Heredity and Development: Second Edition (1972)

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HEREDITY AND DEVELOPMENT: SECOND EDITION 87 5 Morgan and Drosophila During the first ten years following the rediscovery of Mendel’s experiments, the progress of genetics was slow though steady. It was found that Mendel’s scheme worked for many organisms and not for peas alone. To be sure some crosses gave ratios that were different from those expected in the Mendelian scheme. These proved difficult to analyze. During this same decade Sutton had suggested that the chromosomes might provide the physical basis for inheritance but those biologists concerned with breeding experiments were unable to appreciate the force of his arguments and data. In 1910 the American geneticist, Thomas Hunt Morgan (1866–1945), together with his associates Alfred H.Sturtevant (1891–1970), Calvin B. Bridges (1889–1938), and Herman J.Muller (1890–1967), began a remark- able series of experiments. In one decade their efforts changed genetics into the most highly conceptual branch of biology. It sometimes appears that much of the progress in science is due to fortu- nate accidents. One of these accidents was the choice by Morgan of the small fly, Drosophila melanogaster (Fig. 5–1), for genetic work. If Drosophila had never been used, the progress of genetics would have been very much slower. This species is common in nature and it frequents orchards, grocery stores, and other places where there is ripe fruit. It can easily be bred in the labora- tory in simple containers such as vials or half-pint milk bottles. A layer of ‘fly food,’ consisting of cream of wheat, molasses, and yeast, is placed on the bottom of the container. The yeast, which grows on the other substances, is the main food of the Drosophila. If a pair of flies is placed in such a bottle, 

HEREDITY AND DEVELOPMENT: SECOND EDITION 88 5–1 Drosophila melanogaster. Male left and female right. (From T.H.Morgan. 1919. The Physi- cal Basis of Inheritance. Lippincott.) several hundred young will be produced in about two weeks. Though small, these flies are large enough so that many of the external characteristics can be seen with a hand lens and much of Morgan’s early work was done with a no more elaborate magnifying aid. Later it was customary to use low power stereoscopic microscopes to study the flies. So Drosophila melanogaster was easy to collect, simple to maintain in the laboratory, and experimentation was most economical—an important consid- eration at a period when very little money was available to support scientific work. In fact, Morgan used Drosophila because he was unable to obtain the funds to experiment with rabbits, which are far more expensive to maintain. Whoever refused his request for the funds to work with rabbits, must go down in history as one of the truly great benefactors of modern genetics. Had Morgan studied rabbits rather than Drosophila, great progress in genetics would have probably been delayed by at least a generation. The Origin of Hereditary Variation. Morgan began his work with Drosophila to answer a question about the origin of hereditary variation in organisms. Up to this point we have discussed alleles without reference to their possible origin. One of the pairs of alleles that Mendel used was R and r, which determined whether the pea seeds would be round or wrinkled. Mendel obtained the seeds that he used from seed dealers, who in turn proba- bly obtained them originally from farmers. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 89 What was the origin of the round and wrinkled varieties? Were all peas origi- nally round and then did one suddenly become wrinkled? Or perhaps it was the reverse. The Mutation Theory of de Vries. In 1901–3 the Dutch botanist, Hugo de Vries, from his studies of the evening primrose (Oenothera) advanced the hypothesis that abrupt changes can occur in the hereditary material of an organism. He believed that these changes were frequent and tended to be inherited. De Vries would have maintained that the gene causing round seeds in peas could change to an allele that caused wrinkled seeds. He spoke of this process of change as mutation and the new variety as a mutant. The White-eyed Mutant. Morgan began raising Drosophila in the hope of observing the origin of mutants. The first mutant that he found was a male with white eyes. It suddenly appeared in a culture bottle of red-eyed flies. Red is the ‘normal’ or ‘wild type’ color of the eyes in Drosophila melanogaster. He began experiments to determine the mode of inheritance of the white-eyed condition. Morgan mated the white-eyed mutant male with a red-eyed female (the symbols ♂ and ♀ are used for male and female respectively). These were the results, including the actual numbers of individuals obtained in the F2. In addition, the original white male was crossed to one of his F1 daughters. Explaining the Cross: First Hypothesis. These results could not be explained by the usual Mendelian scheme. The peculiar relation of eye color to sex, with the absence of white females in the F2 of the first cross, suggested that sex chromosomes might be involved. Morgan proposed the following hypothesis to explain the data: Let us call the gene that results in white eyes, w, and the gene that results in red eyes, W. The white-eyed male will produce sperm, all of 

HEREDITY AND DEVELOPMENT: SECOND EDITION 90 which will carry w. Half of these sperm will have, in addition, an X chromo- some; the other half will not. The genotype of the original white male could be written wwX. Two types of sperm will be produced, namely, wX and w. The red-eyed female would be WWXX. These symbols represent the two red genes and the two X chromosomes. All of the eggs would be WX. Morgan’s first cross could be represented by the following scheme. This scheme fits the experimental results, but Morgan pointed out that it is necessary to make one assumption about gamete formation in the F1 red males. In these males, which are heterozygous for white eyes, it is necessary to assume that the W gene always goes to the same pole of the spindle with the X and that the w gene never does during the chromosome movements of meiosis. Consequently, there are no wX gametes formed by WwX males. He adds, ‘This all-important point can not be fully discussed in this communica- tion’ (Morgan, 1910). If Morgan’s theory was correct, it should have been possible to make deductions about the behavior of the various genotypes and to test these deductions experimentally. Morgan made four such deductions and tested them by making the appropriate crosses. 1. If the genotype of the white male is wwX and of the white female wwXX, the following would be expected in a cross of these two types: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 91 This cross was made and the results were entirely according to expec- tation, that is, only white-eyed flies were obtained. 2. The hypothesis requires that two genotypes be present in the F2 females, namely, WWXX and WwXX. The two types could be differentiated by crossing to white males. These results would be expected: Tests of the F2 red females showed that these two classes exist. 3. The genotype of the F1 female in the original cross was thought to be WwXX. If this was correct, a cross of the F1 female and a white male would give the same results as cross 2b (above). This cross was made and the prediction verified. 4. The hypothesis requires the F1 male in the original cross to be WwX. If such a male is crossed to a white female, the following results would be expected: Once again, the actual experiment yielded the expected results. Note, how- ever, the assumption that W and X were always together in the same sperm and that no wX sperm could be formed by WwX males. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 92 Nearly all of Morgan’s hypothesis was based on facts or on ideas that seemed quite probable. The role of chromosomes in sex determination, the concept of alleles showing dominance and recessiveness, and the concept of segregation had all been part of biological knowledge for years. He made four deductions from his hypothesis and found that every one could be veri- fied experimentally. He predicted the results expected from crosses before they were made and later found his predictions confirmed. Do you consider that his hypothesis was ‘established beyond a reasonable doubt’? Still another cross was made, but the results were most surprising. A white- eyed female was crossed with a red-eyed male. (The male was from a wild stock that had never been bred with the stock that produced the white-eyed male.) All of the females derived from this cross had red eyes and all of the males had white eyes. One might have expected that all of the F1 would have been red-eyed, since the male should have been of the genotype WWX. This was not the case, so Morgan assumed that all males he used were heterozy- gous for the red eye gene and had the genotype WwX: At this point we should pause and consider these questions: a. Why are there only two classes of sperm, namely WX and w, formed by the heterozygous red males of the genotype WwX? Why is no wX class produced? b. Why are all the wild red-eyed males heterozygous? Why does the WWX type not occur? Morgan’s hypothesis demands that red-eyed males are never homozygous and that they show unusual phenomena in sperm formation. If these basic conditions do not hold, then the hypothesis is either wrong or incomplete. Can you devise other hypotheses for explaining the data? Explaining the Results: Second Hypothesis. It was not long—in fact, only one year—until Morgan devised a simpler hypothesis to account for the white eye case. If one assumes that the gene for white eyes is part of the X chromosome, then the results of all the crosses correspond to what would be expected from the behavior of the X chromosome. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 93 There would then be no need for invoking subsidiary assumptions, such as unusual types of meiosis in some males, or requiring all wild males to be het- erozygous for eye color. Morgan’s second hypothesis has withstood every conceivable test, and there seems to be no reasonable doubt of its correctness. The symbolic representation for the new scheme will be different from that given before. W will continue to mean red and w white, but there will be no need to use X. If we assume that W and w are located on the X chromosomes, ‘W’ should be interpreted as an X chromosome with the W allele. In the same manner, w will indicate an X chromosome with the w allele. The Y chromosome will be indicated by a Y, since by this time it was realized that Drosophila is of the XX ♀—XY ♂ sex chromosome type. The crosses seemed to indicate that the Y chromosomes contain no W or w alleles. (As later work was to show, the Y of Drosophila melanogaster is almost entirely without genes.) The following, then, are the correct diagrammatic representa- tions of the crosses through the F2 generation: The reciprocal P generation cross would be as follows: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 94 This new hypothesis explains the observed results of the genetic experi- ments, and it does not invoke any unknown phenomena. Morgan soon discovered other genes which, from their mode of inheri- tance, he concluded were carried on the X chromosome. All genes that are on the X are said to be sex linked and they always show the type of inheritance just outlined for the white eye crosses. These experiments with white-eyed flies provided additional evidence supporting the hypothesis that chromosomes are the physical basis of inheri- tance. If some genes are parts of the X chromosome, their inheritance must reflect the behavior of the X during meiosis and fertilization. The white eye genes behaves as though it were part of the X. This can mean either that it is part of the X or that it is part of some unknown cell structure that behaves exactly like the X during meiosis, fertilization, and mitosis. Crosses with Sex-linked and Autosomal Genes. Morgan and his asso- ciates discovered mutant genes by the dozens. Some were sex linked. Those genes carried on any chromosome except the sex chromosomes are said to be autosomal genes. The inheritance of autosomal genes followed the usual Mendelian scheme. Sex-linked inheritance follows the scheme that has just been described. The following example of a cross involving sex-linked genes and autoso- mal genes will bring out the difference between the two types. We already know that white eye is a sex-linked gene recessive to red. Our other character- istic in this cross is vestigial wing, which is an autosomal gene recessive to long wing. The F2 which is shown in the checkerboard on the next page consists of the following: ♀ 3/8 red-long; 3/8 white-long; 1/8 red-vestigial; 1/8 white-vestigial. ♂ 3/8 red-long; 3/8 white-long; 1/8 red-vestigial; 1/8 white-vestigial. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 95 What is the ratio of long to vestigial, neglecting the eye-color genes? Is there any difference in the ratios of the autosomal genes between the F2 males and females? The Importance of Morgan’s Work. These first experiments of Morgan are important in several ways. A new experimental animal was introduced to geneticists that was easy to raise in the laboratory and was a producer of large numbers of offspring. In addition, the crosses themselves added considerably to genetic theory in that they were the first well-analyzed cases of sex-linked inheritance. The fact that the genetic results exactly paralleled the behavior of the X chromosome was strong evidence that the gene responsible for white eyes is part of the X chromosome. At least many biologists believed the data to be highly suggestive. Now if it is established that one gene is part of a chromosome, it is a good working hypothesis that other genes are parts of chromosomes. One could even hold to the hypothesis that most or all genes are parts of chromosomes. Scientific Methods. This early genetic work of Morgan is valuable in still another way. The experiments and the way in which they were reported are excellent examples of one of the most important procedures in experimental science, namely, the manner in which ‘cause-effect’ relations are discovered. A scientist is interested in the reason why things behave as they do. In this case, Morgan wondered what was the cause of the peculiar genetics of the white eye gene. He observed the effects and attempted to reconstruct the cause. This reconstruction, according to the philosophers who study scien- tific methods, takes place in well defined if not always explicitly stated steps, which are: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 96 1. Recognition of the problem. In this instance the problem was to interpret the white eye case in genetic and cytological terms. 2. Collection of facts pertaining to the problem. The facts consisted of the data of the first cross together with all that Morgan knew of cytology and genetics. 3. Formulation of a hypothesis. From a consideration of all the particular facts, Morgan formulated a general statement, or hypothesis, that would explain the facts. This logical step from the particular to the general is known as induction. The hypothesis in this case was the symbolic scheme that explained the results of the cross in terms of chromosome behavior. 4. Testing the hypothesis. The correctness of a hypothesis is tested in this manner: First, we assume that the hypothesis is correct and then make certain deductions. These deductions can be tested to see if they are true or false. Morgan made four such deductions and found that the pre- dicted results were always obtained. The more deductions that are veri- fied, the more likely it is that the hypothesis is true. The fate of Morgan’s first hypothesis, which symbolized the white female as wwXX and the white male as wwX, should be a sobering example. It was tested by four deductions and found to be ‘true.’ For most scientists, this might be convincing. It did not, however, offer a convincing explanation of all the data. One had to assume that meiosis in the WwX males was unusual and that all red-eyed males are heterozygous. Morgan found, however, that a second hypothesis would explain the same data and in this case it was not necessary to introduce any qualifications, such as a special type of meiosis in males heterozygous for the eye-color gene or that all red-eyed males are het- erozygous (WwX). The second hypothesis, which symbolized the white female as ww and the white male as wY, was simpler. When one has the choice of two hypotheses, one simple and one complex, one generally selects the first. This is the famous Occam’s razor, which admonishes the scientist to explain his results in the simplest manner possible, and to introduce no unnecessary complexity. It must be realized that both of Morgan’s hypothe- ses explain the data. Subsequent events have shown the first one to be false and the second, and simpler one, to be true. This episode is an example of the self-correcting nature of scientific proce- dures. If deductions are made and tested, the truth or falsity of the hypothesis can be established. If the hypothesis fails to account for all the data, then it must be modified or abandoned. Morgan’s first hypothesis accounted for most but not all of the experimental results. It was not necessary for him to abandon the hypothesis entirely, merely to modify it. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 97 New mutants were discovered rapidly in Morgan’s laboratory and very soon there were more of them than there were pairs of homologous chromo- somes. This was the difficult moment for genetics and cytology that Sutton had predicted (page 78). The Prediction of Linkage. At the time Sutton proposed his hypothesis, he pointed out one situation in which the Mendelian laws could not apply, namely, those cases where two genes are carried on the same chromosome. Clearly, they would not obey Mendel’s law of independent assortment. He foresaw that this problem would arise when more pairs of alleles had been discovered than there are pairs of chromosomes in the species being studied. Let us consider the problem as it applies to Drosophila melanogaster. The diploid cells of this species have four pairs of chromosomes. Let us assume that each of the first four mutant genes discovered are located on a different chromosome pair. If this is the case, each of these four genes will show inde- pendent assortment. What will happen when we discover the fifth pair of genes? Since there is no fifth pair of chromosomes, the fifth pair of alleles must be located on a chromosome that already has one of the first four pairs of alleles. When this situation arises, obviously the two pairs of alleles cannot act in an independent way during meiosis. They would be linked in inheri- tance. This deduction is inevitable, if genes are parts of chromosomes. The Discovery of Linked Genes. In 1906 Bateson and R.C.Punnett (1875–1967) reported a cross involving two pairs of genes that did not show independent assortment. Their cross was with sweet peas, where blue flower color (B) is dominant over red (b) and long pollen grain (L) is dominant over round pollen (l). The scheme of the cross was this: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 98 If there is independent assortment, we would expect the four classes of F1 gametes shown in the diagram to be produced in equal numbers. Each type would account for 25 per cent of the total. The standard genetic way for find- ing gamete percentages is to cross the organism being tested with the pure recessive. This is called the test cross. For the F1 heterozygous plant it would be this: In this test cross, the phenotype of the offspring would be a measure of the gamete frequency of the plant being tested. Thus, 25 per cent of the gametes would be BL and 25 per cent of the offspring would be blue-long. This would be true for all classes of gametes, since they would be combining with a gamete having both recessive genes. (These bl gametes, having only reces- sive genes, cannot alter the expression of genes in the gametes with which they combine.) When Bateson and Punnett made the cross, these were the results: EXPECTED ACTUAL blue-long 25% 43.7% blue-round 25% 6.3% red-long 25% 6.3% red-round 25% 43.7% Clearly these results do not conform to those expected from the Mendelian theory. Two points should be noticed. 1. The two most frequent phenotypes are those of the original parents (blue-long and red-round). 2. The percentages of the original parental types, blue-long and red-round, are the same. The percentages of the two recombination classes, blue- round and red-long, are also the same. An even more disturbing finding was that the F2 ratios depended largely on the genotype of the P generation. On strict Mendelian principles a cross of blue-long×red-round should give the same F2 as blue-round×red-long. This was not so with Bateson’s sweet peas. The blue-round×red-long cross gave the following results (once again the results are compared with what would have been expected if Mendel’s rules applied): 

HEREDITY AND DEVELOPMENT: SECOND EDITION 99 When these results are compared with those of the first cross, the percent- ages for each phenotype are found to be different, but again we notice a pre- ponderance of the parental types. Both crosses suggest an orderly, though non-Mendelian, mechanism of inheritance. Some new principles must be involved. Let us try to explain the results on the basis of Sutton’s hypothesis. If we assume that the two genes, B and L, are parts of the same chromosome, this will be the schematic representation of the cross: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 100 The percentages actually obtained can be compared with the ratios expected on the basis of Mendel’s hypothesis and Sutton’s hypothesis: blue-long blue-round red-long red-round OBSERVED 43.7 6.3 6.3 43.7 MENDEL’S 25 25 25 25 SUTTON’S 50 0 0 50 At first sight it may seem as though Sutton’s explanation is the better since the blue-long and red-round classes are nearer the observed percentages. One difficulty, however, is fatal to Sutton’s hypothesis. Both blue-round and red- long plants were obtained in the cross. Neither of these classes would be pos- sible if both the B and L genes were part of the same chromosome and were inherited in the manner outlined. We may conclude that the results obtained in the cross cannot be understood on the basis of either Mendel’s or Sutton’s hypotheses, as originally stated. Linkage and Crossing Over. Morgan and his associates discovered many new mutants and used them in crosses. Some of the crosses involving two pairs of genes gave the independent assortment expected in the Mendelian scheme. In other crosses, deviations of the sort found by Bateson and Punnett were encountered. Morgan was convinced of the correctness of Sutton’s hypothesis that genes are parts of the chromosomes and assumed that the exceptions to independent assortment were due to the two different genes being on the same chromosome. But that simple assumption could not explain the results satisfactorily; it was necessary to assume that under some circumstances genes could be transferred from one chromosome to another. Was there any cytological evidence for this? A possible cytological mechanism for an exchange of genes had been sug- gested in 1909 by F.A.Janssens (1863–1924). He described a type of behav- ior of chromosomes in meiosis that is now known to be nearly universal in both animals and plants. It is called crossing over and it occurs during the tetrad stage (Fig. 5–2). During synapsis the homologous chromosomes come close together with their long axes parallel. Both chromosomes duplicate and a tetrad of four chromatids is formed. According to Janssens, there is consid- erable coiling of chromatids around one another at this time and in some cases two of the chromatids break at the corresponding place on each. The broken chomatids rejoin in such a way that a section of one chromatid is now joined with a section of the other. As a result, ‘new’ chromatids are produced that are mosaics of segments of the original ones. Janssens’ hypothesis of crossing over could provide the basis of gene 

HEREDITY AND DEVELOPMENT: SECOND EDITION 101 5–2 Janssens’ hypothesis of crossing over. transfer from one chromosome to another. Morgan suspected that it did and wrote in 1911: In consequence, the original materials will, for short distances, be more likely to fall on the same side of the split, while remoter regions will be as likely to fall on the same side as the last, as on the opposite side. In consequence, we find [linkage] in certain characters, and little or no evidence at all of [linkage] in other characters; the difference depending on the linear distance apart of the chromosomal materials that represent the factors. Such an explanation will account for all of the many phenomena that I have observed and will explain equally, I think, the other cases so far described. The results are a simple mechanical result of the location of the materials in the chromosomes, and of the method of union of homologous chromosomes, and the proportions that result are not so much the expression of a numerical system as of the relative location of the factors in the chromosomes. Instead of random segregation in Mendel’s sense we find ‘associations of factors’ that are located near together in the chromosomes. Cytology furnishes the mechanism that the experimental evidence demands. The term linkage was introduced to refer to cases where different genes are located on the same chromosome. Crossing over was the term applying to the coiling, breaking, and rejoining of homologous chromosomes during meiosis. The following is an example of inheritance of two linked genes: In Drosophila gray body color (B) is dominant to black body color (b). Long wing (V) is dominant to vestigial wing (v). The two pairs of genes are located on the same pair of autosomes. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 102 This F1 individual will produce four types of gametes, two the result of crossing over and two non-crossovers. Since crossing over occurs in the tetrad stage we could diagram gamete formation as follows: The middle pair of chromatids will break and recombine. The two meiotic divisions then occur and each resulting gamete will receive one chromosome. The four types of gametes will be as follows: These four gametes are not produced in equal frequency. Morgan sug- gested that the chance of a crossover occurring between two genes is a junc- tion of the distance between them. This suggestion was based on a simple argument. Let us assume that crossing over can occur at any point along a chromosome. Let us assume further that there are three genes, A, B, and C, on the chromosome. A and B are close to one another; B and C are far apart. If this is so, crossing over is more likely to occur between B and C than A and B merely because there is a longer stretch of chromosome where it might occur. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 103 Possibly this analogy will help: We will imagine that we have a string three feet in length and of uniform strength. We will stretch this string until it breaks. The site of breakage is more likely to be at some point in the first 30 inches of the string than in the last six inches. Continuing the experiment, we mate an F1 female of the above cross with a black-vestigial male, the results are as follows: Among the offspring, 17 per cent are derived from gametes that carry chromosomes that had a crossover between the two genes being studied. The remainder, 83 per cent, are from non-crossover gametes. The frequency of crossing over between any two genes is nearly constant. If we cross a black-long×gray-vestigial fly the F1 would be gray-long and heterozygous for both genes. In these two respects the F1 of this cross will be identical with that in the one previously described. In this second cross, how- ever, one of the P generation chromosomes will be bV and the other Bv. If a female of this constitution is crossed with a black-vestigial male the offspring will be as follows: 41.5% black-long 41.5% gray-vestigial 8.5% gray-long 8.5% black-vestigial Compare these percentages with the previous cross and be sure you under- stand the reason for the difference. Subsequent events have shown that Morgan’s explanation satisfactorily accounts for the inheritance of genes located on the same chromosome. Many details were added, such as the occurrence of double or triple crossovers, and, for reasons still not understood, the absence of crossing over in Drosophila males. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 104 

HEREDITY AND DEVELOPMENT: SECOND EDITION 105 These experiments are an example of the mutual checking that the com- bined genetic and cytological approach permits. As we have mentioned before, inheritance must be explained in both fields. If two genes are in the same chromosome they will be linked in inheritance. If they are not com- pletely linked there must be a chromosomal basis for the recombination. A chromosomal basis is to be found in the phenomenon of crossing over. Linkage Groups in Drosophila. Another interesting parallel between genet- ics and cytology was soon apparent. One deduction we could make from Sut- ton’s hypothesis is this: If genes are on chromosomes and all chromosomes have genes, then the number of groups of linked genes would correspond to the number of pairs of homologous chromosomes. This deduction was veri- fied. By 1915 Morgan and his associates had studied more than 100 mutant genes. When these were tested, they were found to comprise four linkage groups. The number of chromosome pairs in Drosophila is also four. The partial list that Morgan published at that time is given in Fig. 5–3. The evidence was becoming almost overwhelming that Sutton’s hypothe- sis was correct, though it was necessary to modify it to take crossing over into account. In Figure 5–3, two sets of data are given: the genetic list of linked genes and a drawing of the chromosomes. As we have seen, there is good reason to believe that a relation exists between the two sets of data. If we accept the hypothesis that the linkage groups correspond to the pairs of homologous chromosomes, how could we determine which linkage group corresponds to each of the four pairs of chromosomes? It will be worthwhile for you to consider this problem. Another matter should be mentioned. Mendel believed that in crosses involving two pairs of alleles there is always independent assortment. This is to be expected if each pair of alleles is situated on a different pair of homolo- gous chromosomes. Mendel worked with seven pairs of alleles and there are seven pairs of homologous chromosomes in the garden pea. Each pair of alle- les could have been on different homologues but later work has shown this not so. In some cases two pairs of alleles are on the same chromosome and might be expected to show linkage, 5–3 A total of 85 genes of Drosophila melanogaster were reported in 1915. These fell into 4 link- age groups. Cytological investigations showed that this species has 4 pairs of chromosomes. This parallelism between the number of chromosomes and the number of linkage groups suggested that the genes were situated on the chromosomes (T.H.Morgan, ‘The Constitution of the Heredi- tary Material,’ Proc. Amer. Phil. Soc. 54:143–53. 1915). 

HEREDITY AND DEVELOPMENT: SECOND EDITION 106 yet Mendel reports only data that suggest independent assortment. The answer to this paradox lies in the relative positions of the alleles on the chro- mosomes. If they are far apart, the amount of crossing over may be so great that the different alleles appear to be inherited independently. Relation of Genes to Characteristics. The data in Figure 5–3 showing the linkage groups of Drosophila are instructive in another connection. Notice that many different genes affect the same character: 13 influence eye color and 33 modify the wings in some manner. The question arises, what determines the normal red eye color? The answer is that the wild type alleles of all of these 13 eye color genes, together with many undiscovered in 1915 when Morgan published his list, act together to produce the wild type red eye color. If an individual is homozygous for the mutant allele of any one of these genes, then the eye is not red but some other color such as white, sepia, or peach. We should think of the normal red eye color as the end product of a series of gene actions. If any of these actions is altered, the eye color will be different. Figure 5–3 is misleading in one respect. Each mutant gene appears to have a single effect. It usually does have a single main effect, but most of the genes that have been studied intensively are found to have many different effects. Thus the white eye color gene in Drosophila is responsible not only for the absence of color in the compound eyes but also for the absence of color in the simple eyes and in some of the internal organs as well. It is called an eye color gene simply because the most obvious effect of the gene is on the color of the compound eyes. Genes that affect more than one structure are said to be pleiotropic. THE CYTOLOGICAL PROOF OF CROSSING OVER With the data so far given, the concept of crossing over as a mechanism for genetic recombination might be regarded as a good working hypothesis and nothing more. The hypothesis was invented to account for the results of genetic crosses. Thus, the absence of recombination of genes in some crosses suggested that these genes were parts of the same chromosome. This explana- tion did not account for all the data, however, since in a definite percentage of the individuals recombination did occur. Now if the genes in question are parts of chromosomes, these recombinations among genes of the same link- age group could only be explained on the basis of some exchange of genes between homologous chromosomes. After the Morgan group had analyzed the situation to this extent they sought some possible cytological basis for the postulated interchange of genes between chromosomes. It was then that they came across the work 

HEREDITY AND DEVELOPMENT: SECOND EDITION 107 of Janssens, who had described a phenomenon that might be interpreted in terms of the breaking and rejoining of chromatids during the tetrad stage of meiosis. It should be emphasized that Janssens did not actually observe the breaking and rejoining of chromosomes and no one has to this day. The diffi- culty is this. Crossing over is assumed to occur between homologous chro- matids. Since they are homologous, they are of identical appearance when viewed under the microscope. Furthermore, the act of crossing over is assumed to occur when the four chromatids of the tetrad are tightly coiled around one another. Crossing over cannot be seen in living cells, and in fixed and stained cells there is no direct way of telling whether a chromatid has exchanged portions with another chromatid or not. Figure 5–2 is a diagram of crossing over. The two homologous chromo- somes undergoing synapsis are drawn differently, but it must be remembered that in living or in fixed and stained material they would be of identical appearance. In the four chromatids shown after crossing over there is no diffi- culty in distinguishing the chromatids that have crossed over and those which have not, since the strands have been shaded differently by the artist. Once again this is impossible to observe in the actual material. One could obtain critical cytological evidence for crossing over if there was some visible or detectable difference between the members of a homolo- gous pair of chromosomes. Such evidence was not available for animals until the work of Curt Stern (born 1902) in 1931. This was nearly 20 years later than the time Morgan’s group postulated the existence of crossing over. We shall consider Stern’s work out of turn, so to speak, but by so doing we can complete the analysis. By the time Stern began his work, Drosophila geneticists had a large vari- ety of strains with different types of chromosome abnormalities. He was able to use a female that had the necessary chromosomal and genetic characteris- tics to demonstrate convincingly whether or not crossing over involves a transfer of material from one chromosome to another. The female used had two structurally and genetically different X chromo- somes (Fig. 5–4). One of the X chromosomes was in two portions: one por- tion behaved as an independent chromosome and the other was attached to one of the tiny fourth chromosomes (Fig. 5–3 shows the chromosomes of a normal ♀; the fourth chromosomes are the pair of dot-shaped structures). The other X of this female was unusual in that a piece of a Y chromosome was attached to it. These structural differences were so great that they could be seen easily in fixed and stained nuclei. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 108 5–4 Stern’s Experiment The two X chromosomes, in addition to being structurally different, were also genetically different. The divided X had in one portion of it the recessive gene carnation (c), which when homozygous produces a dark ruby eye color, and the dominant gene bar (B), which reduces the eye to a narrow band. The other X, which had the piece of the Y attached to it, contained the wild type alleles, C and b, which when homozygous result in red eyes of normal shape. The essential point about this female is that she had two X chromosomes that could be distinguished from one another on both cytological and genetic grounds. She was crossed to a male fly carrying the genes for carnation color (c) and normal eye shape (b). The ova of the female would be of two types if no crossing over occurred: one type of ova would contain the short X chromo- some with the genes c and B; the other type would have the C and b genes on the X that had the piece of Y chromosome attached to it. If crossing over occurred between the two marker genes, two other types of gametes would be produced. One of these crossover 

HEREDITY AND DEVELOPMENT: SECOND EDITION 109 types would have the c and b genes on an X chromosome of normal size; the other crossover type would have the C and B genes on a short X chromosome to which the piece of Y chromosome was joined. Stern studied only the female offspring of the cross. One can determine from Fig. 5–4 that four types of daughters are to be expected, showing all combinations of the phenotypic characters. These flies should also have four different chromosome configurations, and if the theory is correct it should be possible to predict the chromosome configuration for each phenotypic class. Thus, the flies that give genetic evidence of coming from crossover gametes will be either carnation-normal or red-bar. The carnation-normal flies, alone among the offspring, should have two normal-shaped X chromosomes. The red-bar flies, again alone among the offspring, should have one short X with a piece of Y chromosome attached and an X of normal proportions. Stern studied the cytology of his flies and saw that the phenotype corre- sponded to the expected chromosomal configuration. This was a brilliant demonstration of the hypothesis that chromosomal material can be inter- changed between homologous chromosomes. MAPPING THE GENES Morgan and his fellow workers made numerous crosses involving linked genes. It was found that, in successive experiments, the amount of crossing over between two particular genes was always the same. Depending on the genes used, this might be less than 1 per cent or nearly 50 per cent. Morgan suggested that the different values were the result of the relative positions of the genes on the chromosome: If the amount of crossing over between hypo- thetical genes A and B was small and between genes C and D large, one would predict that A would be closer to B than C would be to D. The development of this concept, that linkage data could be used to map the relative positions of the genes on the chromosomes, was attempted in 1913 by Alfred H.Sturtevant when he was a graduate student of Morgan. He made crosses involving five genes carried on the X chromosome: yellow body (y), white eyes (w), vermilion eyes (v), miniature wings (m), and rudi- mentary wings (r). From the data obtained, he constructed a genetic map showing the ‘positions’ of these genes on the X chromosome. This was his basic assumption: ‘It would seem…that the proportion of “crossovers” could be used as an index of the distance between any two factors. Then by deter- mining the distances (in the above sense) between A and B and between B and C, one should be able to predict [the amount of crossing over in the inter- val] AC. For, if proportion of crossovers really represents distance, 

HEREDITY AND DEVELOPMENT: SECOND EDITION 110 AC must be approximately, either AB plus BC, or AB minus BC, and not any intermediate value.’ The percentage of crossovers between y and v was found to be 32.2 and between y and m 35.5 On the basis of the hypothesis we would expect v to be closer to y than in to y. What can we conclude about the relative positions of m and v? According to Sturtevant this should be 67.7 (35.5+32.2) or 3.2 (35.5 −32.2). The reason for this is as follows: The chromosome is a long and very thin structure so we can represent it as a line. On this line we shall put y and v, as follows: Now m can be either to the right or to the left of y, as shown here: If v and m are on the same side of y, we would expect the amount of cross- ing over between v and m to be 3.2 per cent. If they are on opposite sides of y the value should be 67.7 per cent. When Sturtevant measured the amount it was found to be 3 per cent, which indicated that v and m were on the same side of y. This close correspondence between the actual and expected result was strong support for his hypothesis. In this manner, the relative positions of the five genes were determined and a genetic map constructed. The y gene was taken as the reference point and the distances measured from it. This was the result. It was found that the most reliable values were obtained when crossover values for adjacent genes were used. This was due to the occurrence of dou- ble crossover, which introduced an error into the results. Let us consider the three genes y, w, and m (Fig. 5–5). Sturtevant raised 10,495 flies to test the linkage relations. He found that in 6,972 flies there was no crossing over between the three genes. In 3,454, crossovers occurred between w and m; in 60, crossovers between y and w were detected; and in nine a double crossover occurred. That is, there was one crossover between y and w and another between w and m. This would result in y and m being on the same chromosome as they were before the two crossover occurred. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 111 5–5 Sturtevant’s Experiment In the case of the double crossovers it should be noted that three genes are always necessary to detect the event. Thus, if only the genes y and m were used, any double crossover between them would be undetected since y and m would still be together after the chromatids had broken and rejoined. When genes are far apart, double crossovers are likely. If they are not detected they will introduce an error in the positions assigned to the genes, for the data would suggest that the genes are closer to one another than they really are. For this reason Sturtevant suggested that chromosome maps be based on crossover values of genes close to one another and not those far apart. Now the question arises, What is the relation of the chromosome map to the position of these genes on the chromosome? Sturtevant has this to say: Of course, there is no knowing whether or not these distances as drawn repre- sent the actual relative spatial distances apart of the factors. Thus, the distance wv may in reality be shorter than the distance yw, but what we do know is that a break is far more likely to come between w and v than between y and w. Hence, either wv is a long space, or else it is for some reason a weak one. The point I wish to make here is that we have no means of knowing that the chromosomes are of uniform strength, and if there are strong or weak places, then that will prevent our diagram from representing actual relative distances—but, I think, will not detract from its value as a diagram. Sturtevant’s chromosome map was a graphic way of expressing linkage data. Once constructed, these maps proved useful in predicting the results of untried crosses. The most important induction from the data 

HEREDITY AND DEVELOPMENT: SECOND EDITION 112 is that the genes are arranged in a linear order on the chromosomes, analo- gous to the sequence of beads on a string. The position occupied by a gene is its locus. THE ‘FINAL PROOF’ THAT GENES ARE PARTS OF CHROMOSOMES Beginning in 1884 with Hertwig and others, we have seen that some biolo- gists thought the evidence indicated that the hereditary factors were parts of the chromosomes. It is probable that a minority held this view prior to 1910. After 1910 the Drosophila data collected by Morgan and his associates made it increasingly probable that genes are parts of chromosomes, and more and more biologists came to accept this view. Geneticists generally credit the work of Calvin Bridges, another of Morgan’s students, published in 1914 and 1916 as being the ‘final proof’ that the genes are parts of chromosomes. The material that will now be covered should be studied more to learn the type of evidence constituting a ‘final proof,’ and less for the genetic details. Bridges’ experiments dealt with the inheritance of sex-linked genes in Drosophila. The hypothesis that he sought to prove was that ‘sex-linked genes are located on the sex chromosomes.’ Normal Inheritance of Sex Chromosomes. In order to understand Bridges’ experiments, it is necessary to have clearly in mind the normal inher- itance of sex chromosomes in Drosophila. The X chromosome of a male is transmitted only to his daughters and his Y only to his sons. The X chromo- somes of a female are transmitted to both sons and daughters. Looked at from the point of view of the offspring, a daughter receives one X from her father and one from her mother. The sons receive an X from the mother and a Y from the father. The following diagram depicts this. In it, the sex chromo- somes of the female are indicated in large letters and those of the male in small letters. Inheritance in Non-disjunction Females. Bridges noticed that in some strains of Drosophila the inheritance of sex-linked genes was most unusual. Thus, in a cross between a white-eyed ♀ and a red-eyed ♂, some white-eyed daughters and red-eyed sons were obtained. These daughters 

HEREDITY AND DEVELOPMENT: SECOND EDITION 113 had inherited their sex-linked genes solely from the mother, and the sons had inherited their sex-linked genes solely from the father. This would be impos- sible if (a) the sex-linked genes were located on the X chromosome, and (b) the sex chromosomes were inherited as shown in the diagram. Bridges realized that the unexpected breeding results could be explained on the assumption that the female parent giving the unexpected offspring had two X chromosomes plus one Y. We could designate her XXY, in contrast with a normal female, which is XX. During meiosis a normal female pro- duces only one class of ova so far as the sex chromosomes are concerned: those with a single X. An XXY female would produce four types of ova dur- ing meiosis. These would be XY, X, XX, and Y. There was no way of predict- ing the frequency of each type of gamete, but we will anticipate the breeding results where Bridges found that the proportions were: 46 per cent XY; 46 per cent X; 4 per cent XX; 4 per cent Y. Females of the XXY type were called non-disjunction females. The term refers to the fact that in some of the ova produced by these females there is no separation, or disjunction, of the two X chromosomes. In a normal female there is regularly a disjunction of the two X chromosomes with the result that a single X is present in each ovum. The cross of a white-eyed non-disjunction female to a normal red-eyed male according to Bridges’ hypothesis would be as shown in Figure 5–6. It must have taken considerable courage to postulate such a seemingly pre- posterous hypothesis, although some such hypothesis was necessary to explain the results, if one were to continue to hold the belief that genes are located on chromosomes. The hypothesis could be verified, however, since deductions could be made and tested by observation and experiment. These were the main deductions: 1. If the hypothesis is true, we would expect 50 per cent of the daughters (classes 1 and 7 of Fig. 5–6) to be non-disjunction females. Breeding experiments showed this to be the case. 2. If the hypothesis is true, we would expect the exceptional ♂ (class 4) not to transmit the power of producing exceptions in later generations. It should behave like a normal male. Breeding experiments showed this to be true. 3. If the hypothesis is true, we would expect 46 per cent of the males to be XYY. These would produce sperm of four genotypes, namely, X, YY, XY, and Y. If a male of this type were crossed to a normal female, there should be no exceptional offspring (i.e. males inheriting 

HEREDITY AND DEVELOPMENT: SECOND EDITION 114 5–6 Bridges’ Experiment their sex-linked characteristics only from the father, and females inherit- ing theirs only from the mother). However, every XY sperm that entered a normal X-containing egg would produce an XXY daughter, which should be a non-disjunctional female. Breeding experiments confirmed all these predictions. 4. If the hypothesis is true, we would expect that 50 per cent of the daugh- ters (classes 1 and 7) would have two X chromosomes and one Y. It should be possible to verify this deduction by cytological examination of the F1 females. Bridges did this and found that approximately half of the daughters that he examined had two X chromosomes plus one Y (Fig. 5–7). The other half had two X chromosomes only. This was the crucial test of the hypothesis, since the test was of a very different sort, namely, cytological. (We can ignore the rare XXX females.) Bridges’ conclusion was as follows: ‘…there can be no doubt that the com- plete parallelism between the unique behavior of the chromosomes and the behavior of the sex-linked genes and sex in this case 

HEREDITY AND DEVELOPMENT: SECOND EDITION 115 means that the sex-linked genes are located in and borne by the X- chromosomes.’ It seemed equally probable that the autosomal genes were likewise parts of chromosomes, though this was not proved by his experi- ments. A further extension was made to other species, and the hypothesis advanced that the genes of all organisms are parts of chromosomes. This extrapolation from the data was done because it appeared that inheritance was the same in all organisms being studied. It might be of interest to inquire about the nature of this ‘final proof,’ in 1914, that genes are located on chromosomes. It must be apparent that it is the same type of evidence that had been offered ever since 1902. Sutton pointed out the parallel behavior of chromosomes in meiosis and fertilization with the behavior of Mendelian factors. This evidence probably convinced a few that Mendelian factors were on the chromosomes. Morgan’s analysis of the white eye case in Drosophila showed that inheritance of the gene was an exact parallel to the inheritance of the X chromosome. This study convinced more biologists. The discovery that the number of linkage groups is the same as the number of chromosome pairs was further support for the theory. These and many other experiments showed that either the genes were parts of chro- mosomes or the genes were parts of structures that behaved precisely like the chromosomes. Bridges’ evidence was of the same type, though it differed in degree. The inheritance of eye color in his non-disjunction experiments was completely different from any other type of inheritance. If he assumed genes were car- ried on chromosomes, then he had to postulate some most unusual chromo- some phenomena. Cytological studies verified the predictions made from the genetic data. There could no longer be a 5–7 Bridges’ drawings of the chromosomes of the females in his non-disjunction experiment. Approximately half of the females (class 2) had the normal chromosome complement as shown in a. The remaining females (classes 1 and 7) have two X chromosomes and a Y as shown in b (C. Bridges, ‘Non-disjunction as proof of the chromosome theory of heredity,’ Genetics 1:1–51; 107–63. 1916). 

HEREDITY AND DEVELOPMENT: SECOND EDITION 116 ‘reasonable doubt’ that the genes were on chromosomes. More elaborate evi- dence was still to come, but for many biologists this evidence of Bridges was sufficient. There are few cases of inheritance that seemed unrelated to chromosomes. There were grouped under the term cytoplasmic inheritance, since it seemed that some non-nuclear factor was responsible. Over the course of the years, many cases first thought to be due to cytoplasmic inheritance were found to be misinterpretations of the data. A few instances of cytoplasmic inheritance are well established. For the other thousands of analyzed cases, there is no doubt that the genes are parts of chromosomes. The chromosomes form the physical basis for 99.9+ per cent of inheritance. THE CHROMOSOME BALANCE THEORY OF SEX DETERMINATION The problem of sex determination as it was understood in the first decade of the twentieth century was discussed in Chapter 4 where it was shown that there is a constant relation between the sex of an organism and its chromo- somes. The cells of Drosophila melanogaster females contained three pairs of autosomes and two X chromosomes. In the cells of males of this species there were three pairs of autosomes plus one X and one Y chromosome. Genetic work with Drosophila melanogaster revealed that the Y chromo- some contained very few genes, although it is essential for fertility in males. The Y came to be looked upon as a nearly inert chromosome genetically in all organisms; a view strengthened by the discovery that in many animal species the males have a single X and no other sex chromosome. These data led to the concept that in species with a XX ♀ -XY ♂ sex chromosome consti- tution, the presence of a single X determined that the individual be a male and a pair of X chromosomes determined that the individual be a female. (Excep- tions to this rule were eventually discovered—man being one of them.) This hypothesis was strengthened further by some remarkable observa- tions on gynandromorphs in Drosophila. Gynandromorphs are individuals in which part of the body has the morphological features of a male and the other part has the morphological features of a female. Morgan and Bridges discov- ered some gynandromorphs that were female on one side and male on the other. Analysis showed that these individuals began development as females. Due to some developmental accident in the early embryo, one X chromo- some was lost from the cells that were to form one-half of the body. As a result the cells of one side 

HEREDITY AND DEVELOPMENT: SECOND EDITION 117 of the body remained normal and contained the three pairs of autosomes and two X chromosomes. This side had the external structure typical of females and internally an ovary might be present. The cells of the other side of the body where one X was lost contained three pairs of autosomes and a single X chromosome. This side had the external structure typical for males and inter- nally a testis might be present. These observations made more probable the hypothesis that a fly was a- male or female depending on the number of X chromosomes in its cells. This concept seemed to be an adequate explanation of the data and geneticists were willing to accept it until further tests were possible. The work of Bridges on non-disjunction showed that a remarkable amount of chromosome juggling was possible in Drosophila. As a consequence it became feasible to further test the hypothesis that one dose of X resulted in a male and two doses of X resulted in a female. This concept was found to be inadequate to explain all of the data. Some Drosophila were obtained that were triploid, that is, there were three X chromosomes and three members of each autosome type in every cell. These individuals were females. Bridges obtained flies with various combinations of autosomes and sex chromosomes (Fig. 5–8) and proposed the hypothesis that sex is due to a bal- ance between the number of X chromosomes and the number of autosomes. In a normal ♀ there are two X chromosomes and 5–8 The various combinations of X chromosomes and autosomes obtained by Bridges and oth- ers. (The lowest circle at ratio 1.00 is a haploid female. No such fly has been obtained but some diploid flies have been observed that have haploid areas in their bodies. If these areas include sex structures, they are of the female type.) 

HEREDITY AND DEVELOPMENT: SECOND EDITION 118 two haploid sets of autosomes (a haploid set of autosomes consists of one of each of the three different kinds of autosomes). We could express this as 2X/2A=1.0=♀. A normal male would be 1X/2A=0.5 =♂. The triploid female would be 3X/3A=1.0=♀. When a triploid female is crossed with a diploid male, some of the off- spring have two X chromosomes and three members of each autosome. We could write these as 2X/3A=0.67. Now this ratio is intermediate between the value for normal males (0.5) and that for normal females (1.0) and it was observed that these individuals were intermediate in appearance between males and females. They are described by Bridges as follows: The ‘intersexes,’ which were easily distinguished from males and from females, were large-bodied, coarse-bristled flies with large roughish eyes and scolloped wing-margins. Sex-combs (a male character) were present on the tarsi of the fore-legs. The abdomen was intermediate between male and female in most characteristics. The external genitalia were preponderantly female. The gonads were typically rudimentary ovaries; and spermathecae were present. Not infrequently one gonad was an ovary and the other a testis; or the same gonad might be mainly ovary with a testis budding from its side. The intersexes were sterile. It was possible to obtain individuals with ratios of X chromosomes to auto- somes that were below the normal male value or above the normal female value. Some individuals had three X chromosomes and two sets of auto- somes. The ratio for these would be 3X/2A=1.5. This ratio is higher than that of a normal female, and the resulting imbalance in X chromosomes and auto- somes produces a sterile and somewhat abnormal female. Bridges was able to obtain a value below the 0.5 ratio of normal males in individuals with a single X and three autosome sets. These would be X/3A=0.33. Such flies were structurally abnormal and sterile males. These and many other combinations of chromosomes were obtained by Bridges and others. The results formed a consistent pattern, there being a rela- tion between the ratio of X chromosomes to autosome sets and the sex charac- teristics of the flies. RATIO X/A MORPHOLOGICAL TYPE 0.33 abnormal male 0.50 male 0.67 intersex 0.75 intersex 1.00 female 1.33 abnormal female 1.50 abnormal female 

HEREDITY AND DEVELOPMENT: SECOND EDITION 119 The significance of these ratios is to be found in the differential effective- ness of genes on the autosomes and on the X chromosomes. The net effect of the autosomal genes is a male-forming tendency. The net effect of the X chromosome genes is a female-forming tendency. In a normal male the genes of the two autosome sets overbalance the genes of the single X to produce the male. In the normal female the X chromosome genes are in a double dose and this is sufficient to produce the female body type. MULTIPLE ALLELES AND HUMAN BLOOD TYPE GENES In all the cases considered so far, a gene has existed in only two states. It might be involved in the production of red or white eyes, long or vestigial wings, round or wrinkled peas. The white eye allele appeared in a stock of red-eyed flies. Since the stock had been under observation for many genera- tions, it is reasonable to assume that in one X chromosome the gene, which in the normal condition is involved in the production of red eyes, changed in such a way as to produce white eyes. One question this suggests is: If the orig- inal red eye gene can change to white, might it not change in another way to produce a still different result? Continued observations answered this ques- tion in the affirmative. A mutant known as eosin was discovered. Its pheno- typic expression was a diluted red eye color. In its linkage relations and crossover behavior, it was found to occupy the same place on the X chromo- some as the gene for white. In crosses involving the red, white, and eosin alle- les, it was never possible to have more than two of the three in the same female. All of the data were consistent with the hypothesis that red, white, and eosin were different states of the same gene. Phenomena of this sort are known as multiple alleles. At the present several dozen alleles have been dis- covered at the white locus. The A, B, O Blood Type Alleles. A well-known case of multiple alleles in man is the ABO blood type series. The blood types, A, B, AB, and O are determined by the interaction of three autosomal alleles, A, B, and o. (Other designations frequently used are IA, IB, and Io; furthermore, additional alleles are now known for this locus.) A and B are dominant over o. When A and B are present in the same individual neither gene is dominant and the individual is type AB. The phenotypes and possible genotypes are as follows: PHENOTYPE GENOTYPE Type A AA or Ao Type B BB or Bo Type AB AB Type O oo The inheritance of these alleles follows the usual Mendelian scheme. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 120 A cross of a heterozygous type A with a heterozygous type B would be as follows: Digression on the Importance of Blood Types in Transfusions. Some years before the genetic basis of blood groups had been worked out, Karl Land- steiner found that human blood could be classified into the four types just described. The types proved to be of great importance in connection with blood transfusions. In some cases death resulted when the donor and recipi- ent were of different types. Experimentation and observation revealed that this incompatibility was due to the interaction of antigens on the surface of the red blood corpuscles with antibodies in the plasma. There are two types of antigens, A and B, and two types of antibodies, α and β. The distribution of these substances is as follows: BLOOD TYPE ANTIGEN IN CORPUS- ANTIBODY IN PLASMA CLE A A β B B α AB A and B none O none α and β The corpuscles are agglutinated (clumped) if those containing A antigen come in contact with α antibody, or if those containing B antigen come in contact with β antibody. The important factor is the type of corpuscle intro- duced in a transfusion; the introduced plasma has little or no effect on the recipient’s corpuscles. Any interaction, therefore, will be between the donor’s antigens on the corpuscles and the recipient’s antibodies in the plasma, resulting in clumping of the introduced corpuscles. The possible combinations are as follows: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 121 A ‘o’ in the table signifies no reaction while a ‘+’ indicates agglutination of corpuscles. It can be seen from the chart that the blood of an O type person can be used in any transfusion. For this reason type O is spoken of as a univer- sal donor. A type AB individual can receive blood of any of the four types. For this reason type AB is spoken of as a universal recipient. These interrelations are no longer quite so simple as described; it is now realized that more alleles are involved than the original three. INDUCED MUTATIONS The origin of mutants had been a mystery since the early days of genetics. In the initial work of Morgan and his associates, stocks of wild-type Drosophila might be kept for generations, and thousands of individuals examined, before a new mutant was discovered. The occurrence of mutations was a sponta- neous event that could neither be predicted nor controlled. Attempts to induce hereditary changes in the chromosomes were made from the very beginnings of Drosophila genetics. Various agents were tried, such as exposure of the flies to radium, X rays, and many different chemical agents. In one of Morgan’s first papers he reported the appearance of several new mutants in the offspring of flies that had been exposed to radium. Sev- eral other investigators reported similar results. Difficulties in Studying Induced Mutation. None of these early experi- ments was conclusive because of the difficulty of distinguishing induced from spontaneous mutations. This was the problem. Mutants were appearing in stocks not exposed to unusual radiations or to special chemical treatment. Their appearance could not be correlated with any known ‘cause,’ so they were termed ‘spontaneous.’ Spontaneous mutations were of very rare occur- rence. In the experiments attempting to produce mutations by physical or chemical means, mutations occurred but they also were very rare. Thus, if we expose flies to radium in an effort to produce mutations, and if a mutant form appears among the offspring or later descendants of the irradiated flies, we could not be sure whether radium was the cause or whether it ‘just happened.’ Since new mutant genes appear infrequently and most of them are reces- sive, the mere detecting of them becomes a problem. Assume, for example, that one autosomal gene in a sperm nucleus mutates. If this sperm then enters an egg the new individual will have one mutated allele from the father and one unmutated allele at the same locus from the mother. The mutant will, consequently, be masked by its dominant allele and the observer will see no evidence that a mutation has occurred. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 122 Appropriate crosses could be made to produce an individual homozygous for the new mutation if there was some way of knowing which individuals to cross. Since there is no way of knowing this, the alternative would be to make innumerable crosses in the hope of having at least one fly heterozygous for the new mutant allele. An appreciation of this problem will be gained if you determine the num- ber of crosses that would have to be made to secure homozygous flies starting with a single adult heterozygous for a new mutant gene. If you then wished to measure the mutation rate per one million flies, what would the total number of necessary crosses be? Muller’s ClB Method. H.J.Muller (1927) was the first person to give a practical solution to the problem. He was able to do so because he devised an ingenious experiment that gave an easy and accurate measure of mutation rate. His experiments were designed to test the effects of X rays on the induc- tion of mutations. As a control, it was necessary for him to know the sponta- neous mutation rate as well. After considerable experimental manipulation, Muller developed the ClB strain of Drosophila. A ClB ♀ contains the C inversion on one of her X chro- mosomes, a recessive lethal gene l and a dominant mutant bar-eye B, both of the genes being within the inverted section of the chromosome. An inversion is a region of the chromosome that has been reversed. If the normal order of genes is a b c d e f g, a chromosome with genes in the order a b e d c f g would contain an inversion. An inversion is caused by a double break of the chromosome, in this case between b and c and between e and f. Following this there is a rotation of the central section, c d e, through 180° and a subsequent fusion with the two ends of the chromosome. Inversions were discovered by Morgan’s group, and it was found that they have the important effect of reducing or even preventing crossing over between genes in the inverted section of one chromosome and in the corresponding normal sequence of its homologue. In the C inversion, crossing over is entirely pre- vented. This means that the C inversion with the l and B genes will be inher- ited as a unit. The B gene has the sole purpose, in this experiment, of serving as a ready means of recognizing a fly heterozygous for the ClB chromosome, since every fly that has bar eyes must have one ClB chromosome. It could not have two ClB chromosomes since the l gene, if homozygous, would result in the death of the fly. If a female heterozygous for a ClB chromosome is crossed with a normal male the results are as follows: 

HEREDITY AND DEVELOPMENT: SECOND EDITION 123 Those males that inherit the ClB chromosome from their mothers will have the lethal gene, l. Since there is no normal gene on the Y to counteract the effects of this lethal, these males will die. Therefore, the sex ratio will be 2 ♀:1 ♂. It was well known at the time Muller performed his experiment that many separate gene loci can mutate in such a way as to lead to death. These lethal genes were usually recessive. Since many genes can do this, the chance of getting some lethal mutation is greater than the chance of observing muta- tions at a specific locus. Thus, if we studied the rate of mutations to the lethal condition on the X chromosome we would be measuring the sum of the rates for all of its genes that can form lethals by mutation. The rate for a specific locus would be much smaller. With ClB flies it is possible to measure the mutation rate for lethal genes on the X of sperm. Once again it must be emphasized that this will not be a measure of rate for one locus, but of all the loci on the X that can mutate to a lethal condition. Should a lethal mutation occur on the X in one of the sperm, it could be detected by the cross shown at the top of page 124. The * will rep- resent the new lethal. If the F1 ClB ♀ is then mated with a normal ♂, the marked chromosome that we are searching for will pass to the ♂ offspring and be revealed as shown in the cross at the bottom of page 124. One class of the daughters will be normal in appearance and heterozygous for the new lethal mutant gene. Another class will be ClB females. One of the males will carry the new lethal mutant gene and die as a result. Another class of males will inherit the ClB chromosome and die because of the lethal 

HEREDITY AND DEVELOPMENT: SECOND EDITION 124 gene in the C inversion. Therefore, only females will appear. Thus, if a lethal gene was present on the X of the original sperm, there will be no sons in the F2. This fact can be ascertained by a quick examination of the F2 flies. This point is of considerable significance, since it makes it feasible to check many crosses in a short period of time. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 125 When Muller used normal untreated males in crosses of this type, he found that approximately one cross in a thousand gave solely females in the F2. This means that the chance of a lethal mutation occurring at some locus on the X is 1 in 1,000 or 0.1 per cent. This is the natural, or spontaneous, mutation rate. If the males are first exposed to about 4,000 r-units of X rays the results are strikingly different: approximately 100 crosses in every 1,000, or 10 per cent, have only females in the F2. This amount of radiation, therefore, increased the mutation rate 100 times. Muller’s results were not only of great theoretical importance in showing that mutations could be experimentally produced, but they gave geneticists a practical means of securing new mutants for their work. Later it was found that radiations would induce not only gene mutations but also cause inver- sions, translocations (the attachment of a piece of one chromosome to another), or deficiencies (elimination of a section of a chromosome). SALIVARY GLAND CHROMOSOMES The concepts of genetics were developed mainly on data derived from breed- ing experiments. That is, the localization and behavior of genes were studied without the gene ever being seen. With standard cytological techniques, the chromosomes appear as uniformly-staining structures with no differentia- tions recognizable as genes. By 1930 geneticists felt that genes were proba- bly some type of protein. If this were so, it would be impossible to see them even under the most powerful compound microscopes available since protein molecules are too small to be observed with these instruments. Geneticists became resigned to investigating their invisible genes just as the chemist stud- ies his invisible molecules and the physicist his invisible sub-atomic particles. The Discovery of Salivary Gland Chromosomes. It was against this background that Theophilus S.Painter (1889–1969), in 1933, and somewhat later Calvin Bridges, made discoveries of the first importance concerning the finer structure of chromosomes. It was found that the chromosomes in the salivary glands of larvae of Drosophila were of enormous size, being about 100 times longer than those of ordinary body cells. Of even greater interest and importance was the presence of cross bands on the chromosomes. Figure 5–9, from Painter’s first paper on the subject, shows the appearance of the salivary chromosomes. The salivary glands are diploid, but instead of the expected eight chromo- somes, Painter found only four. This is due to the fact that homologous chro- mosomes have fused together. They are so close to one 

HEREDITY AND DEVELOPMENT: SECOND EDITION 126 5–9 Painter’s first drawing of the salivary gland chromosomes of Drosophila melanogaster. The chromosomes radiate out from the chromocenter. The X is attached to the chromocenter by one end so it appears as a single long structure. Both the II and III chromosomes are attached by their middle portions. Consequently both of these chromosomes have two arms extending from the chromocenter. The tiny IV chromosome is attached by its end to the chromocenter. The approxi- mate location of several X chromosome genes (B, f, sd, etc.) is shown. (T. S.Painter, ‘A New Method for the Study of Chromosome Aberrations and the Plotting of Chromosome Maps in Drosophila melanogaster,’ Genetics 19:175–88. 1934.) another that the line of separation cannot be seen in the illustration. The line of separation can be seen in some microscopic preparations, however. The pairing of the two chromosomes is so exact that the cross bands extend across both chromosomes as though they were a single structure. In different sections of the chromosomes the bands were found to vary in size, distinctness, shape, and distance from adjacent bands. These regional differences were found to be constant for the chromo- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 127 somes in different cells of the same larva and in different larvae. For the first time it became possible to recognize regional differences in chromosomes. The concepts of chromosome structure previously based solely on the genetic data could be confirmed. Was there a close relation between the genetic map of a chromosome, which was nothing more than a way of describing linkage groups and crossover percentages, and the real chromosome? There were many other special situations that needed to be checked. For example, geneticists some- times resorted to seemingly preposterous hypotheses to explain the outcome of their crosses: genes jumping from one sort of chromosome to another (a translocation); the loss of an entire gene (a deletion); a chromosome might seem to have two loci for the same gene (a duplication); and still other results were explained on the basis of a reversed order of genes (an inversion). Critics of the Morgan school were delighted to point out that one can man- ufacture hypotheses to explain anything. What is needed is a means of testing hypotheses. If one postulated a genetic inversion, it was up to him to show that a section of the chromosome was, indeed, reversed. But that was impos- sible so long as one could not distinguish the sections of chromosomes: a chromosome with an inversion would look exactly like one without the inver- sion. The discovery of salivary chromosomes made it possible to test these hypotheses. Inversions and Translocations. In the early days of Drosophila genetics, Morgan’s group encountered some instances where the amount of crossing over was less than expected. For example, on the basis of previous work it might have been observed that the amount of crossing over between genes A and B was 10 per cent. Such data would have been used to fix the position of these genes on the chromosome map. But what should one conclude if, in still other experiments, the amount of crossing over between A and B was zero? This would suggest that the hypothesis that the positions of genes was con- stant and could be determined from the amounts of crossing over is invalid. A new hypothesis was developed to account for the exceptional results. If crossing over was reduced or prevented, possibly this was a consequence of something interfering with synapsis. If the homologous chromosomes could not synapse, there would not be an opportunity for crossing over to occur. It had always been assumed that synapsis was possible only because the homol- ogous chromosomes were identical in the order of their genes. Could it be that in the flies giving the exceptional results the order of the genes was dif- ferent? If a section of a chromosome became inverted, then normal synapsis and crossing-over would 

HEREDITY AND DEVELOPMENT: SECOND EDITION 128 5–10 Inversions in Drosophila pseudoobscura. In a the standard band sequence in the third chro- mosome is shown. The chromosome shown in b is identical to the one in a except for the region between the two lines, which has the band 

HEREDITY AND DEVELOPMENT: SECOND EDITION 129 be prevented. This could happen if a chromosome broke in two places, to give three pieces, and the middle piece rotated 180° and then rejoined the two end sections of the chromosome. It was quite an intellectual achievement to make this hypothesis, which at first must have seemed most improbable. Direct evidence for this hypothesis could not be obtained so long as it was impossible to detect regional differences in chromosomes. When salivary chromosomes were discovered, a method of checking became possible. Figure 5–10 gives an example. In Drosophila pseudoobscura the ‘stan- dard’ arrangement of bands is shown in a. In this species many inversions have been discovered. One of these is known as ‘arrowhead.’ A chromosome with the arrowhead inversion is shown in b and it can be seen that the sequence of bands is reversed. Cytology confirmed the genetic hypothesis of chromosomal inversions. An interesting situation arises when an individual has one standard and one arrowhead chromosome. During synapsis these two chromosomes will pair, with corresponding bands being adjacent as shown in c. Since a section of one chromosome has a reversed order of regions, considerable gyrations are necessary for matching of the corresponding regions to be achieved. The way it is accomplished is shown in Fig. 5–10d. The occurrence of translocations, which had been predicted on the basis of genetic results, was also verified by an examination of the salivary chromo- somes. In those individuals suspected of having translocations, it was found that a piece of chromosome, with its characteristic set of bands, was no longer in its customary place, but instead it was joined to another chromosome. How did Painter decide which salivary chromosome corresponded to a particular linkage group? It was done largely by means of inversions and translocations. Painter had studied the salivary gland chromosomes of nor- mal flies so carefully that he was able to recognize the various regions. Once he could do this, he studied flies that were known on genetic grounds to have an inversion in one chromosome—for example, the X. Invariably, he found that in one of their salivary sequence reversed. This is a cytological demonstration of an inversion. If an individual fly has one of each of these chromosome types, the salivary gland picture will be as in c. Pairing is achieved by one chromosome forming an inverted U and the other a loop. As a result of these contortions, it is possible for the corresponding bands of the two chromosomes to be situated opposite to one another. In d the two chromosomes are separated slightly to show more clearly the manner of pairing. (Modified from Dobzhansky and Sturtevant, ‘Inversions in the chromo- somes of Drosophila pseudoobscura,’ Genetics 23:28–64. 1938.) 

HEREDITY AND DEVELOPMENT: SECOND EDITION 130 chromosomes there was a region of reversed bands. This chromosome was, therefore, the X. He had numerous inversions and translocations for study, so it was possible to identify each salivary chromosome with the corresponding linkage group. The Gene Locus. So much for the gross problems: Now we can ask, Where are the genes? Chromosomal structural aberrations were the basis for solving this problem, small deficiencies being especially useful. The follow- ing example, from the work of Milislav Demerec (1895–1966) and M.E. Hoover, will show how it was done (Fig. 5–11). They studied Drosophila with deficiencies near one end of the X chromosome. Most deficiencies, except those that are very small, are lethal when homozygous. In the het- erozygous condition they do not cause death, but 5–11 Diagrammatic representation of the experiment of Demerec and Hoover showing how the positions of genes on the salivary gland chromosomes can be determined. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 131 they do have a characteristic genetic effect, which can be brought out by the following consideration. Let us assume that a fly is heterozygous for a deficiency including the locus of the gene A. That is, there will be no A on the chromosome with the deficiency, but in the normal chromosome the locus will be present. The result is that the genes at the A locus on the normal chromosome will be the one determining the character of the individual. If the dominant A allele is present, it will have its usual effect. If the recessive a allele is present it will produce its effect, since there is no gene on the chromosome with the defi- ciency to influence its action. These results should not be too surprising. All the X chromosome characters of the Drosophila male that we have studied behave in a similar way. The Y lacks nearly all loci normally present on the X and, therefore, acts as one giant deficiency. Demerec and Hoover used three stocks, each with a different small defi- ciency at one end of the X chromosome. The locations and extents of the defi- ciencies were determined by study of the salivary chromosomes. Genetic crosses had previously established that the genes y (yellow), ac (achaete), and sc (scute) were close to the end of the X chromosome. Demerec and Hoover made their crosses in such a way that a fly would receive one normal X chromosome carrying the recessive genes y, ac, and sc and another X carry- ing a deficiency but no mutant genes. If the deficiency included the locus of any of these genes, then the fly would exhibit the recessive character since there would be no normal allele to counteract its action. The first deficiency tried was a small one. It removed the first 4 bands of the X chromosome (Fig. 5–11). When this deficient chromosome was present with the X carrying y, ac, and sc, the flies were normal. This means that none of these genes is in the part of the X having the first 4 bands. The second deficiency removed the 8 terminal bands. When this chromo- some was present with an X carrying y, ac, and sc, the flies were yellow and achaete. This experiment showed that y and ac were in that part of the chro- mosome having bands 1 through 8. The previous cross showed they were not in the region covered by bands 1 through 4. Therefore, the genes y and ac must be in the region marked by bands 5 through 8. This cross also showed that the locus for sc is not in the area covered by the first 8 bands. The third deficiency removed the 10 terminal bands. When one of these chromosomes was present with an X carrying y, ac, and sc, the flies were yellow, achaete, and scute. This indicated that y, ac, and sc were in the area covered by the first 10 bands. In the previous experi- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 132 ment we saw that sc was not in the first 8 bands. The present cross shows it is somewhere in the first 10. Combining these results, we can conclude that the sc gene is in that region of the chromosome containing bands 9 and 10. In this manner it was possible to give an approximate location for many genes. In a few cases genes were localized to a portion of the chromosome having but a single band. No genes were found in areas without bands. These observations led to the tentative hypothesis that the bands are gene loci. If the bands are gene loci, it should be possible to determine the number of genes in Drosophila by counting the number of bands. This attempt was made but one difficulty made an exact determination impossible. Not all of the bands are equally distinct: they vary from those that stain distinctly to those so indistinct as to be at the limit of visibility. Approximately 5,000 bands could be seen and this figure was taken as the tentative minimum num- ber of genes in Drosophila. Once genes were located on salivary chromosomes, a comparison could be made with the genetic maps. One example is given in Figure 5–12, which shows corresponding parts of a small section of the salivary second chromo- some and the genetic map. The point of greatest importance is the close resemblance of the two. The genetic map is 5–12 Corresponding points in the salivary chromosome and linkage map for the tip of the second chromosome of Drosophila melanogaster. (Modified from C. B.Bridges, ‘Correspondence between linkage maps and salivary chromosome structure, as illustrated in the tip of chromosome 2R of Drosophila melanogaster,’ Cytologia. Fujii Jubilee Volume, pages 745–55, 1937.) 

HEREDITY AND DEVELOPMENT: SECOND EDITION 133 based on breeding experiments and the arrangement of loci is based on the percentages of crossing over. These data suggest that the genes are in a linear order and in a certain sequence. When it became possible, with the salivary gland techniques, to determine the actual position of genes on the chromo- somes, it was found that the order and sequence predicted by genetic means was verified by cytology. Once again, these mutually supporting fields had established a hypothesis as ‘true’ beyond a reasonable doubt. Salivary gland chromosomes are of great importance in many genetic prob- lems being studied today. One of their more spectacular applications has been in the field of evolution. As an interesting historical footnote we might add this bit of information: The banding of salivary chromosomes in flies had been observed and recorded by cytologists in 1881, but this was not known to geneticists. If the Morgan group had been aware of this, their efforts would have been made much easier. Their prediction of chromosomal aberrations such as inver- sions, translocations, and deficiencies, largely on genetic data, was a tremen- dous intellectual achievement. All this time a simple method for demonstrat- ing these cytological phenomena was buried and forgotten in the archives of biological literature. Cases such as this, which are not infrequent, make all scientists wonder what important facts have been discovered, forgotten, and now await redis- covery and a realization of their worth. Suggested Readings Morgan’s Croonian Lecture (1922) before the Royal Society of London and his Presidential Address (1932) before the Sixth International Congress of Genetics are reprinted in Readings in Heredity and Development. A more complete bibliography is also provided there. The development of ideas in genetics, to produce what Morgan called ‘The Theory of the Gene,’ is discussed in: CREW, F.A.E. 1966. The Foundations of Genetics. New York: Pergamon. DUNN, L.C. Editor. 1951. Genetics in the 20th Century. Essays on the Progress of Genet- ics During Its First 50 Years. New York: Macmillan. DUNN, L.C. 1965. A Short History of Genetics. New York: McGraw-Hill. STURTEVANT, A.H. 1965. A History of Genetics. New York: Harper & Row. As recalled by one who did so much to make genetics an exact science. Chapter 7 is a fascinating account of life and science in the “Fly Room” at Columbia University, where so much of the research was done. STURTEVANT, A.H., and G.W.BEADLE. 1939. An Introduction to Genetics. Philadel- phia: W.B.Saunders. Reprinted by Dover Books, New York. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 134 Much of the material presented in this chapter will be found, differently presented, in the standard books on genetics. Some of the more notable ones are: BEADLE, GEORGE and MURIEL. 1967. The Language of Life. An Introduction to the Science of Genetics. Garden City, New York: Doubleday Anchor Book. Elementary. Paperback. BURNS, GEORGE W. 1969. The Science of Genetics. An Introduction to Heredity. New York: Macmillan. GARDNER, ELDON J. 1968. Principles of Genetics. New York: Wiley. HERSKOWITZ, IRWIN H. 1965. Genetics. Boston: Little, Brown. KALMUS, H. 1964. Genetics. Garden City, New York: Doubleday Anchor Book. Paperback. KING, ROBERT C. 1965. Genetics. New York: Oxford University Press. PAPAZIAN, HAIG P. 1967. Modern Genetics. New York: W.W.Norton. SINNOTT, EDMUND W., and L.C.DUNN. 1925–. Principles of Genetics. New York: McGraw-Hill. Subsequent editions: 1932, 1939, 1950, and 1958. Th.Dobzhansky joined as an author for the two latest editions. Throughout its long history this has been the clas- sic textbook of classical genetics. STRICKBERGER, MONROE W. 1968. Genetics. New York: Macmillan. SRB, ADRIAN M., RAY D.OWEN, and ROBERT S.EDGAR. 1965. General Genetics. San Francisco: W.H.Freeman. WHITEHOUSE, H.L.K. 1969. Towards an Understanding of the Mechanism of Heredity. New York: St. Martin’s Press. WINCHESTER, A.M. 1966. Genetics. A Survey of the Principles of Heredity. Boston: Houghton Mifflin. The principal research papers referred to in the chapter are: BRIDGES, C.B. 1914. ‘Direct proof through non-disjunction that the sex-linked genes of Drosophila are borne by the X-chromosome.’ Science 40:107–9. BRIDGES, C.B. 1916. ‘Non-disjunction as proof of the chromosome theory of heredity.’ Genetics 1:1–52, 107–63. BRIDGES, C.B. 1921. ‘Triploid intersexes in Drosophila melanogaster.’ Science 54: 252– 54. BRIDGES, C.B. 1939. ‘Cytological and genetic basis of sex.’ In Sex and Internal Secre- tions, edited by E.Allen.Williams and Wilkins. DEMEREC, M., and M.E.HOOVER. 1936. ‘Three related X-chromosome deficiencies in Drosophila.’ Journal of Heredity 27:206–12. MORGAN, T.H. 1910. ‘Sex-limited inheritance in Drosophila.’ Science 32:120–22. MORGAN, T.H. 1911. ‘Random segregation versus coupling in Mendelian inheritance.’ Science 34:384. MULLER, H.J. 1927. ‘Artificial transmutation of the gene.’ Science 66:84–87. MULLER, H.J. 1928. ‘The production of mutations by X-rays.’ Proceedings of the National Academy of Sciences 14:714–26. PAINTER, T.S. 1934. ‘A new method for the studying of chromosome abberrations 

HEREDITY AND DEVELOPMENT: SECOND EDITION 135 and the plotting of chromosome maps in Drosophila melanogaster.’ Genetics 19:175–88. PAINTER, T.S. 1934. ‘Salivary chromosomes and the attack on the gene.’ Journal of Heredity 25:465–76. STERN, C. 1931. ‘Zytologisch-genetische Untersuchungen als Beweise für die Morgan- sche Theorie des Faktorenaustauschs.’ Biologisches Zentralblatt. 51:547–87. STURTEVANT, A.H. 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. Questions 1. In the light of what you now know about genetics, try to explain the ten types of observations that Darwin attempted to account for by his The- ory of Pangenesis (Chapter 1). 2. Can you suggest an explanation for Darwin’s observations on color blindness (page 12)? 3. The ‘porcupine man’ transmitted his defect only to his male descendants (p. 9). Assume that this is the true pattern of inheritance. What genetic explanations are possible? 4. What beliefs concerning the importance of the nucleus were held by Schwann, Flemming, and Morgan? 5. Compare the evidence available in 1900 and 1920 for the participation of the nucleus in inheritance. Do you believe that the evidence of 1900 would have convinced you? 6. Discuss the experiment of Bridges that is referred to as the “final proof” that genes are parts of chromosomes. How did his proof differ from ear- lier proofs? 7. In Drosophila, vestigial (v) is an autosomal recessive to long wings (V). Sepia (s), another autosomal character, is recessive to red eyes (S). These two genes are located on different chromosomes. Describe (geno- type and phenotype) the F1 and F2 of a cross between a vestigial female and a sepia male. Also the F1 and F2 of a cross between a sepia female and a vestigial male. In all these questions assume homozygosity for the normal allele unless you are told otherwise. 8. Two flies, both heterozygous for sepia and vestigial, are crossed. Describe the offspring of this mating. 9. A normal-appearing fly is crossed with one which is both sepia and ves- tigial. The offspring are: 1/4 sepia-vestigial 1/4 sepia-long 1/4 red-vestigial 1/4 red-long What is the genotype of the normal parent? Hint. When doing genetics problems of this type, it is best to solve for one character at a time. It is also necessary to keep in mind how the usual ratios 

HEREDITY AND DEVELOPMENT: SECOND EDITION 136 are obtained. Thus, a 3:1 ratio results from the crossing of two heterozy- gous individuals (Aa×Aa). A 1:1 ratio results from the cross of a het- erozygous individual with a homozygous recessive individual (Aa×aa). 10. In an F1 the following results were obtained: 3/8 red-long 1/8 sepia-long 3/8 red-vestigial 1/8 sepia-vestigial What were the genotypes and phenotypes of the parents? 11. A female with sepia eyes and long wings was crossed with a male fly. The F1 gave the following ratios: 3/4 red-long 1/4 red-vestigial What were the genotypes of the parents? 12. In Drosophila the character vestigial wings (v) is an autosomal recessive to long wings (V). White eyes (w) is a sex-linked recessive to red eyes (W). Describe the F1 and F2 of a cross between a white-eyed female and a red-eyed male. 13. Describe the F1 and F2 of a cross of a homozygous red-eyed female and a white-eyed male. 14. A white-eyed, long-winged female is crossed to a red-eyed, vestigial male. Describe the F1 and F2. 15. In an F1 the following results were obtained: Males—1/2 white-vestigial; 1/2 white-long. Females—1/2 red-vestigial; 1/2 red-long. What were the genotypes of the parents? Hint. In doing problems of this nature, involving both autosomal and sex-linked genes, solve for the autosomal gene first. When you begin with the sex-linked gene remember these points: a. a female has two X chromosomes, which are distributed to both sons and daughters. b. the male has one X chromosome, which goes to the daughter, and one Y which is transmitted to the son. c. the son, therefore, receives his X only from his mother. The pheno- type of the sons will, therefore, identify the genotype of the mother. d. the daughter receives one X from the father and one from the mother. With the genotype of the mother already established, the genotype of the father can be determined by considering the known genotype of the mother and the phenotypes of the daughters. 16. In an F1 the following results were obtained: Males—1/4 white-long; 1/4 white-vestigial; 1/4 red-long; 1/4 red-vestigial. Females—1/2 red-long; 1/2 red-vestigial. What were the genotypes of the parents? 17. In an F1 the following results were obtained: Males—3/8 white-long; 3/8 red-long; 1/8 white-vestigial; 1/8 red-vestigial. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 137 Females—3/8 white-long; 3/8 red-long; 1/8 white-vestigial; 1/8 red-vestigial. What were the genotypes of the parents? 18. In an F1 the following results were obtained: Males—3/8 white-long; 3/8 red-long; 1/8 white-vestigial; 1/8 red-vestigial. Females—3/4 red-long; 1/4 red-vestigial. What were the genotypes of the parents? 19. In man hemophilia (h) is a sex-linked recessive to normal clotting blood (H). Apparently hemophilia is an embryonic lethal when homozygous in females. At least no homozygous females have been recorded. Brown eyes (B) is autosomal and dominant to blue eyes (b). Describe the off- spring of a female heterozygous for hemophilia and a normal male. 20. Describe the offspring of a female heterozygous for hemophilia and a hemophiliac male. 21. A blue-eyed female heterozygous for hemophilia marries a male het- erozygous for brown eyes and with normal blood. Describe the possible offspring. 22. A case is recorded of a couple living in Outer Mongolia who were the parents of 193 children. Of the children, 99 were females and 94 were males. Of the 94 males, 12 were blue-eyed and had hemophilia; 10 were blue-eyed and normal; 33 were brown-eyed and had hemophilia; 39 were brown-eyed and with normal blood. Of the 99 females all had nor- mal blood but 26 were blue-eyed and 73 had brown eyes. What were the genotypes of these fortunate parents? 23. In man brown eyes (B) is dominant over blue (b). The gene is autoso- mal. Colorblindness (c) is a sex-linked recessive to normal vision (C). What are the probable genotypes and phenotypes of two individuals who had the following children? Daughters: 1 blue-normal 4 brown-normal Sons: 2 blue-color blind 5 brown-color blind 24. The blood groups in man, A, B, AB, and O are determined by the interac- tion of three multiple alleles, A, B, and o. A and B when present together result in type AB. Both A and B are dominant over o. Type A is either AA or Ao. Type B is either BB or Bo. Type O is the homozygous recessive oo. These are autosomal genes. What types of children would be expected from the following crosses: AB×oo; AB×AA; AB×Bo; Ao×Bo. 25. Two mothers, one whom we shall call M (husband is N) and the other R (husband is S), delivered two children, X and Y, in an obscure British hospital during a power failure. In the confusion no one remembered which baby belonged to which mother. It was therefore necessary for all individuals concerned to submit to a blood test. Baby X was found to be of type O and 

HEREDITY AND DEVELOPMENT: SECOND EDITION 138 baby Y of type A. Mother M was of type A and her husband N was also of type A. Mother R was type AB. Her husband, S, being on a business trip to Ceylon at the time, could not be tested. With this information, was it possible for the hospital to make the proper allocation of babies? 26. In chickens a common type of comb, single, occurs when two different recessive genes, p and r, are homozygous. Rose comb is formed when one or two dominant R genes are present. Pea comb is formed when one or two of the dominant P genes are present. If one or two R genes and one or two P genes are present in the same individual, the result is a wal- nut comb. The loci for P and R are on different chromosomes. What will be the phenotypes and genotypes in the F1 and F2 of a cross of a homozygous rose and a homozygous pea chicken? 27. Let us assume that an organism has a pair of autosomes and a pair of sex chromosomes (♀XX and ♂XY) and that crossing over occurs in the female only. In the ♀, one of the autosomes has the genes a and B and the other A and b; one of the X chromosomes has the genes c and D and the other C and d. Assume that crossing over between the a and b loci occurs in 40 per cent of the cases and between c and d loci in 20 per cent of the cases. This ♀ is crossed with a ♂ homozygous for a and b and with c and d on his X chromosome. Assume that there are no loci of c and d on the Y chromosome. Diagram the chromosomal events, begin- ning with the diploid cells in the ovary and testis and ending with the first division of the zygote. 28. In Drosophila, ebony (e) and stripe (s) are recessive autosomal genes located on the same chromosome. They show 8 per cent crossing over. Describe the offspring of a cross of a female heterozygous for both genes (her father was ebony-stripe and her mother homozygous-normal) with an ebony-stripe male. There is no crossing over in Drosophila males. 29. In Drosophila bar eyes (B) is a sex-linked dominant to normal eyes (b). Red eyes (W) is a sex-linked dominant to white eyes (w). The two loci are 50 cross-over units apart. Cross a white-normal female with a red- bar male. Then cross the F1 male with a white-bar (heterozygous) female. What will be the genotypic and phenotypic ratios of the result- ing offspring? 30. A cross is made of a Drosophila female with white eyes and a red-eyed male. One of the eggs was fertilized with a sperm carrying an X chromo- some. Something happened in the very young embryo and the father’s X was eliminated from the cells that were destined to form the entire left side of the body. What would be the eye color of this fly when it reached the adult stage? 31. Bar eye is a dominant sex-linked gene of Drosophila that reduces the normal circular eye to a bar-shaped structure. Solely on the basis of genetic crosses, Sturtevant and others advanced the hypothesis that the bar gene is a duplication. That is, it differs from a normal gene in having two loci, which are adjacent, on the same chromosome. At the time it was impossible to test this hypothesis by studying the chromosomes. Years later when the salivary 

HEREDITY AND DEVELOPMENT: SECOND EDITION 139 gland techniques were known, Bridges examined the region of the X chromosome of normal flies and of bar-eyed flies where the bar locus was thought to be located. This is what he saw (modified from Bridges 1936; Science 83: 210). Was Sturtevant correct? 32. If volume 44 of Science (1916) is available to you, read Bateson’s review (pages 536–543) of ‘The Mechanism of Mendelian Inheritance’ by Morgan, Sturtevant, Muller, and Bridges. Do you agree with his caution? 33. Darlington in ‘Genetics and Man’ (page 118), mentions the reluctance of most European biologists to accept Morgan’s Theory of the Gene. Read what he has to say and see if you understand the biological and philosophical problems involved.