Mendel’s Law of Independent Assortment
In a series of experiments first reported in 1865, Austrian botanist Gregor Mendel
established the first principles of genetics. Mendel showed that the units of heredity were inherited as particles that maintained their identity across the generations; these units of heredity are now known as genes. These genes exist as pairs in all the body’s cells except for the egg and sperm cells. When Mendel studied two traits at a time (dihybrid inheritance), he discovered that different genes were inherited independently of one another, a principle that came to be called the law of independent assortment. For example, if an individual inherits genes A and B from one parent and genes a and b from the other parent, in subsequent generations the combinations AB, Ab, aB, and ab would all occur with equal frequency. Gene A would go together with B just as often as with b, and gene B would go with A just as often as with a. Mendel’s results were ignored for many years after he published his findings, but his principles were rediscovered in 1900 by Erich Tschermak von Seysenegg in Vienna, Carl Erich Correns in Tübingen, Germany, and Hugo de Vries in Amsterdam. Organized research in genetics soon began in various countries in Europe and also in the United States.
Sutton’s Hypothesis
Mendel’s findings had left certain important questions unanswered: Why do the genes exist in pairs? Why do different genes assort independently? Where are the genes located? Answers to these questions were first suggested in 1903 by a young American scientist, Walter Sutton, who had read about the rediscovery of Mendel’s work. By this time, it was already well known that all animal and plant cells contain a central portion called the nucleus and a surrounding portion called the cytoplasm. Division of the cytoplasm is a very simple affair: The cytoplasm simply squeezes in two. The nucleus, however, undergoes mitosis, a complex rearrangement of the rod-shaped bodies called chromosomes, which exist in pairs. Sex cells (eggs or sperm) are “haploid,” with one chromosome from each pair. All other body cells, called somatic cells, have a “diploid” chromosome number in which all chromosomes are paired. During mitosis, each chromosome becomes duplicated; then the two strands (or chromatids) split apart and separate. One result of mitosis is that the chromosome number of each cell is always preserved. Sutton also noticed that eggs in most species are many times larger than sperm because of a great difference in the amount of cytoplasm. The nuclei of egg and sperm are approximately equal in size, and these nuclei fuse during fertilization, a process in which two haploid sets of chromosomes combine to make a complete diploid set. From these facts, Sutton concluded that the genes are probably in the nucleus, not the cytoplasm, because the nucleus divides carefully and exactly while the cytoplasm divides inexactly. Also, if genes were in the cytoplasm, one would expect the mother’s contribution to be much greater than the father’s, contrary to the repeated observation that the parental contributions to heredity are usually equal.
Of all the parts of diploid cells, only the chromosomes were known to exist in pairs. If genes were located on the chromosomes, it would explain why they existed in pairs (except singly in eggs and sperm cells). In fact, the known behavior of chromosomes exactly paralleled the postulated behavior of Mendel’s genes. Sutton’s hypothesis that genes were located on chromosomes came to be called the chromosome theory of heredity. According to Sutton’s hypothesis, Mendel’s genes assorted independently because they were located on different chromosomes. However, there were only a limited number of chromosomes (eight in fruit flies, fourteen in garden peas, and forty-six in humans), while there were hundreds or thousands of genes. Sutton therefore predicted that Mendel’s law of independent assortment would apply only to genes located on different chromosomes. Genes located on the same chromosome would be inherited together as a unit, a phenomenon now known as linkage.
In 1903, Sutton outlined his chromosomal theory of heredity in a paper entitled “The Chromosomes in Heredity.” Many aspects of this theory were independently proposed by Theodor Boveri, a German researcher who had worked with sea urchin embryos at the Naples Marine Station in Italy.
Linkage and Crossing Over
Sutton had predicted the existence of linked genes before other investigators had adequately described the phenomenon. The subsequent discovery of linked genes lent strong support to Sutton’s hypothesis. English geneticists William Bateson and Reginald C. Punnett
described crosses involving linked genes in both poultry and garden peas, while American geneticist Thomas Hunt Morgan
made similar discoveries in the fruit fly (Drosophila melanogaster). Instead of assorting independently, linked genes most often remain in the same combinations in which they were transmitted from prior generations: If two genes on the same chromosome both come from one parent, they tend to stay together through several generations and to be inherited as a unit. On occasion, these combinations of linked genes do break apart, and these rearrangements were attributed to “crossing over” of the chromosomes, a phenomenon in which chromosomes were thought to break apart and then recombine. Some microscopists thought they had observed X-shaped arrangements of the chromosomes that looked like the result of crossing over, but many other scientists were skeptical about this claim because there was no proof of breakage and recombination of the chromosomes in these X-shaped arrangements.
Genetic Mapping
Sutton had been a student of Thomas Hunt Morgan at Columbia University in New York City. When Morgan began his experiments with fruit flies around 1909, he quickly became convinced that Sutton’s chromosome theory would lead to a fruitful line of research. Morgan and his students soon discovered many new mutations in fruit flies, representing many new genes. Some of these mutations were linked to one another, and the linked genes fell into four linkage groups corresponding to the four chromosome pairs of fruit flies. In fruit flies as well as other species, the number of linkage groups always corresponds to the number of chromosome pairs.
One of Morgan’s students, Alfred H. Sturtevant, reasoned that the frequency of recombination of linked genes should be small for genes located close together and higher for genes located far apart. In fact, the frequency of crossing over between linked genes could serve as a rough measure of the distance between them along the chromosome. Sturtevant assumed that the frequency of recombination would be roughly proportional to the distance along the chromosome; recombination between closely linked genes would be a rare event, while recombination between genes further apart would be more common. Sturtevant first used this technique in 1913 to determine the relative positions of six genes on one of the chromosomes of Drosophila. For example, the genes for white eyes and vermilion eyes recombined about 30 percent of the time, and the genes for vermilion eyes and miniature wings recombined about 3 percent of the time. Recombination between white eyes and miniature
wings took place 34 percent of the time, close to the sum of the two previously mentioned frequencies (30 percent plus 3 percent). Therefore, the order of arrangement of the genes was:
white ← 30 units →vermilion ← 3 units →miniature
Since the distances were approximately additive (the smaller distances added up to the larger distances), Sturtevant concluded that the genes were arranged along each chromosome in a straight line like beads on a string. In all, Sturtevant was able to determine such a linear arrangement among six genes in his initial study (an outgrowth of his doctoral thesis) and many more genes subsequently. Calvin Bridges, another one of Morgan’s students, worked closely with Sturtevant. Over the next several years, Sturtevant and Bridges conducted numerous genetic crosses involving linked genes. They used recombination frequencies to determine the arrangement of genes along chromosomes and the approximate distances between these genes, thus producing increasingly detailed genetic maps of several Drosophila species.
The use of Sturtevant’s technique of making linkage maps was widely copied. As each new gene was discovered, geneticists were able to find another gene to which it was linked, and the new gene was then fitted into a genetic map based on its linkage distance to other genes. In this way, geneticists began to make linkage maps of genes along the chromosomes of many different species. There are now more than one thousand genes in Drosophila whose locations have been mapped using linkage mapping. Extensive linkage maps have also been developed for mice (Mus musculus), humans (Homo sapiens), corn or maize (Zea mays), and bread mold (Neurospora crassa). In bacteria such as Escherichia coli, other methods of genetic mapping were developed based on the order in which genes
were transferred during bacterial conjugation. These mapping techniques reveal that the genes in bacteria are arranged in a circle or, more precisely, in a closed loop resembling a necklace. This loop can break at any of several locations, after which the genes are transferred from one individual to another in the order of their location along the chromosome. The order can be determined by interrupting the process and testing to see which genes had been transferred before the interruption.
Confirmation of the Chromosome Theory
The first confirmation of the chromosome theory was published in 1916 by Bridges, who studied the results of a type of abnormal cell division. When egg or sperm cells are produced by meiosis, only one chromosome of each chromosome pair is normally included in each of the resultant cells. In a very small proportion of cases, one pair of chromosomes fails to separate (or “disjoin”), so that one of the resultant cells has an extra chromosome while the other cell is missing that chromosome. This abnormal type of meiosis is called nondisjunction. In fruit flies, as in humans and many other species, females normally have two X chromosomes (XX) and males have two unequal chromosomes (XY). Bridges discovered some female fruit flies that had the unusual chromosome formula XXY; he suspected that these unusual females had originated from nondisjunction, in which two X chromosomes had failed to separate during meiosis. Bridges studied one cross using a white-eyed XXY female mated to a normal, red-eyed male. (The gene for white eyes was known to be sex-linked; it was carried on the X chromosome.) Bridges was able to predict both the genetic and chromosomal anomalies that would occur as a result of this cross. Among the unusual predictions that were verified experimentally was the existence of a chromosome configuration (XYY) that had never been observed before. Using the assumption that the gene for white eyes was carried on the X chromosome in this and other crosses, Bridges was able to make unusual predictions of both genetic and chromosomal results. These studies greatly strengthened the case for the chromosomal theory.
In 1931, Harriet Creighton and Barbara McClintock
were able to confirm the chromosomal theory of inheritance much more directly. Creighton and McClintock used corn plants whose chromosomes had structural abnormalities on either end, enabling them to recognize the chromosomes under the microscope. One chromosome, for example, had a knob at one end and an attached portion of another chromosome at the other end, as shown in the figure headed “Creighton and McClintock’s Cross.” Creighton and McClintock then crossed plants differing in two genes located along this chromosome. One gene controlled the color of the seed coat while the other produced either a starchy or waxy kernel. The parental gene combinations (C with wx on the abnormal chromosome and c with Wx on the other chromosome) were always preserved in noncrossovers. However, a crossover between the two genes produced two new gene combinations: C with Wx and c with wx.
In this cross, Creighton and McClintock observed that the chromosomal appearance in the offspring could always be predicted from the phenotypic appearance: Seeds with colorless seed coats and starchy kernels had normal chromosomes, seeds with colored seed coats and waxy kernels had chromosomes with the knob at one end and the extra interchanged chromosome segment at the other end, seeds with colorless seed coats and waxy kernels had the interchanged segment but no knob, and seeds with colored coats and starchy kernels had the knob but not the interchanged segment. In other words, whenever the two genes showed rearrangement of the parental combinations, a corresponding switch of the chromosomes could be observed under the microscope. The interchange of chromosome segments was always accompanied by the recombination of genes, or, in the words of the original paper,
cytological crossing-over . . . is accompanied by the expected types of genetic crossing-over. . . . Chromosomes . . . have been shown to exchange parts at the same time they exchange genes assigned to these
regions.
In short, genetic recombination (the rearranging of genes) was always accompanied by crossing over (the rearranging of chromosomes). This historic finding established firm evidence for the chromosomal theory of heredity. Later that same year, Curt Stern published a paper describing a similar experiment using fruit flies.
Physical Mapping and Further Confirmation
Other evidence that helped confirm the chromosome theory came from the study of rare chromosome abnormalities. In 1933, Thomas S. Painter called attention to the large salivary gland chromosomes of Drosophila. Examination of these large chromosomes made structural abnormalities in the chromosomes easier to identify. When small segments of a chromosome were missing, a gene was often found to be missing also. These abnormalities, called chromosomal deletions, allowed the first physical maps of genes to be drawn. In all cases, the physical maps were found to be consistent with the earlier genetic maps (or linkage maps) based on the frequency of crossing over.
When Bridges turned his attention to the “bar eyes” trait in fruit flies, he discovered that the gene for this trait was actually another kind of chromosome abnormality called a “duplication.” Again, a chromosome abnormality that could be seen under the microscope could be related to a genetic map based on linkage. Larger chromosome abnormalities included “inversions,” in which a segment of a chromosome was turned end-to-end, and “translocations,” in which a piece of one chromosome became attached to another. There were also abnormalities in which entire chromosomes were missing or extra chromosomes were present. Each of these chromosomal abnormalities was accompanied by corresponding changes in the genetic maps based on the frequency of recombination between linked genes. In cases in which the location of a chromosomal abnormality could be identified microscopically, this
permitted an anchoring of the genetic map to a physical location along the chromosome. The correspondence between genetic maps and chromosomal abnormalities provided important additional evidence in support of the chromosomal theory. Other forms of physical mapping were developed decades later in mammals and bacteria. The increasingly precise mapping of gene locations led the way to the development of modern molecular genetics, including techniques for isolating and sequencing individual genes.
The discovery of restriction endonuclease enzymes during the 1970s allowed geneticists to cut DNA molecules into small fragments. In 1980, a team headed by David Botstein measured the sizes of these “restriction fragments” and found many cases in which the length of the fragment varied from person to person because of changes in the DNA sequence. This type of variation is generally called a “polymorphism.” In this case, it was a polymorphism in the length of the restriction fragments (known as a restriction fragment length polymorphism, or RFLP). The use of the RFLP technique has allowed rapid discovery of the location of many human genes. The Human Genome Project
(an effort by scientists worldwide to determine the location and sequence of every human gene) would never have been proposed had it not been for the existence of this mapping technique.
Key terms
crossing over
:
the breakage of chromosomes followed by the interchange of the resulting fragments; also, the recombination of genes that results from the chromosomal rearrangement
genetic mapping
:
the locating of gene positions along chromosomes
independent assortment
:
the inheritance of genes independently of one another when they are located on separate chromosomes
linkage
:
the frequent inheritance of two or more genes together as a unit if they are located close together on the same chromosome
linkage mapping
:
a form of genetic mapping that uses recombination frequencies to estimate the relative distances between linked genes
physical mapping
:
a form of genetic mapping that associates a gene with a microscopically observable chromosome location
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