Molecular Genetics Chapter 7: Linkage and Chromosome Mapping in Eukaryotes.

Sturtevant constructed a chromosome map of the three genes on the X chromosome, setting 1 map unit (mu) equal to

1 percent recombination between two genes In the preceding example, the distance between yellow and white is thus 0.5mu, and the distance between yellow and miniature is 35.4mu. It follows that the distance between white and miniature should be 35.4-0.5=34.9 mu. This estimate is close to the actual frequency of recombination between white and minature (34.5 percent). The fact that these do not add up perfectly is due to the imprecision of mapping experiments.

To execute a successful mapping cross of three or more genes, three criteria must be met:

1. The genotype of the organism producing the crossover gametes must be heterozygous at all loci under consideration 2. The cross must be constructed so that genotypes of all gametes can be determined accurately by observing the phenotypes of the resulting offspring. This is necessary because the gametes and their genotypes can never be observed directly. To overcome this problem, each phenotypic class must reflect the genotype of the gametes of the parents producing it. 3. A sufficient number of offspring must be produced in the mapping experiment to recover a representative sample of all crossover classes.

Theoretically, if we consider only single exchanges and observe 20 percent recombinant gametes, crossing over actually occurred in ________ percent of the tetrads

40 percent Under these conditions, the general rule is that the percentage of tetrads involved in an exchange between two genes is twice the percentage of recombinant gametes produced. Therefore, the theoretical limit of observed recombination due to crossing over is 50 percent

When two linked genes are more than ______ mu apart, a crossover can theoretically be expected to occur between in 100 percent of tetrads. If this prediction were achieved, each tetrad would yield equal proportions of the four gametes shown in figure 7.6, just as if the genes were on different chromosomes and assorting independently. However, this theoretical limit is seldom achieved.

50

When a single crossover occurs between two nonsister chromatids, the other two chromatids of the tetrad are not involved in this exchange and enter the gamete unchanged. Even if a single crossover occurs 100 percent of the time between two linked genes, recombination is subsequently observed in only ___________________ of the potential gametes formed.

50 percent

A read on the importance of Sturtevants work

Although many refinements in chromosome mapping have been developed since Sturtevant's initial work, his basic principles are considered to be correct. These principles are used to produce detailed chromosome maps of organisms for which large number of linked mutant genes are known. Sturtevant's findings are also historically significant to the broader field of genetics. In 1910, the chromosomal theory of inheritance was still widely disputed-even Morgan was skeptical of this theory before he conducted his experiments. Research has now firmly established that chromosomes contain genes in a linear order.

Figure 7.1 c) Results of gamete formation when two heterozygous genes are on the same pair of homologs, but with an exchange occurring between two non-sister chromatids. Linkage: two genes on a single pair of homologs; exchange occurs between two nonsister chromatids.

As the picture shows, this crossover involves only two nonsister chromatids of the four chromatids present in the tetrad. This exchange generates two new allele combinations, called recombinant or crossover gametes. The two chromatids not involved in the exchange result in noncrossover gametes. The frequency with which crossing over occurs between any two linked genes is generally proportional to the distance separating the respective loci along the chromosome. In theory, two randomly selected genes can be so close to each other that crossover events are too infrequent to be detected easily demonstrating complete linkage. On the other hand, if a small but distinct distance separates two genes, few recombinant and many parental gametes will be formed. As the distance between the two genes increases, the proportion of recombinant gametes increases and that of the parental gametes decreases

Double exchanges of genetic material result from ___________________

Double crossovers (DCOs)

Why should the relative distance between two loci influence the amount of recombination and crossing over observed between them?

During meiosis, a limited number of crossover events occur in each tetrad. These recombinant events occur randomly along the length of the tetrad. Therefore, the closer two loci reside along the axis of the chromosome, the less likely any single-crossover event will occur between them. The same reasoning suggests that the farther apart two linked loci are, the more likely a random crossover event will occur between them.

T/F linkage and crossing over are restricted to X-linked genes and cannot be demonstrated with autosomes

F Linkage and crossing over are NOT restricted to X-linked genes but can also be demonstrated with autosomes.

Figure 7.7 Consequences of a double exchange occurring between two nonsister chromatids.

For a double exchange to be studied, three gene pairs must be investigated, each heterozygous for two alleles. As we have seen, the probability of a single exchange occurring between the A and B or the B and C genes relates directly to the distance between the respective loci. The closer A is to B and B is to C, the less likely a single exchange will occur between either of the two sets of loci. In the case of a double crossover, two separate and independent events or exchanges must occur simultaneously. We can then apply the product law. Suppose that crossover gametes resulting from single exchanges are recovered 20 percent of the time (p=0.20) between A and B, and 30 percent of the time (p=0.30) between B and C. The probability of recovering a double-crossover gamete arising from two exchanges (between A and B, and between B and C) is predicted to be (0.20)(0.30)=0.06 or 6 percent. It is apparent from this calculation that the frequency of double-crossover gametes is extremely low. If three genes are relatively close together along one chromosome, the expected frequency of double-crossover games is extremely low. -For example, suppose the A-B distance in this figure is 3mu and the B-C distance is 2mu. The expected double-crossover frequency is (0.03)(0.02)=0.0006 or 0.06 percent. This translates to only 6 events in 10,000. Thus, in a mapping experiment where closely linked genes are involved, very large numbers of offspring are required to detect double-crossover events. In this example, it is unlikely that a double crossover will be observed even if 1000 offspring are examined. Thus, it is evident that if four or five genes are being mapped, even fewer triple and quadruple crossover can be expected to occur.

Figure 7.1 b) Results of gamete formation when two heterozygous genes are on the same pair of homologs but with no exchange occurring between them. Linkage: two genes on a single pair of homologs; no exchange occurs.

If no crossing over occurs between the two genes, only two genetically different gametes are formed. Each gamete receives the alleles present on one homolog or the other, which is transmitted intact as the result of segregation. This case demonstrates complete linkage, which produces only parental or noncrossover gametes. The two parental gametes are formed in equal proportions. Though complete linkage between genes seldom (rarley) occurs, it is useful to consider the theoretical consequences of this concept.

Figure 7.5 Two examples of a single crossover between two nonsister chromatids and the gametes subsequently produced.

In (a), a single crossover occurs between two nonsister chromatids but not between the two loci; therefore, the crossover is not detected because no recombinant gametes are produced. In (b), the two loci are quite far apart and a crossover does occur between them, yielding recombinant gametes which are detectable.

Figure 7.3 Background and explantion

In his studies, Morgan investigated numerous Drosophilia mutations located on the X chromosome. When he analyzed crosses involving only one trait, he deduced the mode of X-linked inheritance. However, when he made crosses involving two X-linked genes, his results were initially puzzling. For example, female flies expressing the mutant yellow body (y) and white eyes (w) alleles were crossed with wild-type (remember wild type is dominant) males (gray bodies and red eyes). The F1 females were wild type, while the F1 males expressed both mutant traits. In the F2 generation, the vast majority of the offspring showed the expected parental phenotypes-either yellow-bodied, white-eyed flies or wild-type flies (gray-bodied, red eyed). However, the remaining flies, less than 1.0 percent, were either yellow-bodied with red eyes or gray-bodied with white eyes. It was as if the two mutant alleles had somehow separated from each other on the homolog during gamete formation in the F1 female flies. This cross is illustrated in cross A of the figure using data later complied by Sturtevant (undergrad student). (b) When Morgan studied other X-linked genes, the same basic pattern was observed, but the proportion of the unexpected F2 phenotypes differed. For example, in a cross involving the mutant white eye (w), miniature wing (m) alleles, the majority of the F2 again showed the parental phenotypes, but a much higher proportion of the offspring appeared as if the mutant genes had separated during gamete formation. This is illustrated in cross B of figure 7.3, again using data subsequently complied by Sturtevant

When the loci of two linked genes are far apart, the number of recombinant gametes approaches, but does not exceed, 50 percent. If 50 percent recombinants occur, the result is a 1:1:1:1 ratio of the four types (two parent and two recombinant gametes). What does this mean?

In this case, if 50 percent recombinants occur, transmission of two linked genes is indistinguishable from that of two unlinked, independently assorting genes. That is, the proportion of the four possible genotypes is identical, as shown in figure 7.1 (a) and (c).

These criteria are met in the three-point mapping cross from Drosophilia shown in Figure 7.8. Explanation of figure 7.8

In this cross, three X-linked recessive mutant genes-yellow body color (y), white eye color (w), and echinus eye shape (ec)-are considered. To diagram this cross, we must assume some theoretical sequence, even though we do not yet know if it is correct. In this figure, we initially assume the sequence of the three genes to be y-w-ec. If this assumption is incorrect, our analysis will demonstrate this and reveal the correct sequence. In the P1 generation, males hemizygous for all three wild-type alleles are crossed to females that are homozygous for all three recessive mutant alleles. Therefore, the P1 males are wild type with respect to body color, eye color, and eye shape. They are said to have a wild-type phenotype. The females, on the other hand, exhibit the three mutant traits- yellow body color, white eyes, and echinus eye shape. This cross produces an F1 generation consisting of females that are heterozygous at all three loci and males that, because of the Y chromosome, are hemizygous for the three mutant alleles. Phenotypically, all F1 females are wild type, while F1 males are mutant. The genotype of the F1 females fulfills the first criterion for mapping; that is, it is heterozygous at the three loci and can serve as the source of recombinant gametes generated by crossing over. -Note that because of the genotypes of the P1 parents, all three mutant alleles in the F1 female are on one homolog and all three wild-type alleles are on the other homolog. With other females, other possible arrangements are possible that could produce a heterozygous genotype. For example, a heterozygous female could have the y and ec mutant alleles on one homolog and the w allele on the other. This would occur if, in the P1 cross, one parent was yellow, echinus and the other parent was white. In this cross, the second criterion is met by virtue of the gametes formed by the F1 males. Every gamete contains either an X chromosome bearing the three mutant alleles or a Y chromosome, which is genetically inert for the three loci considered. Whichever type participates in fertilization, the genotype of the gamete produced by the F1 female will be expressed phenotypically in the F2 male and female offspring derived from it. Thus, all F1 noncrossover and crossover gametes can be detected by observing the F2 phenotypes With these two criteria met, we can now construct a chromosome map from the crosses shown in this figure. -First, we determine which F2 phenotypes correspond to the various noncrossover and crossover categories. To determine the noncrossover F2 phenotypes, we must identify individuals derived from the parental gametes formed by the F1 female. Each such gamete contains an X chromosome unaffected by crossing over. As a result of segregation, approx equal proportions of the two types of gametes and, subsequently, the F2 phenotypes, are produced. Because they derive from a heterozygote, the genotypes of the two parental gametes and the resultant F2 phenotype complement one another. For example, if one is wild type, the other is completely mutant. This is the case in the cross being considered. In other situations, if one chromosome shows one mutant allele, the second chromosome shows the other two mutant alleles, and so on. They are therefore called reciprocal classes of gametes and phenotypes. The two noncrossover phenotypes are most easily recognized because they exist in the greatest proportion. This figure shows that gametes 1 and 2 are present in the greatest numbers. Therefore, flies that express yellow, white, and echinus phenotypes and flies that are normal (or wild-type) for all three characters constitute the noncrossover category and represent 94.44 percent of the F2 offspring. The second category that can be easily detected is represented by the double-crossover phenotypes. Because of their low probability of occurrence, they must be present in the least numbers. Remember that this group represents two independent but simultaneous single-crossover events. Two reciprocal phenotypes can be identified: gamete 7, which shows the mutant traits yellow, echinus but normal eye color; and gamete 8, which shows the mutant trait white but normal body color and eye shape. Together these double-crossover phenotypes constitute only 0.06 percent of the F2 offspring. The remaining four phenotypic classes represent two categories resulting from single crossovers. Gametes 3 and 4, reciprocal phenotypes produced by single-crossover events occurring between the yellow and white loci, are equal to 1.50 percent of the F2 offspring; gametes 5 and 6, constituting 4 percent of the F2 offspring, represent the reciprocal phenotypes resulting from single-crossover events occurring between the white and echinus loci. The map distance separating the three loci can now be calculated. The distance between y and w or between w and ec is equal to the percentage of all detectable exchanges occurring between them. For any two genes under consideration, this includes all appropriate single crossovers as well as all double crossovers. The latter are included because they represent two simultaneous single crossovers. For the y and w genes, this includes gametes 3, 4, 7, and 8, totaling 1.50% + 0.06% or 1.56mu. Similarly, the distance between w and ec is equal to the percentage of offspring resulting from an exchange between these two loci: gametes 5, 6, 7, and 8, totaling 4% + 0.06+ or 4.06mu. The map of these three loci on the X chromosome is shown at the bottom of the figure.

Because the chromosome, not the gene, is the unit of transmission during meiosis, linked genes are not free to undergo independent assortment. What happens instead?

Instead, the alleles at all loci of one chromosome should, in theory, be transmitted as a unit during gamete formation. However, in many instances this does not occur. During the first meiotic prophase, when homologs are paired or synapsed, a reciprocal exchange of chromosome segments can take place. This crossing over event results in the reshuffling, or recombination, of the alleles between homologs, and it always occurs during the tetrad stage.

If complete linkage exists between two genes because of their close proximity, and organisms heterozygous at both loci are mated, a unique F2 phenotypic ratio results, which we designate the ___________________

Linkage ratio A 1:2:1 phenotypic and genotypic ratio is characteristic of complete linkage if the two organisms are heterozygous at both loci.

Morgan's student, Alfred H. Sturtevant, was the first to realize that his mentor's proposal could be used to map the sequence of linked genes. How did he realize this

Sturtevant complied data from numerous crosses made by Morgan and other geneticists involving recombination between the genes represented by the yellow, white, and miniature mutants. These data are shown in Figure 7.3: (1) yellow-white 0.5% (2)white-miniature 34.5% (3) yellow-miniature 35.4% Because the sum of (1) and (2) approximately equals (3), sturtevant suggested that the recombination frequencies between linked genes are additive. On this basis, he predicted that the order of the genes on the X chromosome is yellow-white-miniature. In arriving at this conclusion, he reasoned as follows: the yellow and white genes are apparently close to each other because the recombination frequency is low. However, both of these genes are much farther apart from miniature genes because the white-miniature and yellow-miniature combinations show larger recombination frequencies. Because miniature shows more recombination with yellow than with white (35.4 percent versus 34.5 percent), it follows that white is located between the other two genes, not outside of them. Sturtevant knew from Morgan's work that the frequency of exchange could be used as an estimate of the distance between two genes or loci along the chromosome.

T/F It is highly improbable that two randomly selected genes linked on the same chromosome will be so close to one another along the chromosome that they demonstrate complete linkage. Instead, crosses involving two such genes almost always produce a percentage of offspring resulting from recombinant gametes.

T

T/F It is possible that in a single tetrad, two, three, or more exchanges will occur between nonsister chromatids as a result of several crossover events

T

Based on the results in figure 7.2 Morgan was faced with two questions: (1) What was the source of gene separation (2) Why did the frequency of the apparent separation vary depending on the genes being studied What answers did he propose to these questions

The answer he proposed for the first question was based on his knowledge of earlier cytological observations made by F.A. janssens and others. Janssens had observed that synapsed homologous chromosomes in meiosis wrapped around each other, creating chiasmata (sing chiasma) where points of overlap are evident. Morgan proposed that chiasmata could represent points of genetic exchange. In the crosses shown in figure 7.3, morgan postulated that if an exchange occurs during gamete formation between the mutant genes on the two X chromosomes of the F1 females, the unique phenotype will occur. -He suggested that such exchanges led to recombinant gametes in both the yellow-white cross and the white-miniature cross, in contrast to the parental gametes that have undergone no exchange. -On the basis of this and other experiments, Morgan concluded that linked genes exist in a linear order along the chromosome and that a variable amount of exchange occurs between any two genes during gamete formation. To answer the second question, Morgan proposed that two genes located relatively close to each other along a chromosome are less likely to have a chiasma form between them than if the two genes are farther apart on the chromosome. Therefore, the closer two genes are, the less likely a genetic exchange will occur between them. Morgan was the first to propose the term crossing over to describe the physical exchange leading to recombination.

Figure 7.1 a) Results of gamete formation when two heterozygous genes are on two different pairs of chromosomes. Independent assortment: Two genes on two different homologous pairs of chromosomes. Note in this figure and in the following two, members of homologous pairs of chromosomes are shown in two different colors.

These are the results of independent assortment of two pairs of chromosomes, each containing one heterozygous gene pair. No linkage is exhibited. When a large number of meiotic events are observer, four genetically different gametes are formed in equal proportions, and each contains a different combination of alleles of the two genes.

The correlation between the distance between two loci on a single chromosome and the frequency at which those loci crossover allows what

This correlation allows us to construct chromosome maps, which give the relative locations of genes on chromosomes.

To illustrate this linkage ratio, we are going to look at Figure 7.2: A cross involving two genes located on the same chromosome and demonstrating complete linkage. Also goes over a new notation

This cross involves the closely linked, recessive, mutant genes heavy wing vein (hv) and brown eye (bw) in Drosophilia melanogaster. The normal, wild-type alleles hv+ and bw+ are both dominant and result in thin wing veins and red eyes respectively. In this cross, flies with normal thin wing veins and mutant brown eyes are mated to flies with mutant heavy wing veins and normal red eyes. If we extend the system of genetic symbols learned in chapter 4, linked genes are represented by placing their allele designations above and below a single or double horizontal line. Those above the line are located at loci on one homolog, and those below are located at the homologous loci on the other homolog. Thus, we represent the P1 generation as follows: P1: hv+ bw/hv+ bw x hv bw+/hv bw+ thin, brown heavy, red In the F1 generation, each fly receives one chromosome of each pair from each parent. All flies are heterozygous for both gene pairs and exhibit the dominant traits of thin wing veins and red eyes: F1: hv+ bw/hv bw+ thin, red As show in (a) when the F1 generation is interbred, each F1 individual forms only parental gametes because of complete linkage. After fertilization, the F2 generation is produced in a 1:2:1 phenotypic and genotypic ratio. Such a 1:2:1 ratio is characteristic of complete linkage. (b) demonstrates the results of a testcross with the F1 flies. Such a cross produces a 1:1 ratio of thin, brown and heavy, red flies. Had the genes controlling these traits been incompletely linked or located on separate autosomes, the testcross would have produced four phenotypes rather than two.

In the preceding example, the sequence (order) of the three genes along the chromosome was assumed to be y-w-ec. Our analysis shows this sequence to be consistent with the data. However, in most mapping experiments the gene sequence is not known, and this constitutes another variable in the analysis. In our example, had the gene sequence been unknown, it could have been determined using a straight-forward method. What is this method

This method is based on the fact that there are only three possible arrangements, each containing one of the three genes between the other two (I) w-y-ec (y in the middle) (II) y-ec-w (ec in the middle) (III) y-w-ec (w in the middle) Use the following steps during your analysis to determine the gene order: 1. Assuming any one of the three orders, first determine the arrangement of alleles along each homolog of the heterozygous parent giving rise to the noncrossover and crossover gametes (the F1 female in our example) 2. Determine whether a double-crossover event occurring within that arrangement will produce the observed double-crossover phenotypes. Remember that these phenotypes occur least frequently and are easily identified. 3. If this order does not produce the predicted phenotypes, try each of the other two orders. one must work! In the figure posted, the above steps are applied to each of the three possible arrangements. A full analysis can proceed as follows: 1. Assuming that y is between w and ec, the arrangement I of alleles along the homologs of the F1 heterozygote is w y ec / w+ y+ ec+ We know this because of the way in which the P1 generation was crossed: The P1 female contributes an X chromosome bearing the w, y, and ec alleles, while the P1 male contributes an X chromosome bearing the w+, y+, and ec+ alleles. 2. A double crossover within that arrangement yields the following gametes w y+ ec and w+ y ec+ Following fertilization, if y is in the middle, the F2 double-crossover phenotypes will correspond to these gametic genotypes, yielding offspring that express the white, echinus phenotype and offspring that express the yellow phenotype. Instead, determination of the actual double-crossover phenotypes reveals them to be yellow, echinus flies and white flies. Therefore, our assumed order is incorrect. 3. Repeat with other possible arrangements.

___________________________ serves as the basis of determining the distance between genes during mapping

crossing over

Crossover frequency between linked genes during gamete formation is proportional to the ______________________, providing the experimental basis for ________________________________________

distance between genes mapping the location of genes relative to one another along the chromosome

The study of single crossovers between two linked genes provides the basis of determining the distance between them. However, when many linked genes are studied, their sequence along the chromosome is more difficult to determine. Fortunately, the discovery that multiple exchanges occur between the chromatids of a tetrad has facilitated the process of producing more extensive chromosome maps. When three or more linked genes are investigated simultaneously, it is possible to determine first the ___________ and then the _______

first the sequence of the genes and then the distances between them.

Genes that are part of the same chromosome are said to be _____________ and to demonstrate ______________ in genetic crosses

linked linkage

Early investigations by geneticts showed that certain genes segregate as if they were somehow joined or linked together. Further investigations showed that such genes are

part of the same chromosome and may indeed be transmitted as a single unit.

The frequency of crossing over between any two loci on a single chromosome is ______________________ to the distance between them

proportional. Therefore, depending on which loci are studied, the percentage of recombinant gametes varies.

T/F Complete linkage is usually observed only when genes are very close together and the number of progeny is relatively small

t

T/F most chromosomes contain a very large number of genes

t

The percentage of offspring resulting from recombinant gametes is variable and depends on

the distance between the two genes along the chromosome.