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MCB 142 MAJOR ADVANCES IN UNDERSTANDING EVOLUTION AND HEREDITY FALL 2015 Week 5: October 6 and 8 OCTOBER 6: LINKAGE AND CROSSING-OVER. GENETIC MAPS. Chiasmata. Linkage and Recombination. Linearity of genetic maps. OCTOBER 8: MUTATION Mutation rates. Artificial mutation. Reversion. Implications for the nature of the gene. (a) LINKAGE AND CROSSING-OVER Readings to be discussed Tuesday October 6 Thomas Hunt Morgan (1911) Random segregation versus coupling in Mendelian inheritance. Science 34: 384. Read the article. Excerpts from Frans A. Janssens (1909) The chiasmatype theory. A new interpretation of the maturation division. Céllule 25: 387-411. Translation by Romain Koszul, Karine Van Doninck & Matthew Meselson. (Plus a schematic diagram of a chiasma.) Read the posted excerpts and inspect the diagram. Alfred Henry Sturtevant (1913) The linear arrangement of six sex-linked factors in Drosophila, as shown by their mode of association. Journal of Experimental Zoology 14: 43-59. Read the article. (b) MUTATION Readings to be discussed Thursday October 8 Hermann J. Muller and Edgar Altenburg (1919) The Rate of Change of Hereditary Factors in Drosophila, Proceedings of the Society for Experimental Biology and Medicine 17: 10-14. Read the article. Hermann J. Muller (1922) Variation Due to Change in the Individual Gene, American Naturalist 56: 32-50. Read the article. Hermann J. Muller (1923) Mutation. Mutation, Eugenics, Genetics and the Family 1: 106-112 (Proceedings of the Second International Congress of Eugenics, New York, September 1921). Read the article. -1- Study Questions Due Tuesday October 6 1) What classes of gametes will be produced by Drosophila females heterozygous at two loci A/a and B/b if the two loci are far apart on the same chromosome? Which of these classes would be reduced if the two loci were closer on the same chromosome? 2) In calculating the “percent of cross-overs” for the interval PM, Sturtevant uses only some of the data in Table 1. What numbers from the table does he use and why does he use only these numbers? 3) What findings of Sturtevant show that there can be more than one cross-over on the X-chromosome in an individual meiosis? 4) Is strict additivity of recombination frequencies required in order to determine the linear order of three separate factors along a chromosome? Explain. 5) Sturtevant notes that flies with rudimentary wings have low viability, an effect seen in the low numbers in which they are found in the progeny of crosses, as compared with the frequency of normalwinged flies. If the same proportion of flies with rudimentary wings is lost from each class of flies having them, will the measured recombination frequency be lowered, increased, or unaffected? Explain. Notation Sturtevant denotes the six mutations used in his crosses by letters no longer used for these mutations. His notation and the modern notation are given below, with Sturtevant’s notation first, followed by the modern term and symbol for the mutation. Wild-type flies have red eyes, gray bodies, and long wings. b yellow body = y; c, white eye = w o, white-eosin colored eye = we p, vermillion eye = v r, miniature wing = m m, rudimentary wing = r w and we are alleles of the same gene. 6) Describe the basis for the second (non-sex ratio) test employed by Altenburg and Muller (1919) for detecting newly arising X-linked lethals. Why is it superior to the first test they describe, based on measuring sex ratios? 7) What phenomena in which a new genetic type suddenly appears does Muller (1923) exclude in defining the term mutation. What, then, is his definition of a mutation? 8) In Muller's (1922) discussion of the nature of genes there are certain remarkable properties of chromosomes and genes that he believes may hold the key to the problem of how genes replicate. What are these properties? Now that we know how genes replicate, which of these properties would you say misled Muller in his consideration of the problem of gene replication? -2- Linkage and Crossing-Over: Some Background Recall that Mendel had found in the two and three factor crosses he did in Pisum that the allele pair for one factor (for example round/wrinkled) assorted independently with respect to the allele pair for any another factor (say yellow/green). Thomas Hunt Morgan, founder of the Drosophila group, had by 1911 found departures from independent assortment in crosses involving Mendelian factors for eye color, body color and wing shape, factors all known by then to be inherited as though they were on the X chromosome, being inherited by sons only from their mothers. But instead of assorting independently, factors that entered a cross together, say yellow body color and white eye color, tended to remain together in the progeny, an effect called linkage—a departure from independent assortment. Thomas Hunt Morgan 1866-1945 In the UK, William Bateson, working with the moth Abraxas, had also encountered departures from the independent assortment of Mendelian factors. Unlike Morgan, who initially did not accept the chromosome theory but did so after 1910, Bateson continued to disbelieve that Mendelian factors reside on chromosomes and instead attempted to explain the departures from independent assortment as the result of attractions and repulsions between factors supposed not to be on chromosomes. Morgan, however, saw that his own findings of departures from Mendelian assortment as well as Bateson’s could most readily be explained by assuming that the hereditary determinants are physically located on chromosomes. Morgan found that linkage between the X-linked factors he studied was only partial. For example, in a cross of yw (yellow-body, white-eye) males with yw/++ females, he found that most of the male progeny had the same combination of factors as were present in their mothers, either yw or ++, an example of linkage. But a small percentage of the male progeny was recombinant, +w or y+. Moreover, the proportion of recombinants varied, depending on what pairs of loci were crossed. When white-eye, miniature-wing males were crossed with wm/++ females, for example, the percentage of male progeny that were recombinant was much greater than for the pair white-eye and yellow-body. To explain this, Morgan assumed that the hereditary factors are linearly arranged along the length of the chromosome and invoked the hypothesis of the Belgian cytologist Alfons Janssens proposed two years earlier that in meiosis during chromosome synapsis when homologous maternal and paternal chromosomes lie side-by-side, they break and rejoin at one or a few places along their length (at what Janssens called “chiasmata”, for the cross-shape of such an exchange), thereby exchanging homologous segments. As Morgan wrote in his 1911 paper: "If the materials that represent these factors are contained in the chromosomes, and if those factors that "couple" be near together in a linear series, then when the parental pairs (in the heterozygote) conjugate like regions will stand opposed. Frans Alfons Janssens There is good evidence to support the view that during the strepsinema (i.e. 1865-1924 zygotene-pachytene) stage homologous chromosomes twist around each other, but when the chromosomes separate (split) the split is in a single plane, as maintained by Janssens… In consequence, we find coupling in certain characters, and little or no evidence at all of coupling in other characters; the difference depending on the linear distance apart of the chromosomal materials that represent the factors.” -3- Based on Morgan’s proposal that the frequency of recombination between a pair of factors reflects the distance they are apart on a chromosome, Alfred Sturtevant, then an undergraduate student working in Morgan’s laboratory at Columbia, realized that if, as suggested in Morgan's 1911 paper, the frequency of recombination between genes depends on "the linear distance apart of the chromosomal materials that represent the factors", recombination frequencies could be used to construct a map reflecting the order of genes on the chromosome. In his 1965 History of Genetics, Sturtevant described his realization as follows: "I went home and spent most of the night (to the neglect of my undergraduate homework) in producing the first chromosome map, which included the sexlinked genes y, v, m, and r, in the order and approximately the relative spacing that they still appear on the standard maps." Alfred Henry Sturtevant 1891-1970 Using recombination frequencies for various combinations of six X-linked "factors" in Drosophila melanogaster, Sturtevant constructed the first genetic map, found it to be linear, and discovered double crossing-over and interference (the tendency of cross-overs not to occur close together). Within the next ten years, more than a hundred genes had been mapped by Morgan and his students on the Xchromosome and the autosomes of Drosophila melanogaster. Mutation: Some Background A central problem in evolution theory is: What is the source of the heritable variation upon which natural selection acts? As we have seen, Charles Darwin attributed the source of such variation to the effects of "conditions of life" on the reproductive system and to the inherited effects on the soma of "use and disuse". Darwin and Alfred Wallace both thought that the variation on which natural selection acts is not the sporadic variation that occurs in a single individual, the so-called "sport of nature". Given the prevailing belief in blending, such variations would be diluted to insignificance during successive generations within any sizeable randomly-mating population. Therefore rejecting sports, it was supposed that natural selection can be effective only on variation common to a substantial proportion of individuals at the same time. (Confusingly, Darwin called such common variation "individual variation".) Because populations show more or less continuous variation for many attributes, for example height, arm strength, visual acuity, etc. This view of heritable variation did not seem unreasonable at the time. It was only after Mendel's demonstration of particulate (i.e. nonblending) inheritance came to be accepted as the general rule that the idea of blending was abandoned, and then only gradually. As we have seen, August Weismann, who at first shared Darwin’s generally-held belief that acquired characteristics could be inherited, from 1883 onwards argued powerfully to the contrary, proposing instead that all heritable variation arose in the germ line, with no input from the soma. Always a strong supporter of Darwin’s theory of natural selection but needing to look elsewhere for a source of heritable variation upon which natural selection could act, Weismann in an 1886 essay entitled "On the significance of sexual reproduction in the theory of natural selection", proposed that all variation arises from the effect of sexual reproduction in producing new combinations of already existing genetic determinants. At first proposing that all such determinants originated in the earliest life-forms and that sexual reproduction merely reassembles them in various combinations, Weismann had by 1891 correctly concluded that, while sexual reproduction does indeed produce new combinations of existing -4- determinants, new or modified determinants continuously arise in the germline. Although Weismann lived until 1914, more than a decade after the “rediscovery” of Mendel’s 1865 paper in 1900, he did not incorporate Mendelism into his model of heritable variation. Nor was Mendelian inheritance generally accepted as more than a special case until well into the 20th century. The period from 1900 until about 1930 was one of intense disagreement about the nature of heritable change and its relevance to evolution. Genetics, as William Bateson had defined the word in 1905, was the study of two central topics, heredity (inheritance) and variation. While the mechanism of inheritance including its chromosomal basis and the production by meiosis of recombinant combinations of alleles already present in a population was more or less understood by 1920, the study of mutation, i.e. allelic change, had barely progressed at all. When the American geneticist and student of Morgan, Hermann Muller, took up the problem of mutation almost nothing was known about it. In fact, the rate of appearance of new mutations was found to be so low—only about 1 in 50,000 fruit flies were found to have a new visible mutation—that it was difficult to conceive of how one might study the problem at all. Muller saw the study of mutation informative not only as regards the frequency and nature of mutations but as a way to probe the nature of the gene itself and to discover the Hermann J. Muller mechanism by which genes duplicate. This problem was a special passion of Muller, the 1890-1967 solution of which came, not from formal genetics but from the sort of approach that Muller foresees near the end of his 1922 paper. After discussing recent findings regarding bacteriophage reproduction, he wrote "Must we geneticists become bacteriologists, physiological chemists and physicists, simultaneously with being zoologists and botanists? Let us hope so." Reasoning that recessive lethal mutations would be likely to be the most frequent class of mutation (Do you see why this is a reasonable expectation?), Muller and Edgar Altenburg, conducted experiments designed to detect and measure the frequency of newly arising recessive lethal mutations in the Drosophila X-chromosome. Drosophila females have two Xchromosomes (XX) while males have only one (XY). On the average therefore, mothers heterozygous for a recessive lethal mutation will produce only half as many sons as will wild-type mothers. But the ratio of males to females among the progeny of any particular female depends on the ratio of X-bearing sperm to Y-bearing sperm with which her eggs happen to be fertilized. Sex determination is therefore a random process so that the sex ratio in the Edgar Altenburg progeny of only about 30 flies from any particular female would be expected to 1888-1967 fluctuate considerably from female to female, making it difficult to distinguish departures from a 1:1 sex ratio caused by the mother being heterozygous for an X-linked redcessive lethal from a deficiency of males caused by statistical variation. Nevertheless, Muller and Altenburg determined the frequency of males in the progeny of each of 385 females, each of which was the daughter of a female which had produced males and females in approximately equal numbers, showing her to not to be carrying an X-linked lethal. But newly arising X-linked lethals present in the eggs produced by such a female would give heterozygous daughters each of which would therefore produce, on average, only half the normal frequency of sons. Among the broods from 385 females, 13 had only about half as many males as females, corresponding to a frequency of 13/385 or 3.38 percent of the mothers appearing to be heterozygous for a recessive X-linked lethal mutation. The implication was therefore that about 3 percent of the eggs produced by the grandmothers carried a newly arising Xlinked recessive lethal mutation. -5- In order to avoid errors resulting from random variation in the sex ratio, Muller and Altenburg then devised a more sophisticated genetic screen that allowed the detection of newly arising X-linked recessive lethals without relying on sex ratios. This was accomplished by employing females heterozygous for visible X-linked markers that could easily be recognized at a glance. If an X-linked lethal was present in the egg from which such a female comes, she would produce only one kind of sons. (This ignores the minor frequency with which the lethal could be separated from the markers by cross-overs within such a female). Even with this more discriminating protocol, such sons could be missing from a particular brood simply by chance but this should not be a major effect. Nevertheless, for reasons regarding which we can only speculate, perhaps infection with an active transposable genetic element, the frequencies obtained by Altenburg and Muller using this method were too high by a factor of about 10 when compared with the value subsequently obtained using a new and particularly reliable approach, known as the ClB-method. Using that method, Muller found a mutation rate of about 0.001 recessive X-linked lethal mutations per generation, a rate not greatly different from modern measurements, corresponding to an average mutation rate for recessive lethals among the ~3,000 genes on the Drosophila X-chromosome of about 3X10-7 per generation. The measured rates of lethal mutation, although small, were nevertheless much greater than the rate of appearance of visible mutations, supporting Muller’s expectation that most mutations are deleterious. [Much later was it discovered that some mutations, detectable as nucleotide sequence changes, are neutral or nearly so, having little if any effect on phenotype. Even most of these, however, may be very slightly deleterious, a matter of ongoing investigation today.] As Drosophila work continued, it was found that certain visible mutations reverted to wild phenotype, making it clear that mutations are not exclusively losses, although losses (deletions) clearly do occur. Furthermore, a gene can mutate to more than one mutant form. The eye color mutant eosin, for example, is a spontaneously-occurring partial reversion of the white mutant, as were the eye-color mutations buff, coral, blood, and ivory. Still, it was thought that the different alleles of a given gene could not be separated by crossing-over and therefore that a chromosome is like a string of beads, with the genes as beads and crossing-over possible only in the strings between beads. That recombination does, in fact, take place within as well as between genes, and that the map linearity first discovered by Sturtevant extends down to the smallest dimensions of the genetic material was not known until much later, as we shall see in Week 6. -6-