Sex Linkage and Recombination (Practical 1)

An experiment to determine sex linkage or independent assortment of three different alleles of Drosophila melanogaster and the map of the corresponding chromosome.

 

NAME of STUDENT

Stock: #4

DATE

 

Purpose:

To learn about Drosophila melanogaster and some of it’s mutations. To study sex linkage, recombination and independent assortment. To offer possible genetic maps for the traits studied if the genes corresponding to the traits are on the same chromosome and show linkage[1].

Introduction, theories, and background:

Drosophila has been used as a model organism for research for almost a century, and today, several thousand scientists are working on many different aspects of the fruit fly. So much is already known about it that it is easy to handle; it is a small insect, with a short life cycle of just two weeks, and is affordable and easy to keep large numbers. Mutant flies, with defects in any of several thousand genes are available, and the entire genome has recently been sequenced.

The Drosophila egg is about half a millimeter long. It takes about one day after fertilization for the embryo to develop and hatch into a worm-like larva. The larva eats and grows continuously, molting one day, two days, and four days after hatching (first, second and third instars). After two days as a third instar larva, it molts one more time to form an immobile pupa. Over the next four days, the body is completely remodeled to give the adult winged form, which then hatches from the pupal case and is fertile after another day. (timing is for 25°C; at 18°, development takes twice as long.)

Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes2, 3, and 4. The fourth chromosome is quite tiny and rarely heard from.

It is important to note that for the most part of these experiments, a virgin female fly is crossed with a male. The reason for this is that female fruit flies store sperm and fertilize their eggs with it over time. Virgin flies are needed to make sure that that the crosses are being appropriately made with the females using the desired sperms to fertilize their eggs. Female Drosophila are considered virgin eight to ten hours after they hatch from their pupa because during that time they are not receptive to male companionship and mating. Virginity of the male flies is irrelevant. Female flies that are virgin are indicated by the symbol .

In this experiment a stock of mutant flies with unknown mutations were given to each student. The goal of the experiment was to first identify the mutations and then through proper crossing of the flies, and analyzing the results of the crosses, find out whether the traits were sex linked, or autosomal, whether the genes for those traits are on the same or different chromosomes, and to construct a possible genetic map of the chromosome on which the genes for the traits are located.

The mutations present in stock number four were the following:

a) The mutant flies had lighter than normal body colors (yellow body color).

b) The mutants had unusual bristles on their back; the mutated bristles were shorter and crooked.

c) The mutant flies had wings that lacked the normal cross bridges or veins in them.

d) The mutants had brighter than normal eye colors.

The first three of these four mutations were chosen to be investigated. The reason for choosing the first three and not the fourth one was the relative ease of distinguishing the mutants from the normal, wild type flies with each of the mutations.

Throughout the experiment, the flies are labels as following

a) The files with normal body color are labeled “c+”, and the mutants are labeled as “c”.

b) The files with normal bristles are labeled “b+”, and the mutants are labeled as “b”.

c) The files with normal wing veins are labeled “v+”, and the mutants are labeled as “v”.

the first part of the experiment involves determining (or ruling out) the possibility of the sex-linkage of the traits. Two crosses are made: a) a normal/wild type virgin female with a mutant male, and b) a mutant virgin female with a normal male. Any traits that are transferred exclusively from mothers to sons are considered to be sex-linked. The reason for this is that male flies receive their only X-chromosome from their mothers. The female progeny will have wild type phenotypes because they also received a normal X-chromosome from their fathers which overrides the mutant allele. The first crosses are summarized as follows:

Cross #1=>	Females	Males	cross #2=>	Females	Males
	bvc/bvc	b+v+c+/ b+v+c+	AND	b+v+c+/ b+v+c+	bvc/bvc

In this step of the experiment, conclusions regarding the sex linkage of the traits are made.

The second part of the experiment will involve looking at independent assortment and recombination. Mendel’s principle of independent assortment states that “segregation of the members of any pair of alleles is independent of the segregation of other pairs in the formation of reproductive cells” (Hartl 101). What this means is that the genes encoding for say lighter body color segregate independently and that its assortment has nothing to do with the assortment of say, gene for mutant bristles. The genes that show independent assortment are called to be unlinked. There are, however, many genes that are linked together and would usually manifest themselves together. This is caused by a phenomenon called crossing over which is physical exchange of pieces of chromosome which contain homologous genes. Crossing over occurs during prophase I of meiosis. If two genes encoding for two different traits are very close together on the chromosome, they would usually move and be exchanged together (they are linked together) and would show up together in the phenotype of the individual. Based on the frequency of this happening, that is, based on seeing the frequency of two genes appearing together, one can construct a map of the area of the chromosome which contains those genes. This is done by tabulating the number of flies with the different possibilities of recombination of the genes under study (see table 1), calculating the percentage of the frequency of those recombinations and converting those percentages to map unites. The order of these genes can also be determined by looking at these results, and by looking at the lowest frequency of the recombinants which constitute the double crossover.

Materials and Methods:

Materials used in this experiment included: a stock of mutant flies (stock number 4), a stock of wild-type flies, plastic vials with plugs, Drosophila food (dehydrated food with food coloring which turned blue upon re-hydration), incubators with constant temperature at 22.1 degrees Centigrade. Also used were dissection microscopes with fiber optic instruments providing source of light, small painting brushes and an index card for handling the anesthetized flies, carbon-dioxide pads and CO2 gas as anesthetizing agent.

All the vials of flies were clearly labeled with the type of flies crossed, the initials of whom they belong to and the date of the cross. No vials of flies were kept in the incubator for longer than one month. If after one month the flies were still needed, they were transferred to a new vial of food and were properly labeled.

In order to obtain a significant number of F2 flies in a relatively short amount of time, several crosses were set up in different vials at the same time. Generally for each cross, three virgin female and five male flies were used (if more virgin flies were available, the ratio was increased).

The process of isolating virgin flies entailed emptying the vial from which we want to isolate virgins, waiting for new flies to hatch out of the pupa and examining them for their gender. For the experiment to be accurate, female flies which had hatched more than eight hours before examination with male flies, were considered not to be virgin.

Just as it was important to isolate virgin females for the experiments, it was important to make sure that parents of the crosses were removed before ten days after the initiation of the crosses in order to make sure that the parental generation did not mate with the F1 generation contaminating the results.

Results and Calculations:

The results of the first cross (true breading[2] wild type with true breading mutant stock number 4) showed that all four of the mutations of the mutant stock were sex linked. This is because in the cross where virgin female mutants were crossed with male wild type flies, all of the males of the F1 generation had all four of the mutations and none of the females of the F1 generation had any of the mutations. This shows that all the traits were transferred from the mothers to the sons only, showing that all four traits were sex-linked and are located on the X-chromosome. As mentioned above, out of the four mutations, tree of them were chosen for an investigation of degree of recombination between the genes.

The result of the cross of virgin female wild-type flies with mutant males was that all the progeny (male and female) were all wild type, this is because the X-chromosomes of the mothers would manifest themselves over the one X-chromosome of the mutant fathers.

For the second part of the experiment (second cross), the F1 progeny of the cross between virgin female mutants and male wild types were crossed together without needing to isolate virgin females. This is because all the male progeny of this cross had all of the mutations (were homozygous recessive for all of the mutations) and this is what we needed to cross our F1 females to. The following illustrates the results of the first cross:

Parents	Females	Males	=> F1 progeny	Females	Males
	bvc/bvc	b v+c+	AND	b v+c+ (all females wild type)	bvc (all males mutants)

The results of the second cross are illustrated in the following table (table #1):

Table 1:

Based on the table, we can see that there are clearly more parental types (numbers 1 and 2) than the recombinants, this is expected since crossing over of the genes under investigation does not occur all the time and since no crossing over of the X-chromosome occurs in the male Drosophila. We can also see that the frequency of progeny with double cross over (numbers 7 and 8) are the lowest because the probability of occurrence of double cross over is less than the probability of single gross over (numbers 3-6) simply because two acts of crossing over and exchange of physical pieces of chromosome need to be done. Based on the results of table one, we can also figure out the order in which the genes are in. this is done by looking at the type of progeny with the fewest members. This category would contain the progeny with the double crossovers this means that among the three traits that we chose (b, v, and c), two of them should have moved together and third one that moved by itself is the one gene that is in the middle. The correct order of the genes is illustrated in the table as bvc.

Another observation that we can make from the table is regarding linkage or whether or not two separate genes are linked together and to what degree. We can see that the gene coding for body color (c) and that coding for the veins of the wing (v) are more linked than the genes coding for body color and those coding for bristles (b). This is because there are more progeny that have mutated body color and veins of the wing, than there are progeny that have both mutations for body color and bristles together. This would mean that the genes v and c are closer together than v and b or b and c.

The next step in the experiment is to calculate the distances between these genes and to propose possible maps of the chromosome region containing these three genes. To do this we have to do the following calculations:

* Frequency of recombination/crossover between bristles mutation and vain mutation: 32.5%+3.5%=36%, this means that the distance between these two genes on the chromosome is 36 map units or centimorgans. (This is because the category of flies that had 32.5 percent of the flies and the one that had 3.5 percent of the flies, both had recombination between these two genes (b and v)).
* Frequency of recombination/crossover between bristles mutation and body color mutation: 32.5%+6%=38.5%, this means that the distance between these two genes on the chromosome is 38.5 map units or centimorgans.
* Frequency of recombination/crossover between body color mutation and vein mutation: 6%+3.5%=9.5%, this means that the distance between these two genes on the chromosome is 9.5 map units or centimorgans.

Based on these observations and calculations we can propose two possible genetic linkage maps of the section of X-chromosome containing the three genes as follows:

OR

Another phenomenon that we can see from the data in the table has to do with the fact that the number of double crossovers that we see is less than the number that we expected to see. We expect to see 38.5% X9.5%=3.65% of the progeny to have gone through double crossovers. But the actual percentage of flies that have gone through double crossovers for these genes is 3.5%. The reason for this deficiency is a phenomenon called chromosome interference “in which crossing-over in one region of a chromosome reduces the probability of a second crossover in a nearby region” (Hartle 194). From this information we can calculate the coefficient of coincidence which is the observed number of double-recombinant chromosomes divided by the expected numbers: 3.5/3.65=.958; and from this interference is calculated: i=1-.958=.042.

Discussion

First of all it is important to point out the fact that on the proposed maps, the distance between b and c genes do not add up to 38.5 as it was calculated. This can be explained by noting the possibility that some experimental error might have caused an inaccuracy in the crossing, handling or counting the flies and that the distance between the c-v and v-b genes are actually lass than calculated. Another possibility is that one or two of these genes maybe located near the centromere or telomere area of the chromosome and that affects the linkage of the genes. Any error in the results can be attributed to one of several factors. First of all in the laboratory, there were many free-flying fruit flies that have mistakenly been released in the lab by other students and these free flying fruit flies could have possibly entered the vials containing the crosses and contaminated them (even though utmost effort was made to keep all the vials plugged at all times that they were not in use). Another possibility is that while separating the flies based on their mutations, it is possible that several flies during the several sessions of counting became entangled in the brush and were transferred to the wrong quadrant of the CO2 pad and caused mistakes in the counts.

Another significant source of error could be attributed to the fact that since there was no time to count the F2 flies as they hatched, most of the F2s were transferred to an empty vial, terminally anesthetized, and placed in a freezer to be counted later. Freezing had two noticeable effects on the flies: one was that since they were dehydrated in the freezer, some of their body colors became darker than usual and flies with a lighter body color might have been identified as having wild type body color; to solve this problem, any fly that there was some doubt about it’s body color or that had some other problem that caused uncertainty in terms of identifying its characteristics, was discarded and was not counted. Another effect that freezing the flies had was that some of the flies became stuck together and when attempt was made to separate them, the now more brittle wings (because of freezing) became shattered rendered those flies impossible to identify in terms of their wing vein alleles; again, such flies were mostly excluded from the count.

Another possible source of error could be that even though effort was made to remove all the F2 as soon as they hatched, to prevent formation of F3 flies, it is possible that due to the labs being closed for holidays or other reasons, few F3 were formed and some of the these flies affected the numbers reported in table 1.

Summery:

The purpose of this experiment was to learn about Drosophila melanogaster and some of its mutations, to study sex linkage, recombination and independent assortment. To see whether the traits under investigation showed sex linkage or independent assortment, and to offer possible genetic maps for the traits studied if the genes corresponding to the traits are on the same chromosome.

This was done by doing two sets of crosses. The first sets of crosses were reciprocal crosses between the all-mutant flies and all wild-type flies to obtain F1 generation flies. At this point, any sex-linked traits were identified (all four of the traits in the mutant stock number 4 which was under investigation in this experiment were sex-linked.)

The second cross was done between the females of F1 generation and the mutant males (which were also F1­s because of sex linkage) to obtain F2 flies. The F2 flies were counted and categorized into the possible eight categories of phenotypes (three traits were under investigation: 2^3=8), and based on the number of flies in each category, and calculating the distances between the genes corresponding to each trait, a genetic map pertaining to the traits were proposed.

Bibliography

Hartl, Daniel L., Elizabeth W. Jones. Genetics Analysis of Genes and Genomes, Fifth Ed. Jones and Bartlett Publishers, 2000.

[1] When genes are very close to each other on the same chromosome and are transmitted together to the progeny most or all the time, these genes are said to show linkage.

[2] True breeding means that the flies were homozygous for the alleles and so that all the progeny from those flies crossed with each other would have the exact same alleles as their parents.