Download MCB 142 Week 5: October 6 and 8

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.
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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?
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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.”
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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
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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.
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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.
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