in humans and selected mammalian species has advanced impressively during the past decade. In humans, approximately 1,400 genes of known function have been mapped to some degree of precision, and another 1,000 DNA markers of unknown function have been localized. A preliminary low-resolution genetic map of the human genome has already been constructed.

Restriction Fragment Length Polymorphism DNA Markers Have Increased the Power of Mendelian Techniques for Mapping Genes

RFLPs are naturally occurring variants in the nucleotide sequence of DNA that can be used as genetic markers. They are transmitted from one generation to the next in the same way as the genes that govern eye or flower color. The advantage of RFLPs is that they can be detected through the use of only a few cells of the organism by the techniques of molecular biology. In addition, RFLPs relate the genetic linkage map to the physical (DNA) map. In essence, RFLPs add a large set of new markers for mapping any genetic locus of interest and for obtaining a high-resolution genetic map of any chromosomal region of particular interest.

The Value of a Human Gene Map Has Become Apparent in the Past Several Years

The map serves as a body of data that can be used to address a broad range of biomedical questions. It becomes more useful in generating and resolving hypotheses as the data base grows. For example, the map has been useful in establishing the connection between chromosome rearrangement and proto-onco gene activation. Proto-oncogenes are normal genes generally involved in cellular growth control, but they have the potential to cause cells to become cancerous when inappropriately expressed. In some cases inappropriate activation has been associated with rearrangement of the chromosome bearing the proto-oncogene. The gene map has also enabled investigators to detect linkage relationships between RFLP markers and specific genes that carry a risk for genetic disease. This can now be accomplished for many genetic diseases, such as various anemias, phenylketonuria, hemophilia, and Huntington's disease.

The identification of linkage relationships between genes causing genetic disease and RFLP markers is an important first step in physically isolating genes whose mutants cause genetic disease. A start in this direction has been made for a number of human heriditary diseases, such as Duchenne's muscular dystrophy, Huntington's disease, and cystic fibrosis. Isolation of these disease-causing genes will be the first step in analyzing the molecular basis of these diseases. The use of this approach to isolate genes and determine their products can reveal the underlying molecular basis of more than 3,000 human genetic conditions and provide crucial insights into human developmental processes.

If the number of human genes is approximately 100,000, only 1 percent of them have been mapped to date. Currently, new genes are being mapped at the rate of one every day. Two important new mapping activities are just now beginning: the formulation of maps based on overlapping cloned DNA segments and the DNA sequencing of the nucleotides that constitute these segments (see Chapter 3 ).

• Cytoplasm: Organelles and Functions

In a eukaryotic cell, the nucleus is surrounded by the cytoplasm, which in animal cells usually accounts for nine-tenths of the cell's volume; the cytoplasm is surrounded in turn by a cell membrane, or plasmalemma. The cytoplasm contains within a protein polymer matrix several types of minute, functionally specialized cell organs, or organelles, most of them present in multiple copies. In prokaryotic cells (bacteria), by contrast, the genome is not separated from the cytoplasm, there are no membrane-bounded organelles, and the plasma membrane subserves many of the specialized functions of eukaryotic organelles.

Prokaryotic and Eukaryotic Cells Use Related Strategies to Compartmentalize Their Biosynthetic Reactions

Prokaryotic cells have only one membrane, located at the cell surface (although some bacteria have a double membrane). In contrast, eukaryotic cells, such as those of animals and plants, have, in addition to their plasmalemma, at least a dozen different types of chemically specific membranes that create separate intracellular compartments with different microenvironments required by various processes, such as respiration, photosynthesis, protein synthesis, and intracellular digestion.

In prokaryotic cells, the plasmalemma is the site of a number of important biosynthetic activities, including the synthesis of membrane lipids, adenosine triphosphate, and cell-wall components. This membrane also contains a multitude of transporters, receptors, and sensors for chemotactic movements, which create the interface of the bacterium with its extracellular environment.

In eukaryotic cells, comparable activities are differently distributed. Some of them (such as those of the transporters and receptors) remain in the plasmalemma, but others are relocated to different intracellular membrane systems. Lipid synthesis, for instance, in animal cells occurs only in the endoplasmic reticulum (ER), a network of membrane-bounded channels that pervades the cytoplasm. In plant cells, lipid synthesis occurs in plastids. From the ER or plastids, newly synthesized lipids are distributed to all other cellular membranes. The ER also contains a complex set of enzymes that modify lipid-soluble aromatic compounds imported by the cell from the environment. The modifications increase the water solubility of these compounds, thereby facilitating their elimination. Since sterols, drugs, herbicides, toxins, and chemical carcinogens are among these compounds, the relevant enzymes constitute an intracellular detoxifying system.

The ER membrane is also the site of important steps in protein traffic regulation. Issues awaiting resolution include the means by which proteins and lipids are transported from their site of synthesis in the ER to a multiplicity of destinations and the way differences in lipid chemistry are established and maintained in different membranes. We must also advance our understanding of the ER detoxifying system to shed light on problems related to chemically induced cancers and toxic effects of chemical pollutants in the environment.

Protein Synthesis and Regulation

Protein synthesis and regulation of protein traffic take place in the cytoplasm.

Protein Synthesis . For all cells, protein synthesis is a major, continuous activity needed for the production of intracellular enzymes, contractile and cytoskeletal assemblies, membranes, ribosomes, chromosomes, and many other functionally important macromolecular assemblies. In more complex multicellular organisms, it is also needed for the production of proteins destined for export out of the cell, such as enzymes, hormones, growth factors, blood proteins, antibodies, or components of the extracellular matrix.

In all cells, proteins are synthesized on ribosomes, which translate into amino acid sequences the instructions received from active genes in the form of mRNAs. The ribosomes themselves are macromolecular assemblies of ribosomal RNA and protein molecules. The two sets of components are produced separately in the cytoplasm (proteins) and in the nucleus (ribosomal RNAs) and are modified and assembled in a special compartment in the nucleus (the nucleolus) before being transferred to the cytoplasm as part of a functioning ribosome.

In both prokaryotic and eukaryotic cells, ribosomes are basically similar and protein synthesis proceeds by similar steps. In eukaryotic cells, however, the ribosomes are larger and require more factors for their activity. These differences are probably related to the emergence of more versatile regulatory processes in eukaryotes.

Protein Traffic Control . In eukaryotic cells, ribosomes and protein synthesis occur primarily in the cytosol, to which mRNAs have direct access from the nucleus and in which the pool of amino acids and all ancillary factors required for protein synthesis are located. Only 2 percent of the total protein production is accounted for by small mitochondrial ribosomes whose products are used exclusively in mitochondria. In plant cells a somewhat larger fraction of the cell's protein is produced by chloroplasts (or other differentiated forms of plastids).

From the cytosol, proteins are accurately directed to more than 20 different sites of final functional residence. These sites are membranes or compartment contents, each endowed with chemical specificity.

ER Targeting System . Many proteins are directed to the ER membrane as they are being synthesized. The selectivity is based on mutual recognition between signals (called signal sequences) within the amino acid sequence of the protein to be transported and a signal recognition complex. This complex involves a ribonucleoprotein particle (called a signal recognition particle) and at least one transmembrane protein (its receptor) in the target ER membrane.

The ER targeting system recognizes and processes proteins destined for secretion and lysosomes (storage compartments for degradative enzymes), as well as membrane proteins for many intracellular compartments. Secretory and lysosomal proteins are fully translocated across the ER membrane into the ER cisternal space. Membrane proteins are partially translocated and remain anchored in the membrane. Recent experiments indicate that a membrane protein can be converted into a secreted polypeptide, and conversely, a secretory protein can be convened into a membrane protein by deleting or adding the information for the membrane anchoring sequences from their mRNAs.

The mode of operation of the ER targeting system has been elucidated in reconstituted in vitro systems in which ribosomes are allowed to translate into proteins the genetic information encoded in specific mRNAs in the presence or absence of ER-membrane vesicles in vitro. The results show that many components of the systems are functionally equivalent in different species, phyla, and even kingdoms, which implies that this part of the traffic regulation system originated early in evolution and has been conserved.

Post-ER Steps in Traffic Control . Once past the ER, proteins are moved within the cell through a specialized membrane-bounded compartment called the Golgi complex, where they are further modified by glycosylation, sulfation, and proteolysis; they are sorted while in transit to lysosomes, secretion vacuoles, or different membranes. The Golgi complex itself contains at least three subcompartments. Transport from the ER to Golgi subcompartments, from one subcompartment to another in the Golgi complex, and finally, from the last Golgi elements to the plasmalemma requires energy and is effected by vesicular carriers; thus, past the ER, protein traffic can be regulated at least in part by controlling the movements of vesicular carriers. These carriers apparently recycle continuously among compartments. The best known among these vesicular carriers are the secretion granules or secretion vacuoles of various glandular cells. They transport products to the cell surface and discharge them into the extracellular medium by a process known as exocytosis. Sorting of the proteins to their ultimate destinations probably involves mutual recognition between a signal and a receptor, but so far only the signal for lysosomal proteins has been chemically defined. Its receptor has been isolated and partially characterized and its gene sequenced. Reactants involved in the sorting of other proteins remain unknown, as are the signals and receptors that regulate the traffic of vesicular carriers. Studies to identify them are being actively pursued.

Other Traffic-Control Systems . The ER targeting system (which includes the Golgi complex) is undoubtedly the most complex component of the overall protein traffic-control system of the cell. The other components are simpler, and most of them probably transport the protein in a single step: from the cytosol directly to the final destination. The amino acid sequence of the signal that directs certain proteins to the nucleus is known in a few cases, but the corresponding receptor remains to be identified. A substantial body of information already exists about traffic regulation of proteins targeted to mitochondrial and chloroplast membranes. Among the protein products of plant nuclear genes are some that function in the mitochondria and some in chloroplasts; it is not known how the systems differ.

In a simpler form, protein traffic regulation occurs in prokaryotic cells and has been studied extensively in gram-negative bacteria, which are provided with two concentric membranes at the cell surface. The number of final destinations is considerably fewer—the two membranes and the intervening space and perhaps the outside of the cell. A signal, generally comparable to that found in eukaryotic proteins targeted to the ER membrane, has been identified and analyzed in detail by sequencing and by extensive genetic modifications. This line of work has led to the recognition of functionally critical residues in the signal sequence, but the other components of the system are still unknown.

We can anticipate considerable activity in this fertile and exciting area, especially in relation to the identification and characterization of signals and their receptor partners and to the intracellular location of receptors and sorters (molecules that control the selection and movement of proteins from one compartment to another). Although the picture is already rich in detail, many uncertainties remain to be resolved by further research. The reasons for removing the signal sequence are not clear, nor are the reasons for the complexity of the enzyme that effects the removal. The enzyme may have additional functions since it consists of six different proteins. The translocation process itself is still a mystery.

Structural biology is likely to yield three-dimensional information on such interactions if large enough quantities of relevant proteins can be obtained. The main goal is to understand how cells process their many proteins and how they achieve and maintain the chemical specificity of their membranes.

Another basic process that remains to be understood in molecular terms is membrane fusion. The process is critical for cell division, cell fusion in egg fertilization, and vesicle fusion along different pathways of intracellular transport. Membrane fluidity is a prerequisite for membrane fusion. It is also a prerequisite for membrane growth, which appears to proceed by expanding preexisting membranes. At the same time, intact diffusion barriers need to be maintained in highly dynamic membrane systems. Much remains to be discussed about how these processes are controlled.

• Mitochondria: Function and Biogenesis

Mitochondria Produce Most of the Cell's Main Energy Source, ATP

The mitochondrion is characterized as the power plant of the cell, because it performs the enzyme-catalyzed, stepwise oxidation of nutrients (such as sugars, fats, and amino acids) in a process called respiration. The most interesting product of this process is ATP, the direct source of the energy required for most of the chemical work the cell must perform to power its growth, movement, synthesis of new components, and other functions. A relatively small amount of ATP is produced in the cytosol during sugar catabolism (glycolysis), but by far the largest amount is generated in mitochondria. The mechanism of mitochondrial ATP synthesis has stubbornly resisted full elucidation, but progress continues to be made as a result of our insistent probing into this fundamental energy-transducing process.

Mitochondria are also of interest because they contain their own complement of DNA, which cooperates with the DNA of the nucleus in the control of mitochondrial formation. The origin and evolution of the mitochondrion are linked to the origin and evolution of eukaryotic cells. Understanding of mitochondrial function in turn sheds light on a wide array of fundamental and practical issues, ranging from certain metabolic and genetic diseases to evolution itself.

New Mitochondria Arise from Existing Ones, and They Are Characterized by Unique Functions

Mitochondria have two membranes (inner and outer) that define two concentric separate spaces. The inner space houses hundreds of enzymes, including those involved in the oxidative reactions that supply the energy needed for cell function. The inner mitochondrial membrane contains the energy-conversion apparatus. The majority of the mitochondrial proteins are specified by nuclear genes; they are synthesized on ribosomes in the cytoplasm and then imported into the organelle. A limited set of proteins of the inner mitochondrial membrane, namely, some protein subunits of the oxidative phosphorylation apparatus, are encoded in DNA molecules located within the organelle itself and are synthesized by an organelle-specific translation system. The distinctive structural RNA components of this system—RNAs and transfer RNAs—are also encoded in mitochondrial DNA. Mitochondria do not arise de novo in the cell by self-assembly of their constituent molecules, but are formed by growth and division of existing mitochondria.

The mitochondrial DNA from several organisms, including humans, has been completely sequenced, and much of its informational content has been elucidated. Furthermore, all mitochondrial gene products in humans have been functionally identified. A dramatic outcome of these studies has been the discovery that the mitochondrial genetic system in the organisms studied, except plants, uses a code that differs in several respects from the universal code and, in addition, utilizes for reading the code a novel mechanism, which requires only a restricted set of transfer RNAs.

Excellent Opportunities Exist to Study the Mechanisms of Expression and Replication of Mitochondrial DNA

Studies of the enzymes and ancillary proteins responsible for mitochondrial DNA replication, DNA transcription into RNA, and RNA processing to mature RNA species are making rapid progress, aided by the development of soluble in vitro preparations derived from broken mitochondria and by the use of recombinant DNA technologies. Specific proteins have already been identified and, in some cases, isolated in partially or completely pure form.

As in the case of many nuclear gene transcripts, the coding sequences of several mitochondrial gene transcripts in lower eukaryotes, especially yeast and filamentous fungi, are interrupted by nonfunctional segments, or introns. These introns must be removed to produce the mature RNA. The transcripts of some mitochondrial genes have the remarkable capacity to excise their own introns in vitro in the absence of protein; that is, they function as enzymes acting on themselves. The discovery of mitochondrial introns has opened an active field of research, which is expected to provide deep insights into the mechanisms of RNA splicing in general and into the origin and evolution of introns.

The Formation of New Mitochondria Is Under the Control of Both the Nucleus and Mitochondria

The dual control of mitochondrial formation is most dramatically illustrated by the chimeric structure of nearly all the enzyme complexes of the oxidative phosphorylation system: Each such complex contains some subunits encoded in the nucleus and some encoded in mitochondrial DNA. Because of this dual control, the formation of functional mitochondria requires a quantitative and temporal coordination of expression of the relevant nuclear and mitochondrial genes.

Two main classes of nuclear genes are the object of intensive investigation based on recombinant DNA techniques and on structural and functional approaches: (1) genes coding for subunits of the enzyme complexes of the oxidative phosphorylation system and for mitochondrial carriers used in metabolite transport and (2) genes coding for proteins involved in the expression and replication of the mitochondrial genome. Most of the latter genes are probably distinct from those that code for the homologous components of the nuclear-cytoplasmic apparatus, although interesting exceptions have recently been reported. They concern the possible existence of common nuclear genes for cytoplasmic and mitochondrial components, which could account for at least some of the reported influences of the mitochondrial genome on the remainder of the cell.

Research now under way promises to elucidate the mechanisms and signals involved in the interactions between the nuclear and mitochondrial genomes in the formation of functional mitochondria. Furthermore, research in the area of nuclear-mitochondrial interactions should help us understand how the assembly and function of mitochondria are integrated with those of the rest of the cell and how these processes can be modified in relation to cell respiratory demands, cell differentiation, and senescence.

Proteins Are Imported into Mitochondria

The hundreds of distinct polypeptides that make up a mitochondrion are distributed in a specific way in four compartments: the outer membrane, inter-membrane space, the inner membrane, and inner mitochondrial space. After their synthesis on cytoplasmic ribosomes, the nuclear gene-coded polypeptides are imported to their correct location within the mitochondrion. Biochemical studies and the application of recombinant DNA technology have shown that proteins destined for one of the three innermost compartments are usually made from precursors with extensions at the amino-terminal end; these extensions can be as long as 100 amino acids and function as signals directing the proteins to the proper location.

Still unanswered questions include: Which molecules are involved in the import of polypeptides into the mitochondria? What is the role of cytosolic factors in the import process? How do mitochondrial signal sequences perform their function? Why does translocation of proteins across the mitochondrial inner membrane require a gradient of electrical potential across that membrane? Do contact or fusion points between the two membranes function as ports of entry for protein import?

Crystallographic Studies Should Reveal the Tertiary Structure of the Oxidative Phosphorylation Apparatus

The subunit composition of the enzyme complexes of the oxidative phosphorylation system is largely known, as is the location—nuclear or mitochondrial—of the genes specifying these subunits. From the nucleotide sequence of these genes, the amino acid sequence of the subunits can be inferred. Definitive knowledge about their tertiary structure should eventually come from crystallographic studies now in progress.

This information, together with data derived from other approaches to studies of the spatial relations of the subunits in each enzyme complex, is likely to provide useful models of the three-dimensional structure of each complex and of its topology in the inner mitochondrial membrane. These models will help in interpreting the results of ongoing functional studies on the individual complexes in intact mitochondria and in reconstructed systems.

• Cell Motility and the Cytoskeleton

An Understanding of the Basis of Cellular Motility Is Central to Our Understanding of the Functioning of All Organisms

Cell motility is necessary for the survival of virtually all living species. For example, the egg would not be fertilized without a motile sperm, and every cell division that occurs thereafter requires a degree of motility in some cell parts. Without active changes in cell shape and cellular migrations, embryos would not form. Without cellular motility, white blood cells would neither accumulate at sites of inflammation nor ingest invading microorganisms. Without active and rapid movements of organelles in axons and large plant cells, the peripheral parts of these cells would not be nourished.

Cell Structure

Three types of protein polymers constitute the cytoskeleton and interact with force-producing enzymes to cause cells to move.

Cells of both animals and plants contain three different types of fibers—actin filaments, intermediate filaments, and microtubules—each of which is formed by the polymerization of distinct protein molecules. Together these fibers provide mechanical support for the cell and thus are considered to be a cytoskeleton. The actin filaments and microtubules also participate in cellular movements, including locomotion of whole cells, cell division, and movement of subcellular components. This combined ability to maintain form against mechanical forces and to produce and transmit force means that this cytoskeletal motility system can determine cell shape and hence the structure of both tissues and whole organisms. A clear understanding of this system will be essential for unlocking the secrets of embryology.

This field is still in an explosive growth phase during which investigators have isolated and started to characterize the major molecular components of these systems. The inventory includes not only the protein subunits of the polymers themselves but also a surprising number of regulatory proteins. For example, in the actin system alone, one cell has already been shown to have almost 20 accessory proteins, which together with the actin constitute 25 percent of the total cell protein. In the developing brain, the microtubule system may include a similarly large proportion of the total cell protein. In skin, the keratin molecules that form the intermediate filaments constitute the major protein in the cells.

The Cytoskeleton Provides Form to Cells and Therefore to Organisms

The polymeric nature and intracellular distributions of the filaments and microtubules suggest that they may mechanically stabilize the cytoplasmic matrix. Recent physical studies on purified cytoskeletal fibers and analysis of the mechanical properties of live cells support this conclusion. Other work has shown that some of the glycolytic enzymes bind to actin filaments and that polyribosomes are associated with isolated cytoskeletons. Thus, beyond imparting mechanical integrity, the cytoskeleton may provide scaffolding for enzymes that participate in cellular metabolism and protein biosynthesis. In this way, the cytoskeleton, like membranes, may be an essential integrator of cellular processes.

It is now possible to describe, in broad outline, how these protein polymers assemble and how some of the steps in the assembly process may be regulated, at least for actin and microtubules. To a large extent, the construction of the system of cytoplasmic fibers can be explained by the process of self-assembly, in which the protein subunits are driven by chemical attraction for each other to polymerize without external direction. This spontaneous process is controlled by a variety of regulatory proteins, some of which must react to signals from the external environment that direct the organization of the cytoskeleton. Cells also contain organelles, such as the centrosome, that help to organize the cytoskeleton. The centrosome is the site where the assembly of microtubules is initiated.

Biochemical and cellular experiments indicate that the mechanisms controlling the assembly and organization of these fibers in the cell are both complex and subtle, as befits a system with such an important influence on cell architecture. To gain a better understanding of how form is determined in biology, considerable new work will be required (1) at the molecular level to elucidate the molecular composition, regulation, and dynamics of the cytoskeleton and (2) at the cellular level to determine how external stimuli affect the assembly of the cytoskeleton.

Cell Movement

Research on the mechanisms of cell movements is progressing on a new frontier.

In parallel with studies on the structural elements of the cytoplasm, work on mechanisms of cell movements has pushed forward rapidly during the past 15 years on two main frontiers; during recent years, a third and possibly a fourth frontier have begun to open. In each case a specific motor protein is responsible for movement.

The first frontier involves the microtubule-dynein system found in cilia and flagella—whiplike organelles (such as sperm tails) that beat rapidly. Cilia are found in groups on the surface of epithelial cells such as those lining the air passages in our lungs, where they are responsible for sweeping mucus and inhaled foreign material out of the lungs. If the cilia are not active, serious infection is inevitable. Flagella form the tails of sperm and propel them toward their meeting with the egg. In cilia and flagella, microtubules interact with a giant enzyme molecule called dynein to convert the chemical energy stored in ATP into a force that bends the cilia and flagella. Since the chemical steps in this process have now been identified, studies on the molecular mechanism that produces the motion can now be pursued vigorously. Dynein is also present in the cytoplasm, outside cilia and flagella where it can move particles along microtubules in the direction from the cell periphery toward the cell center.

The second frontier is the characterization of myosin—the force-producing enzyme long known to cause contraction in highly specialized muscle cells and more recently recognized to exist along with actin in most other cells, even those not specialized for contraction. Superficially most myosins are similar, and it seems likely that all myosins produce force by interacting with actin filaments and ATP in the same fundamental way. The steps in this process have been studied in detail in muscle (an especially favorable test system). Investigations using spectroscopy, x-ray diffraction, electron microscopy, and biochemical methods are also under way to locate the site in the myosin molecule where motion is produced. Myosin and actin are widely believed to be responsible for many forms of cell movements in addition to muscle contraction. For cytokinesis (the final step in cell division), direct experimental evidence validates this hypothesis. Other types of movements required similar experiments in order to explain their molecular basis.

In the past, most cell biologists suspected that either dynein or myosin was responsible for most cell movements, including the ubiquitous rapid movements of cellular organelles in the cytoplasm, but it has recently been discovered that a new type of motor protein called kinesin moves particles along microtubules in the opposite direction from dynein. Together these two motors provide a two-way rapid-transit system for organelles through the viscoelastic cytoplasm. This kinesin-dynein-microtubule system can shuttle a vesicle manufactured in a nerve cell body in the spinal cord to the nerve endings in the big toe (and back) in a few days! Even newer evidence suggests that an unusual form of myosin may pull some organelles along actin filaments. Breakthroughs of this type have raised the hope that we may soon understand how the mitotic apparatus works and how the traffic of organelles is directed to the proper destinations in the cell.

Each of these motile systems operates under exquisite controls that allow cells to respond to internal or external stimuli and to produce a coordinated motile response. In skeletal muscles and heart, the contractile proteins are turned on by calcium, which activates regulatory proteins bound to the actin filaments. In the smooth muscle cells found in internal organs and in nonmuscle cells, the myosin is activated chemically by the attachment of phosphate to the protein. It is not yet understood how these chemical reactions are coordinated in the living cell to produce the complex patterns of movement that are essential for life. The purse-string-like contraction that splits two daughter cells apart at cell division is an example of a movement in response to an internal stimulus arising from the poles of the mitotic spindle. The rapid locomotion of white blood cells to sites of infection and their ingestion of bacteria are examples of complex movements in response to external signals. In these examples, the stimuli and the responses are well documented, but little is known about the mechanisms that convert the stimulus into the response. Regulation of microtubule-based movements presents a similar challenge.

If work on cell motility continues with its current momentum, progress is likely in the following areas.

Molecular Inventory and Characterization . The complete catalog of the molecular components of the cytoskeletal motility system should be completed for a few cell types that are particularly favorable for use as model systems. These include the slime molds Dictyostelium discoideum and Physarum , the protozoan Acanthamoeba , sea urchin eggs, macrophages, platelets, and the intestinal epithelial cell. Completion of this molecular inventory may require new functional assays for proteins that have yet to be discovered. The primary structures of the major components need to be determined by sequencing cloned DNA, and the three-dimensional structures of the major components must be determined by x-ray crystallography. The first atomic resolution structure of a cytoskeletal protein (the actin-binding protein called profilin) has been completed, and good progress is being made on actin and myosin.

Cellular Organization and Dynamics . Electron microscopy should lead to more precise localization of the components of these systems inside whole cells. Vastly improved probes consisting of antibodies coupled to colloidal gold, together with better methods to prepare cells, should give us a clearer picture of macromolecular architecture. Furthermore, it should be possible to characterize the dynamics of the cytoskeleton in live cells by using new fluorescence techniques (see Chapter 2 ). Purified protein molecules can be tagged with fluorescent dyes and then injected into live cells.

Functions and Regulation of the Cytoskeletal Motility System . Perhaps the major challenges in the field will be to determine the functions of the various components inside living cells and to learn how these functions are regulated by the cell. One approach is the use of in vitro assays with purified components. It is remarkable that functions as complex as the contraction of actin and myosin, the movement of an organelle on a microtubule, or the growth of microtubules from the centrosome to the kinetochore of a chromosome can all be reproduced today in vitro. These assays should become more sophisticated, enabling cell biologists to test for the ability of purified components to carry out complex functions outside living cells. Producing mutant cells with defects in single components will also be valuable in demonstrating functions and identifying regulatory mechanisms. This may be a laborious process because there are multiple genes for many components, and even where there is a single gene, the protein itself may be part of a highly redundant system that will function nearly normally without any given component. The microinjection of inhibitory antibodies to inactivate a single component inside a cell and the inactivation of relevant genes are also promising approaches. These and other creative new approaches will be necessary to test current ideas regarding the physiological functions of the cytoskeletal motility system. A long-term challenge will be to characterize the mechanisms by which an external stimulus, such as a chemoattractant, causes a cell to move in a particular direction.

Mechanical Properties . Analysis of the mechanical properties of the cytoskeleton and its component molecules is essential, but has only begun, in part because few cell biologists are familiar with the engineering techniques required for the work. This is an area of potential collaboration between cell biologists and engineers.

• Cell Membrane

The Cell Membrane Not Only Forms a Protective Surface But Also Receives Chemical Messages from the Environment

The outer cell membrane is an extremely thin, sheetlike assembly of lipid and protein molecules that provides a boundary to the cell's body. This exquisitely delicate skin, called plasmalemma, is a diffusion barrier for water-soluble substances. In the plasmalemma, the cell assembles all the molecular equipment needed for its exchanges and interactions with the environment.

The Plasma Membrane Shares Many Basic Structural Features with Other Types of Cell Membranes

Membrane structure relies on the use of a continuous bimolecular layer of lipids, the diffusion barrier, which is fluid at the temperature of the environment in which the cell lives. The barrier is traversed by transmembrane proteins that subserve a variety of functions, and it is reinforced by a fibrillar infrastructure made up of other different proteins. Depending on cell type, these infrastructures are built either for imparting tensile strength to a delicate membrane or for controlling the lateral mobility of transmembrane proteins, which if not restrained would move rapidly in the membrane because of the fluidity of the lipid bilayer. The first type of infrastructure has been studied extensively in the red blood cells of humans, and its molecular components and their mode of assembly are well known. Their function is to reinforce the membrane and to give the cell its characteristic shape. More recent work shows that the same or related proteins are used by many other cells to solve similar problems.

Most of the studies on the infrastructure that controls lateral protein mobility have focused on the miniature geodetic cages formed by the protein called clathrin and associated proteins. These clathrins are found on structures called coated pits and coated vesicles associated with the plasmalemma as well as with certain intracellular membrane systems. Coated pits trap functionally important molecules from the environment or from intracellular compartments.

Permeability Modifiers Are Transmembrane Proteins That Facilitate the Transport of Water-Soluble Molecules Across the Lipid Bilayer

Many permeability-modifier proteins transport nutrients such as glucose and amino acids. Others are channels for ions, and still others are energy-driven pumps that move sodium, potassium, hydrogen, or calcium ions in or out of the cells against concentration gradients. Many of these molecular pumps are called ATPases because they obtain the energy needed for their work by splitting ATP. The main function of the pumps is to maintain stable intracellular ionic concentrations at optimal levels for the cell's activities.

During the last few years, many transporters, channels, and pumps have been moved from their previous status as hypothesized physiological entities to that of well-defined protein molecules. Moreover, the amino acid sequence of many of them has been deduced from the nucleotide sequence of the cognate complementary DNAs. Knowledge of the amino acid sequence of these proteins is needed as a first step toward understanding their function and the way they fit into membranes.

Channels and pumps generate differences in molecule and ion concentrations (chemical and electrochemical gradients) as well as electrical charge separations (membrane potentials) across the corresponding membranes. These gradients and potentials are used by cells to propagate signals along the cell surface, as in nerve and muscle cells, and to drive the transport of other molecules and ions across the membrane, as in the cells of the intestine and the kidney.

• The Extracellular Matrix of Animals

The Cells of Multicellular Animals Are Supported and Organized by a Continuous Extracellular Matrix Composed of Fibrous Proteins and Complex Polysaccharides

In specialized connective tissues, such as bone, cartilage, and tendon, the extracellular matrix is predominant, but even in tissues such as muscle, liver, and brain, each cell is surrounded by a fine matrix. Connective tissues provide the avenues through which blood vessels pass to nourish every organ and serve as homes for the body's defensive cells, including phagocytes and lymphocytes. Consequently, most inflammatory diseases such as arthritis occur in the connective tissues.

The extracellular matrix is produced primarily by cells called fibroblasts, but also by epithelial and muscle cells. For years we have known that collagen (the most abundant protein in our bodies) and elastin form the major fibers in the extracellular matrix. During the past 10 years biochemists have identified more than 10 different types of collagen that are specialized for forming bone, cartilage, and basement membrane (a ruglike structure that all epithelia—lining and covering tissues—stand on). Some collagens form flexible fibers with tensile strength similar to that of steel, while others form three-dimensional networks. The regular arrangement of collagen in tendons has been known for some time, but the elucidation of the molecular organization of less-regular collagen structures such as basement membranes opens a fascinating research opportunity. Rubberlike elastic fibers are responsible for the ability of large blood vessels and skin to recoil when stretched. Elastic elements allow blood vessels to modulate the pulsatile flow produced by beats of the heart. The mysterious loss of elastic fibers during aging has generated a multimillion dollar cosmetic industry to combat wrinkles. The amino acid sequence of elastin in known, but its molecular structure and its association with other molecules in elastic fibers are major research challenges that, when solved, should help explain and perhaps prevent some cardiovascular diseases and effects of aging.

Connective tissues are also rich in a variety of organ-specific complex sugar polymers, some of which are chemically linked to proteins. They are called, as a group, glycosaminoglycans, or GAGs for short. The name derives from their repeating component sugars. Together with collagen fibrils they are responsible for making the cartilage covering most joint surfaces tough and elastic. GAGs also fill the eye and are major components of skin, blood vessels, and other organs.

There is now active research on a variety of proteins that confer biological specificity on the extracellular matrix. For example, an elongated protein called fibronectin binds cells to the matrix. It has binding sites for a receptor protein of the plasma membrane of connective tissue cells, collagen, and GAGs, so it can link them all together. The bond to the cell is relatively weak, so that a cell can gain traction on the matrix but still move through it. Another protein, laminin, binds epithelial cells to the basement membrane. Perhaps the most remarkable feature of these and other adhesive molecules is that they recognize and bind to a very small site (as few as three amino acids—arginine-glycine-aspartic-acid) on collagen and other matrix molecules. The attachments of both fibronectin and laminin can be altered in tumors, and this change is thought to contribute to the ability of tumor cells to invade some tissues—these invasive tumors are the major cause of death from cancer. Future research should reveal ways in which these adhesive interactions can be modified in beneficial ways in tumor therapy. Evidence is also growing that binding to the matrix modulates cellular physiology.

Active work is also being done on specific growth factors that promote the formation of specialized connective tissue such as bone and on other proteins that initiate the formation of the calcium-phosphate crystals that make bone hard. It has long been appreciated that physical forces on bones control their formation and that inactivity of the elderly contributes to the thinning of bones in osteoporosis. Here is a major opportunity for multidisciplinary research by cell biologists, biochemists, and engineers to learn how physical forces are transduced into the cellular activities that maintain normal bone and that fail in osteoporosis. Equally fascinating are the questions of how the information specifying the shape of the skeleton is laid down in the genetic code, how the cells of the connective tissue translate this information, and how matrix molecules influence the development of adjacent tissues.

• Cell Regulation

Cellular Activities Are Regulated by a Combination of Information Provided by the Genes and by Extracellular Signals

The timing of major decisions made by cells, such as whether to grow, to divide, to move to one location or another, or even to die, is determined by genetic programs and also by environmental clues, such as hormones and contacts with other cells. To understand cell regulation, one must study the production and effects of signals coming from the nucleus as well as the mechanisms responsible for transducing extracellular signals into cellular actions.

Cell Division

The cell division cycle is the master program of cell regulation that organizes a variety of subroutines.

In a very broad sense, to understand cell division, we need to understand more than its component pans: how membranes are assembled and disassembled during mitosis and cytokinesis, how DNA is replicated and organized, and how the mitotic spindle is assembled and disassembled. Beyond this, we need to understand how controls integrate the behavior of the spindle with the replication and segregation of the chromosome, as well as integrate growth and differentiation with cell division. Most importantly, we need to understand how cells ''decide" whether or not to leave their normal resting state in tissues and go on to grow, replicate their DNA, and divide. An identification of these controls should lead to a real understanding of several important diseases—most notably proliferative diseases such as cancer and degenerative diseases—some of which are likely to result from a failure of normal proliferation.

Controls that integrate growth with division occur in the first growth phase of the cell cycle, called G1 in yeast and animal cells. Although many of the components of this control system have been identified in yeast, the links between nutrition, protein synthesis, and the apparatus for cell division remain unknown even for this unicellular organism.

After a century of study, the mechanisms that move chromosomes during cell division in both somatic cells (mitosis) and germ cells (meiosis) are finally becoming clear. The main elements are well known: the mitotic spindle composed largely of two arrays of microtubules. One set runs from the centrioles at the spindle poles to the centromeres (kinetochores) of the chromosome. The chromosomes are pulled to the poles as the kinetochore moves along these microtubules toward the poles. Remarkably, this movement seems to be powered by energy stored in the microtubules, whose depolymerization at the kinetochore determines the rate of movement. The second set of microtubules runs from one pole toward the other. These microtubules are slid past each other by an ATP-requiring motor to push the poles apart, which helps to separate the two sets of chromosomes.

A centriole is located at each pole and remains one of the most poorly characterized elements from a molecular standpoint. An understanding of the molecular organization of the centriole will be essential in defining its role in chromosome movement, spindle and aster formation, and its other function as the basal body for cilia and flagella.

Ultimately, the mechanical problems of chromosome movement will need to be placed in the overall context of cell-cycle control, including (before the actual separation of chromatids at mitosis) DNA replication, shutdown of RNA transcription, chromosome condensation, and breakdown of the nuclear envelope; and (after mitosis) nuclear envelope reformation, chromosome condensation, and reinitiation of transcription. Specific protein phosphorylations help drive a cell into mitosis, and the recent development of in vitro systems in which some of these processes occur outside the cell holds promise for a detailed molecular analysis in the near future. Remarkably, some of the central components of the control process are nearly identical in cells as disparate as those of humans and yeast; thus, many of the details of the human cell cycle can be worked on in simpler cells such as yeast, which are readily amenable to a combined molecular and genetic analysis.

Mitosis in somatic cells and meiosis in germ cells resemble each other in such respects as the formation of the spindle apparatus and the general breakdown and reformation of the nucleus. However, details of chromosome behavior differ markedly. Numerous questions need addressing. What causes homologous chromosomes to pair before meiosis? How are the molecular events of crossing over controlled? What causes the unique behavior of the centromeres during meiosis? How is DNA synthesis suppressed before the second meiotic division? These special problems of meiosis remain largely unexplored from a molecular standpoint. Again, important information will come from organisms such as yeast.

Cell-to-Cell Communication

Cells have developed mechanisms for interacting with other cells.

Cell-cell interactions are important in simple organisms for such functions as sexual reproduction, colony formation, and attachment to various substrates. In multicellular organisms, cell-cell interactions have become much more complex, since they are essential for the integration of large cell populations into structurally coherent and functionally controlled tissues, organs, and organisms.

Short-range communication depends on direct contact between cells and their neighbors or the surrounding environment. Long-range communication requires the movement of informational molecules (such as hormones and growth factors) from one cell to another through the blood or other extracellular spaces and the binding of these molecules to specific receptors on the surface of the target cell.

Short-Range Communication Requires Plasma Membrane Specializations

The critical importance of cell-cell interactions is illustrated by testing the developmental sequence of multicellular organisms. As the one-celled embryo begins to divide and cells begin to differentiate, mechanisms of short-range cell-cell communications emerge. They consist of gap (or communicating) junctions that link a cell to its neighbors through common transmembrane channels. These junctions create common intracellular environments in cell subpopulations and ensure rapid cell-to-cell propagation of membrane permeability changes and intracellular messengers. Short-range interaction mechanisms also include junctional complexes between cells, which allow the developing organism to build walls of cells, called epithelia, that separate the different parts of its body. In addition, cells in general and epithelial cells in particular participate in short-range interactions with the newly formed extracellular matrix. These interactions are mediated by mutual recognition between cell membrane receptors and specific parts of matrix proteins. Cell membranes have multiple receptors for many matrix proteins, which are large monomeric or polymeric protein molecules with specific sites for binding to the plasmalemma as well as to other matrix proteins. The result is the progressive construction of a mechanically coherent body in which the cells are kept in place by their attachment to one another as well as to structural differentiations (for example, basement membranes and collagen fibers) of the extracellular matrix.

These attachments are effected through rigid plates on the plasmalemma that connect bundles of fibrils from the extracellular matrix to actin filaments or intermediate filaments in the cytoplasm. The rigid plates are maintained in relatively fixed positions by the fibrillar cables, which are under tension because they generally follow the lines of stress propagation within the entire system. This type of construction allows the cells to retain their shape, resist pressure, and recover from deformations.

During embryonic development, the production of matrix proteins follows a sequential program presumably matched by the production of plasmalemmal receptors for specific matrix proteins. Certain cell migrations are controlled by cell-matrix interactions and can be experimentally blocked by antibodies to (or small peptides derived from) relevant matrix proteins. Cell migration is thought to be controlled by a process that activates secretion of matrix proteins and concomitant production of cognate receptors. The cells move along tracks laid down by themselves or by their predecessors.

In the adult organism, gap junctions control the propagation of contraction waves in the heart muscle and in the smooth muscles of the intestine and uterus. Modulations in the construction of junctional complexes also control the permeability of epithelia in the intestine, lung, and kidney as well as the permeability of the vascular endothelium.

Long-Range Communication Requires Messenger Molecules and Receptors

As embryonic development progresses, mechanisms of short-range communication are extended and diversified, but long-range interactions through hormones and growth factors become progressively more important. Long-range communication requires the production of chemical signals, such as hormones and growth factors that react with target cells and modify their activities. The essential elements of any long-range communication system are a chemical messenger molecule, a cellular receptor, and a mechanism that transduces the binding of the messenger molecule to the receptor into a biochemical change in the target cell. The biochemical change then modifies the physiological behavior of the cell.

Messenger Molecules Have a Wide Variety of Chemical Compositions

The chemicals that carry messages from one cell to another are extraordinarily diverse. The classical hormones include derivatives of cholesterol (cortisol, testosterone, estrogens), derivatives of amino acids (thyroid hormone), small peptides (growth hormone), and other types of molecules (epinephrine). More recently, attention has turned to a growing list of protein and polypeptide messengers that regulate the cell cycle of target cells and are grouped together as growth factors.

Growth Factors

Growth factors are ligands that bind to receptors and cause dna synthesis to begin.

Major advances in purification and characterization of the growth factors during the past decade have facilitated an understanding of their modes of action. Until the advent of genetic engineering, studies were limited to those factors available in sufficient quantity from biological sources. Much early work was done with three growth factors: erythropoietin, a glycoprotein from the kidney that stimulates red blood cell production from a common stem cell progenitor of both red and white blood cells; nerve growth factor (NGF), which stimulates the development of neurons; and epidermal growth factor (EGF), which was discovered in part because it induces premature eyelid opening and tooth eruption in neonatal mice by stimulating epidermal cell proliferation. The initial discoveries of these three hormones were made in animal models, which were later used to assay the progress of their purification.

Research on these hormones was accelerated greatly during the past decade through advances in animal cell culture technology. One principal limitation of that technology was a requirement for serum. This was overcome through the realization that serum is a rich source of growth factors, especially those required by mesenchymal cells. That realization led to the identification of platelet-derived growth factor (PDGF), a powerful mitogen for fibroblastic cells that is widespread in nature and that can play multiple roles. Other hormones are present in platelets, including tumor growth factor beta, which is a potent modulator of mesenchymal cell proliferation as well as a powerful growth inhibitor for other cells such as epidermal cells. These observations helped explain the well-documented selectivity for preferred growth of mesenchymal cells in cell culture medium containing serum. Once this major roadblock was overcome, progress in identifying requirements for growing epithelial cells in culture was rapid. Much interest in controlling growth of epithelial cells results from their being the origin of approximately 90 percent of human tumors.

The suspected role of growth factors, growth modulators, and their receptors in cancer has created intense interest in an understanding of their actions and synthesis. Unlike hormones produced by endocrine organs, such as insulin and growth hormone, growth factors are not secreted into the blood. Instead, these paracrine hormones are released at or near their target cells. A functional homologue of EGF, termed tumor growth factor alpha, and growth factors of the insulin-related families are secreted by a variety of cells lines derived from tumors. The normal progenitors of these tumor cells themselves are responsive to these hormones from which the tumor cells have become independent. This class of mechanism, in which a growth factor is produced and utilized by the same cells, has been termed autocrine hormone function. Although uncontrolled autocrine behavior could contribute to tumor progression, it is unlikely that acquisition of a single uncontrolled autocrine mechanism would cause cancer. The stimulation of reproduction of epithelial or mesenchymal cells probably requires the synergistic action of several growth factors acting in a specific temporal manner.

Immunomodulators, such as tumor necrosis factor and interleukins 1 and 2 (IL-1 and IL-2), are substances that influence the expansion of immune cell populations. They are produced and utilized by white blood cells at sites of inflammation, including areas of tumor growth, by a paracrine process. Thus the effective dose of a hormone normally develops only in the area of the cells that release it. A number of paracrine-acting immunoregulatory agents are currently undergoing clinical trials, even though extreme toxicity has frequently been observed at doses sufficiently high to achieve pharmacological effectiveness. Thus the development of drug delivery systems that mimic the local delivery specificity of a physiological paracrine process represents major challenges and new opportunities in current attempts to apply immunomodulators in cancer therapy.

There May Be Even More Receptor Proteins Than Messenger Molecules

Receptors are protein molecules that bind specific hormones or growth factors and relay a signal that converts an extracellular message into a biochemical action inside the cell. There may be more kinds of receptors than messenger molecules because the receptors for a single messenger molecule can be different on different types of cells.

The receptors handle their information-transduction functions in a variety of ways. During the past 20 years, our perception of receptors as molecular entities has changed dramatically. Our thinking has evolved from a picture of relatively inert structures able to bind specific ligands that then induce a signaling function to the concept that these proteins contain structural information that enables more diverse functions. We now understand that receptor systems contain structural elements that enable them to bind ligands, participate in signal transduction, and respond to regulation by various cellular mechanisms.

In the simplest case (S1 in Figure 4-3 ) the hormone (for example, cortisol, sex steroids, vitamin D, or thyroid hormones) is sufficiently soluble in lipids to diffuse across the plasma membrane and act on an intracellular receptor. Alternatively, the receptor protein in the plasma membrane is oriented toward the exterior of the cell, where it can bind the extracellular ligand (S2) and carry its signal across the membrane into the cell (as low-density lipoprotein receptor and transferrin). Some receptors are transmembrane proteins with an extracellular portion that binds a ligand such as insulin, EGF, or PDGF (S3); a transmembrane segment; and an intracellular part with enzyme activity. Binding of ligands to the extracellular site can stimulate the phosphorylation of protein tyrosine groups by the kinase activity of the intracellular domain. Still other extracellular signals (S4), including acetylcholine and γ-aminobutyric acid), act by binding to a transmembrane ion channel; opening of the channel in response to binding of the ligand allows specific ions to cross the membrane and alter the electrical potential across the membrane. Finally, a large number of distinct extracellular signals (S5) are detected by receptors that act through a family of proteins (G proteins) that bind guanosine triphosphate (GTP) to regulate production of intracellular mediators of hormone action, known as second messengers [or signaling mechanisms, which include calcium and cyclic adenosine monophosphate (cyclic AMP)]. These second messengers then diffuse through the cell's interior and initiate various biochemical processes.

Strategies for transmembrane signaling. [Adapted from H. R. Bourne and A. L. DeFranco, in The Oncogenes, R. Weinberg and M. Wigler, eds. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., in press), chapter 3]

These multiple receptor pathways allow a diverse approach to the control of receptor function and cell metabolism, permitting rational approaches in the design of therapeutic drugs to correct altered cell functions.

Transmembrane Signaling

Ion channels mediate the cell's electrical communication with its environment.

It has been long been known that "excitable" cells in the neuromuscular system communicate rapidly by means of electric signals generated by the selective passage of ions through protein channels in their plasma membranes. Now it is clear that virtually all cells use related membrane channels as part of their signal transduction systems. Membrane proteins, called ion channels, form tiny water-filled pores across the plasma membrane that are narrow enough to permit diffusion of one or another of small ions such as sodium, potassium, and calcium between the internal and external solutions. Passage of charged ions through the pore forms an electric current that generates potential changes for signaling. The nervous system generates many kinds of electrical signals by using many types of ion channels, each specific for a few common ions and each opened and closed under the control of specific stimuli, such as membrane potential changes, neurotransmitters, light, or mechanical stimuli.

The study of ion channels has advanced greatly during the past 10 years because of spectacular technical improvements in electrical recording methods and in molecular biology. A new recording method, called the patch clamp, permits routine observations of the opening and closing of single ion channels on a submillisecond time scale. Hardly anywhere else in science has it been possible to monitor conformational changes of one molecule. With the patch clamp, channels have been shown to click abruptly between open and closed conformations. We are now faced with a wealth of recordings of the transitions that need to be understood in kinetic and ultimately molecular terms.

Different channel types can be recognized by their patch-clamp signatures— different ionic preference, different absolute conductance, and different rules for opening and closing. At least 30 types have been recognized in the past 8 years, and new ones are being discovered every month.

The patch clamp reveals that ion channels are present in the plasma membrane of all eukaryotic cells, not only excitable cells like nerves and muscle. Because this ubiquity was not previously suspected, an important task is to determine what roles these signaling molecules play in the housekeeping functions and daily life of cells outside the nervous system.

By far the best-understood ion channel to date is the acetylcholine (ACh) receptor channel that mediates the transmission of signals from nerves to muscles ( Figure 4-4 ). The ACh receptor opens a pore in the muscle membrane when the neurotransmitter ACh is released by the nerve impulse in a nearby nerve terminal. The protein subunits of this pentameric receptor molecule have been sequenced. DNA cloning and sequencing have revealed related ACh receptors in other cells. The cloned messages for various ion channels have been injected into frog oocytes, which then make the proteins and incorporate them into their plasma membranes, where they can be studied by patch clamp-methods. Several groups are selectively mutating these messages to test which parts of the sequence are responsible for each feature of the overall function. The three-dimensional structures of selected channels that should become available in the near future, along with results of ongoing molecular biological and electrophysiological studies, will elucidate the operation of these channels at the molecular level.

Acetylcholine opens receptor channels in muscle, a process that can be measured by the patch-clamp technique. [Bertil Hille, University of Washington]

RECEPTORS CAN ACT AS SPECIFIC GATES FOR DRUG DELIVERY

The high specificity exhibited by cell-surface receptors for hormones and other ligands, combined with the ability of receptors to be internalized into cells, provides an exciting opportunity for drug delivery to the cell's interior. Such an opportunity is being actively investigated in disease states in which particular cell types need to be eliminated. The strategy links potent toxic molecules covalently to specific receptor ligands, thus promoting specific internalization of toxin with resultant cell death. Experiments with intact cells in vitro have unequivocally demonstrated the effectiveness of introducing toxins such as diphtheria toxin and ricin into cells in this way. When these toxins are covalently linked to peptide ligands such as epidermal growth factor or transferrin, receptors internalize them and rapid cell death results. In order for this technique to be useful in vivo, tissue specificity of the killing action is required. Because most well-characterized receptor systems are expressed in many cell types, the utility of this approach must be perfected so that specific cells or tissues are destroyed.

Recent experiments designed to achieve cell specificity for introduction of cell toxins via receptor-mediated endocytosis are based on differences between cells in respect to receptor expression. Thus tumor cells often express much higher numbers of certain receptors than normal cells from which they were derived. This elevated receptor expression (and the resulting receptor internalization rates) may confer increased sensitivity of such tumor cells to the killing action of the toxin-specific ligand conjugates. Alternatively, some tumor cells may express unique surface antigens. For example, expression of the fetal form of a receptor sometimes occurs in cancer cells. Specific ligands or perhaps specific antibodies against these unique receptors could provide the cell-type specificity required for this therapeutic approach. Thus we have new opportunities to test these hypotheses to develop better strategies for utilizing cell surface receptors to allow targeting of specific cells for drug delivery.

Because ion channels are large, exposed macromolecules having important functions, they have become the target of many classes of natural toxins and of many clinically active drugs. Toxins from cobras, scorpions, cone shells, dinoflagellates, puffer fish, frogs, sea anemones, and many plants act directly on channel molecules. The animal toxins are useful specific labels for the biochemical identification and purification of channel macromolecules. Exploration of such neurotoxic molecules continues to be a rewarding approach to obtaining new reagents for research.

Plant neurotoxins have already been the inspiration for systematic development of standard clinical agents. Thus curate led to neuromuscular blocking agents, cocaine led to local anesthetics and a class of antiarrhythmics, and papaverine led to another class of antiarrhythmics. These agents act on ACh receptor channels, sodium channels, and calcium channels, respectively, by mechanisms that are still only partially understood. Many widely used neuroleptics, tranquilizers, and antipsychotics act directly on channels. There is some hope now that forthcoming knowledge of the three-dimensional structure of channels can lead to the rational design of major new classes of clinical agents with specific actions.

Channel Modulation . All organs of the body are innervated by the two major branches of the autonomic nervous system—the sympathetic and the parasympathetic. In classical terms, signals in the sympathetic nervous system prepare each organ for times of stress—fight or flight—whereas signals in the parasympathetic prepare for more vegetative functions such as digestion. The molecular details of how the body responds to these signals are emerging. The sympathetic neurotransmitter (norepinephrine) and the parasympathetic neurotransmitter (ACh) act on several classes of membrane receptors to produce several intracellular second messengers, which in turn modulate the activities of a variety of ion channels. The receptors, the G proteins activated by the receptors, and effector enzymes and channels are being identified, purified, and sequenced.

Modulation by a cyclic AMP-dependent phosphorylation plays interesting roles in disparate activities. Stimulation of the sympathetic nerves to the heart releases norepinephrine, which speeds the heart rate and strengthens the stroke in each beat—the familar response of the heart to exercise. We believe that in the pacemaker cells of the heart, the rate of depolarization in each cycle is set by the rate of opening of voltage-gated calcium channels and that in the ventricle the force of the pump stroke is set by the number of calcium ions entering per beat. Much of the response to sympathetic stimulation can be attributed to the phosphorylation of voltage-gated calcium channels, which increases the probability of their opening. Each step, from activation of the β-adrenergic receptor by norepinephrine to phosphorylation of the channel, has been carefully documented.

Another example is a learninglike response called sensitization in the sea slug Aplysia . Here serotonin released by action of one set of sensory nerve fibers increases the neurotransmitter released by another. The cyclic AMP-dependent phosphorylation of a potassium channel, which here shuts the channel off, seems to account for this sensitization.

A major recent triumph of visual physiology was the discovery of how the light signal modulates the operation of a channel in rods and cones of the retina to initiate the electrical signals leading to vision. The coupling of rhodopsin to transducin to a phosphodiesterase enzyme is described in Chapter 6 . The important point here is that the result of a cascade of reactions is the eventual change of the concentration of cyclic guanosine monophosphate (cyclic GMP), which is the direct regulator of the ionic channel. This final stage of transduction was demonstrated with the patch-clamp technique: The channel opened whenever cyclic GMP was applied to the cytoplasmic side of a patch of membrane pulled off the photoreceptor. The possibility that olfactory or taste transduction also requires a cascade of intermediate intracellular messengers offers opportunities for study in the coming years.

G Proteins Are Crucial in Many Kinds of Signal Transduction

Cyclic AMP was discovered as an intracellular second messenger for epinephrine and glucagon more than 25 years ago. Cyclic AMP is synthesized by hormone-sensitive adenyl cyclase. It mediates the cellular effects of a host of polypeptides, biogenic amines, and lipids that regulate mobilization of stored energy (carbohydrates in liver, triglycerides in fat cells), conservation of water by the kidney, homeostasis of calcium ions, contractility of heart muscle, production of adrenal and sex steroids, and many other endocrine and neural functions. Studies indicate that odorant stimuli activate adenyl cyclase in the olfactory epithelium of the nose, suggesting that cyclic AMP is also the intracellular second messenger that mediates the sense of smell. More recently, biochemical and genetic studies of the regulation of cyclic AMP synthesis led to the discovery of G s , a membrane protein that couples hormone receptors to stimulation of adenyl cyclase.

Although it seemed unlikely that cyclic AMP would be the only intracellular second messenger of hormones, the next system was not identified until very recently. In this system ( Figure 4-5 ), hormone receptors also act through a G protein to stimulate the activity of phospholipase C (PLC), which cleaves phosphatidylinositol bisphosphate (PIP 2 ) to form two distinct second-messenger molecules. One of these is diacylglycerol (DAG), a membrane lipid that binds to and stimulates protein kinase C. The other messenger derived from PIP 2 is inositoltrisphosphate (IP 3 ), which acts on receptors in the endoplasmic reticulum to release stored calcium ions and raise their cytoplasmic concentration. The two arms of this signaling cascade, calcium ion (Ca 2+ ) and protein kinase C, act on other cellular components to produce a set of responses that includes contraction of smooth muscle (stimulated by agents such as norepinephrine), modulation of synaptic responses in the central nervous system, a variety of secretory responses, and some, but not all, proliferative responses of cells to growth factors such as PDGF.

Calcium ion as a second messenger. [Adapted from H. R. Bourne and A. L. DeFranco, in The Oncogenes, R. Weinberg and M. Wigler, eds. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., in press), chapter 3]

The discovery of G s led to the discovery of a whole family of G proteins involved in other kinds of signal transduction. A key feature of the G protein machine is that it allows amplification and regulation of the transduced signal by separating excitation of the receptor from activation of the effector in both time and space. Thus, the encounter of a neuromodulator such as norepinephrine with its receptor may be short-lived. When the encounter generates a GTP-bound G s molecule, however, the duration of activation of adenyl cyclase depends on the length of time GTP stays bound to the G protein rather than on the tenacity with which the receptor holds onto norepinephrine.

In addition to G s , at least five other G proteins have been discovered. They include one that mediates hormonal inhibition of adenyl cyclase, two that mediate retinal phototransduction in rods and cones, and others that mediate hormonal regulation of ion channels and production of second messengers derived from phospholipids. Despite their diversity, each of the G proteins is built on the same general plan, comprising three dissimiliar polypeptide chains, called α, β, and γ. The a chains bind and hydrolize GTP and confer on each G protein its specificities for interacting with particular receptors and effectors. The β and γ polypeptides anchor the protein to the membrane. In addition, the highly conserved GTP binding sites of the α chains closely resemble similar sites in the elongation and initiation factors of ribosomal protein synthesis and in the protein products (p21 ras ) of the ras oncogenes and proto-oncogenes. Thus, the duplication and divergence of genes in evolution have modified a single molecular machine and made it useful to mediate vision, olfaction, control of cell proliferation, ribosomal protein synthesis, and cellular regulation by many hormones and neurotransmitters.

The apparent parsimony of evolution encourages investigators to extrapolate information obtained from one class of G proteins to another as well as to other proteins with the conserved GTP binding site, such as p21 ras . Only a few years ago, scientists would have scoffed at the notion that studies of bacterial protein synthesis or retinal phototransduction could reveal anything useful about the cause of cancer.

In addition to explaining the cellular actions of a number of important hormones and growth factors, the cascade of events elicited by these substrates has made two other general contributions to the understanding of signal transduction. First, the discovery of a second system of intracellular messengers encourages the expectation that more such systems will soon be found. The enormous array of chemically distinct membrane phospholipids provides an almost inexhaustible source of potential precursor molecules for generating second messengers.

Second, understanding this system has enriched our understanding of the interplay between different second messenger systems in individual cells. For example, epinephrine can stimulate the release of glucose from the liver via two different pathways using different receptors and different G proteins to generate both cyclic AMP and IP 3 -DAG in the same cell type. The two second-messenger systems act in parallel to stimulate the breakdown of glycogen in the liver cell. In contrast, the IP 3 -DAG and cyclic AMP work in opposite directions in platelets. By imagining only one or two more second-messenger systems in addition to cyclic AMP and IP 3 -DAG, we could account for almost any degree of complexity in cellular responses to extracellular signals. Regulation of cell proliferation involves just such a complex interplay of transduction pathways; these pathways are just beginning to be understood.

G PROTEINS AND HUMAN DISEASE

Growing knowledge of G proteins has already contributed significantly to molecular understanding of human disease. The pathogenic toxins of Vibrio cholerae (cholera) and Bordetella pertussis (whooping cough) are now known to act as enzymes that attach adenosine diphosphate-ribose (ADP-ribose) to the α polypeptide chains of two specific G proteins, G s and G i , respectively. The modification of G s by cholera toxin stabilizes the G protein in its GTP-bound form and thereby increases activation of adenyl cyclase (AC); the resulting cyclic AMP rise in mucosal cells of the intestine causes the profuse secretion of salt and water responsible for the often fatal diarrhea of cholera. In contrast, attachment by pertussis toxin of ADP-ribose to the a chain of G i produces a G protein that is uncoupled from interaction with hormone receptors; as a result, pertussis toxin blocks hormonal inhibition of AC. In other cell types, pertussis toxin's action on G i or a G i -like molecule block ligand-stimulated phospholipid metabolism and elevation of cytoplasmic calcium (thereby inhibiting, for example, release of allergic mediators from mast cells).

In the other direction, the ability of these toxins to introduce radioactively labeled ADP-ribose into the G protein α chains has made them valuable tools for studying G proteins. Indeed, the G i molecule was discovered by investigators primarily interested in the mechanisms of action of pertussis toxin rather than in hormone action.

The pivotal biological roles of the G proteins suggest that mutations in the corresponding genes should produce inherited disorders of signal transduction. One of these possible diseases has been identified: inheritance of a mutant allele of the gene that encodes the α chain of G s produces an inherited human disorder called pseudohypoparathyroidism, type 1 (PHP-1). The hypocalcemia, mental retardation, convulsions, and other clinical manifestations of PHP-1 result from partial resistance to hormones that utilize cyclic AMP as a second messenger. Many endocrine responses mediated by cyclic AMP are only mildly affected in PHP-1; resistance to parathyroid hormone, the master hormone of calcium ion homeostasis, is a prominent feature of PHP-1, suggesting that the amount or activity of G s is normally more rate-limiting for actions of this hormone than of others.

Cytoplasmic Free Calcium Acts as a Second Messenger

Three mechanisms for raising the free calcium ion concentration in cytoplasm have been found: the voltage-gated calcium channel of the plasma membrane, release of calcium ions from the endoplasmic reticulum of muscle when the surface membrane is depolarized in response to a nerve impulse, and release of calcium ions from the endoplasmic reticulum in response to hormonal signals acting on plasma membrane receptors. We understand how IP 3 is produced, but not what it does to the endoplasmic reticulum to release calcium. Because of the central role of calcium ions in signaling, the mechanisms of excitation-contraction coupling in muscle and that of calcium ion mobilization by IP 3 in many cells needs vigorous investigation.

Two processes regulated by intracellular calcium also need to be studied. One is the release of vesicles of packaged neurotransmitters, hormones, or enzymes. Although we know that calcium entry is required, we do not understand any of the subsequent steps that, for example, cause the transmitter ACh to be released within 0.1 millisecond of the excitation of a nerve terminal. Both botulism and tetanus are bacterial diseases whose lethal consequences are due to toxins that interfere with this calcium-dependent secretion by nerve cells. Another calcium-dependent process is cell motility, including muscle contraction. The calcium-sensitive component molecules of the contractile machinery have been characterized in some detail, but except for muscle the physiological mechanisms coordinating motility are poorly understood.

Progress is now possible in the analysis of calcium-stimulated events with the advent of new optical methods to measure intracellular calcium ion concentrations. Newly synthesized calcium-indicator dyes can be injected or allowed to permeate into cells. The time course of changes within a single cell can now be monitored reliably. Computer-aided spectroscopic techniques permit making accurate maps of the changing free calcium in each pixel of a video image of the cell observed under a high-power light microscope. The technique has already been used to observe the fertilization of eggs and the stimulation of liver cells by peptide hormones.

Transduction Mechanisms Use a Variety of Strategies to Alter Cellular Chemistry in Response to Messenger Molecules

More intracellular second messengers remain to be discovered, and the means by which they and the ''established" second messenger systems integrate control of cell function are still to be elucidated. Thus, a large number of hormones and other chemical signals act on receptors whose transduction mechanisms are unknown. These include growth hormone, many of the lymphokines, nerve growth factor, and tumor necrosis factor. Understanding the actions of these ligands may uncover new molecular mechanisms of signal transduction. Moreover, each transduction system can alter its sensitivity to extracellular stimuli. Determining the molecular mechanisms that regulate sensitivity represents important new opportunities in research.

A New Class of Genes Can Transform Host Cells

An oncogene (the src gene) was first identified in the transforming Rous sarcoma retrovirus. When suitably expressed, this gene alone was capable of transforming avian cells. Normal cellular homologues of oncogenes, or cancer genes, were found subsequently in virtually every organism analyzed, which suggested that transposed DNA from normal hosts had been incorporated into the retrovirus genome, under control of viral promotors, and was capable of initiating and maintaining transformation of infected cells. Subsequently, more than 25 oncogenes have been identified along with the normal cellular counterparts, the cellular proto-oncogenes. Oncogenes may be resolved into families that include elements of many of the cell-regulatory systems reviewed above: (1) the protein products of the src gene family appear to be tyrosine-specific protein kinases (and thus similar to growth-factor receptors) that are located at the plasma membrane or in cytoplasm; (2) other oncogenes are related to growth factors or to their transmembrane receptors; (3) the ras gene proteins bind and hydrolyze GTP and seem to act as signal transducers at the plasma membrane and cytoplasm; and (4) nuclear oncogenes probably act as transcription factors or DNA-binding proteins.

Research opportunities include the identification and functional characterization of additional proteins encoded by oncogenes. Once defined, the pathways by which these proteins influence cell growth and those that regulate proto-oncogenes should provide insight into the cell cycle, cell proliferation, and control by pharmacological means. Research into oncogenes should help us better understand normal cellular proliferation, the abnormal proliferation characteristic of the cancer cell, and pathways of significance to cellular differentiation. Other research using genetically engineered molecular hybrids should provide understanding at the molecular level of the function of each of the protein domains of these gene products responsible for transformation.

• General Plant Cell Biology

Plant Cells Differ from Animal Cells in Many Ways

Cells of plants and animals have many features in common, such as nuclei, endoplasmic reticula, Golgi complexes, mitochondria, plasmalemmas, coated vesicles, microfilaments, and microtubules; but in some features and processes, cells of these two kingdoms are distinct from one another. For example, plant, but not animal, cells contain plastids (the generic term that includes chloroplasts and modified chloroplasts such as amyloplasts that produce and store starch), large vacuoles that serve as the lyric compartment (lysosome-like) and contain most of the plant cell's water; they also have relatively rigid cell walls composed of strands of cellulose embedded in amorphous carbohydrate polymers together with a small amount of protein.

At higher levels of organization, plant cells are connected to one another by strands of cytoplasm (plasmodesmata), rather than by junctional elements of the type found in animal cells. The tissues and organs of plants and animals are not organized along similar functional lines. Leaves, stems, roots, and buds have no conspicuous counterparts in hearts, livers, skins, or lungs.

At many fundamental levels—biosynthetic pathways and structures of proteins and nucleic acids—plant and animal cells are often, but not always, the same. Thus, differences, when they occur in similar processes, often reveal much about the basic nature of a process. It is often profitable to determine whether newly discovered features of plants occur in animals and vice versa.

The features of plant cells and tissues that differ distinctively from those of animals each require direct investigation. For example, in plants, mitochondria perform the same respiratory functions (and perhaps some other, as yet undiscovered functions) as they do in animals, but they generally contain much more DNA. They also seem to use the standard genetic code, whereas the mitochondria of animals and yeast use a nonstandard one.

In addition to photosynthesis, a number of phenomena are unique to plants, and some are targets of active research. For example, each plant cell contains three rather than two gene-containing compartments (the nucleus, mitochondria, and plastids), thus greatly complicating protein targeting and the integration of genome expression. Other research is focused on the plant's rigid cell walls, which are constituted in different ways at different stages of development and for various specific functions of different cell types. The composition of the cell walls is reasonably well understood, but many questions remain unanswered.

Self-incompatibility, the inability of some plants to fertilize themselves, is an important mechanism for regulating outcrossing. The expression of plant genes can be affected by symbiotic or pathogenic microorganisms through complex interactions. In this area, important advances have been made, especially in studies of symbiotic nitrogen fixation, but the total effort is still very small. Some transposable genetic elements have been characterized molecularly, but how they are integrated into and excised from the genome remains to be understood.

Chloroplasts

Photosynthesis is the biological process that connects life on earth to the sun.

Through photosynthesis, light energy is converted to chemical-bond energy stored in sugar molecules. In higher plants and algae, photosynthesis is carried out in chloroplasts—organelles containing vesicles bounded by energy-transducing membranes in which the chlorophyll is localized. In primitive, noncompartmentalized cells—prokaryotes—these vesicles lie free in the cytoplasm.

Progress in understanding photosynthesis has been intertwined with advances in chemistry, photophysics, and biology. The path followed by carbon during photosynthesis from carbon dioxide to sugar—is now known in detail, and most of the enzymes have been identified. The enzymes themselves are being studied, and, surprisingly, a number of them are found to be regulated (in ways yet to be understood) by light and by certain of the small molecules that are intermediates in the biosynthetic chains of photosynthetic carbohydrate production. The regulatory mechanisms and how each enzyme interacts with other proteins and with its substrates are questions currently being addressed.

Chloroplast Genes Are Being Mapped and Sequenced at a Rapid Rate

The chloroplast genomes of tobacco and liverwort have been fully sequenced, and restriction maps of the chloroplast genome have been completed for at least 40 to 50 species of plants. Most gene mapping is limited to genes that were located and initially mapped and sequenced in maize, spinach, tobacco, and the green algae Chlamydomonas and Euglena .

As more and more chloroplast DNA is sequenced, interest will grow in identifying the gene products of unrecognized proteins; their identification should move us toward a better understanding of photosynthesis and plastid metabolism. This DNA sequencing will also reveal features of plastid genes. At least one type of promoter sequence—resembling the prokaryotic type—has been recognized. The existence of other kinds of control sequences remains to be established. The identification of these and other controlling elements of plastid or nuclear-cytoplasmic origin that constitute the parts of the machinery for control of differential gene expression may well be the most interesting and outstanding problem in this research area. Its resolution is likely to illuminate the mechanisms underlying the transcriptional control of chloroplast gene expression and to reveal research approaches leading toward an understanding of intergenomic integration.

Gene Expression in Chloroplasts Is Both Developmentally and Functionally Regulated

Like mitochondria, plastids contain genetic material, but not all of their components are products of these genes. Many proteins of the chloroplast are imported from the cytoplasm, and these are encoded in nuclear genes. For example, the larger of the two subunits of ribulose bisphosphate carboxylase/oxygenase (rubisco) is the product of a chloroplast gene, whereas the smaller subunit is the product of a nuclear gene.

How the photosynthetic apparatus of chloroplasts is produced, that is, its biogenesis, is a question fundamental to understanding the life of a plant or alga cell. A number of related questions are under active investigation: What are the special characteristics of chloroplast genes? What are the enzymes and mechanisms for their replication? What are the mechanisms for controlling the expression of sets of developmentally regulated chloroplast genes? How is the expression of these genes integrated with the expression of nuclear genes for chloroplast components? How are nuclear gene-coded, cytoplasmically synthesized proteins targeted to plastids, and what are the mechanisms for their uptake and integration into the life of the plastid? How does the machinery for chloroplast gene expression interact with the machinery of the nuclear-cytoplasmic compartment? Among the most interesting aspects of the plant eukaryotic cell is the integration of the activities of its multiple compartmentalized genomes—of nuclei, plastids, and mitochondria. The nature of the integrating mechanisms can now be investigated.

The Origin of Plastids Is Still a Mystery

One of the great puzzles of modern biology is how eukaryotic cells originated. A unique characteristic of eukaryotes is the presence of multiple compartmentalized genomes. In plants, these include the nucleus, mitochondria, and plastids. The question of how the expression of these genomes is integrated has already been raised. An older unanswered question is, How did the multiple genomes come into existence? There are two obvious possibilities. One is that membranous compartments formed in the structural equivalent of a modern prokaryotic cell, and some genes then became sequestered in each compartment. A second possibility, favored by most available evidence, is that the major organelles characteristic of eukaryotic cells—mitochondria (which are found in the cells of all but a very few eukaryotes) and chloroplasts—are the descendents of symbiotic bacteria that entered early eukaryotic cells. To account for the genetic organization of contemporary eukaryotic cells it is necessary to assume the movement of genes or gene functions from the symbiont to the host genome. Many researchers have interpreted the available evidence as suggesting multiple origins, involving different groups of bacteria, for chloroplasts and perhaps for mitochondria also. These hypotheses require further analysis.

What evolutionary pressures led to the existence of the different information-processing systems now found in the nuclear-cytoplasmic and organelle compartments? Are the different systems relics of the independent evolution of two progenitor cell types that subsequently joined to become the ancestral form of the modem eukaryotic cell? Alternatively, did two or three information storage and processing systems evolve within a single cell? What we learn about the molecular biology of the organelle and the nuclear-cytoplasmic systems may lead to a better understanding of the origin and evolution of eukaryotic cells as well as of the forces and mechanisms underlying the shifts of genes among genomes.

Evolutionary relations among genomes are much better understood now than they were half a dozen years ago because of the accumulation of nucleic acid sequence data. However, the forces that have led to the segregation of genes in nuclei, mitochondria, and plastids are unknown. Gene-transfer methods are beginning to be used to explore these questions from the focus of the nuclear genome. Efficient organelle gene transfer (transformation) methods that would greatly aid such investigations remain to be developed.

The Plant Cell Wall

Research on the extracellular matrix (cell wall) of plants is crucial to understanding how plants grow.

Lacking a skeletal structure and subjected to a fluctuating osmotic environment, plant cells rely on rigid cell walls to serve as cementing substances between cells, to provide mechanical strength, and to support high internal osmotic pressures. For many years, it has been known that the ultimate shape and strength of such walls are largely determined by the pattern and extent of deposition of extended fibrils composed of cellulose (glucose molecules joined in β-1,4 linkage). In recent years, the concept has evolved that a second framework, composed of cross-linked extended rods of a hydroxyproline-rich glycoprotein called extensin, also contributes to strength and shape in some plant cell types. Interspersed within these frameworks exist a variety of matrix polysaccharides, some having complex structures. One example is the recently discovered, highly branched rhamnogalacturonan II polymer that contains a number of variously linked sugars including a novel monosaccharide called aceric acid, which was previously unknown in nature. Progress in determining the structure of these polysaccharides has advanced considerably in recent years, in part as a result of vastly improved techniques for the analysis of complex carbohydrates. These techniques require the use of expensive instrumentation such as mass and nuclear magnetic resonance spectrometers. Increased access to such instrumentation would hasten progress in the study of extracellular matrices of both plants and animals.

Understanding plant cell-wall structure is crucial for the ultimate understanding of how plants grow. Plants increase in size by expanding their rigid cell walls, but our understanding of the mechanism by which this occurs is still limited. The process is known to be under hormonal control; wall loosening is believed to occur by processes of breakage and reformation of crucial linkages and by turnover of some wall polymers, but the specific processes remain elusive. Recent structural analyses have provided evidence for intra- and interchain linkages between extensin molecules through isodityrosine residues, presumably formed in the wall by a peroxidase-mediated reaction. Similar enzymes may also be responsible for the formation of cross-links between phenolic components attached in ester-linkage to matrix polysaccharides. Other recent studies have implicated hormonally regulated degradative enzymes involved in the turnover of some wall polysaccharides, such as xyloglucan, in the process of wall extension.

On the basis of current knowledge of wall structure, one can estimate that there must be more than a hundred different enzymes required for wall assembly. Not one of these enzymes has yet been purified and characterized in detail, although a number have been detected and assayed in crude membrane preparations isolated from plants. Similarly, no gene responsible for coding for these enzymes has been identified, mapped, or cloned. Exciting progress has been made recently for the cell-wall protein extensin, since a gene for this protein has recently been cloned and characterized. Coupled with previous structural information on the protein, data from the gene sequence now give the entire amino acid sequence of the protein so that we can identify sites of glycosylation, cross-linking, and possible areas of interaction of this polymer with other wall components.

We now recognize that the cell wall is a dynamic structure. Not only do changes occur during normal growth and development, but the wall also responds quickly to external perturbations such as mechanical damage, water, or temperature stress and upon interaction with symbionts, pathogens, or parasites. Recent work has defined some of these "wound" responses in some detail. Specific changes identified are a cessation of cellulose synthesis coupled with induction of synthesis of a related beta-glucan called callose, which seals off areas of assault; induction of lignin or suberin synthesis; and elevation of synthesis of the soluble precursor to cell-wall extensin. This latter compound in some cases seems to serve as an agglutinin for invading pathogens. Most exciting is the recent work implicating fragments of wall polysaccharides as regulatory molecules. One example is an oligogalacturonide released from the wall by invading pathogens; this oligosaccharide serves as a specific inducer of the synthesis of phytoalexins—plant-made antibiotics that are toxic to invading microorganisms. Other examples include small phenolic compounds that may be released from the wall and that signal interactions crucial for the establishment of plant parasites or symbiotic associations with bacteria (see Chapter 11 ).

Recent advances in our ability to analyze the structure of wall components indicate that the time is rapidly approaching when data from structural studies can be integrated with growth physiology studies to achieve an overall understanding of plant growth. Recent development of specific dyes and monoclonal antibodies that interact with specific linkages in the wall should lead to a much better understanding of the localization and interaction of the various polymers in the wall. Cloning of the extensin genes will now allow a study of the regulation of various members of this family of genes; the patterns of expression of these genes should help clarify the various functions of this unusual polymer. The exciting discovery that fragments of wall components serve as regulatory signals will undoubtedly open a whole new area of study on ways plants communicate and interact with other organisms. Finally, recent advances in the biochemistry of membrane proteins may lead to breakthroughs in the identification and isolation of enzymes and their corresponding genes involved in wall synthesis. Since plant cell walls are a major sink for biomass, much of which is only poorly digestible, it is hoped that our ultimate ability to modify wall structure by modifying the expression of genes controlling wall assembly will lead to a greater understanding of just how flexible such wall structure can be, and perhaps also to the development of plants with improved agronomic or nutritional value.

• Cite this Page National Research Council (US) Committee on Research Opportunities in Biology. Opportunities in Biology. Washington (DC): National Academies Press (US); 1989. 4, Genes and Cells.
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