ay in Investigation of Genetic Mutation Linkage in Caenorhabditis elegans
Introduction:
Caenorhabditis elegans, or C. elegans, is the specimen that was used in this experiment. C. elegans is a commonly used organism that has been used in many experiments because it is a model organism. They are cheap, easy to grow in laboratories, small (adults are about 1 millimeter in length), and have a relatively short lifecycle. In the wild, they live in soil, while in laboratories, they live in petri dishes filled with agar and feed on Escherichia coli bacteria. Adults have only around 800 somatic cells while still having most major differentiated tissue. (Hartman & Caudle, 2003)
The body wall is of C. elegans has a covering of a collagen-like cuticle that goes through a series of molts as the nematode ages. It is able to move through whatever medium it is in because C. elegans has two muscle strips located on the bottom side of the nematode. They muscle strips move in concert producing a sinusoidal wave motion. There are two concentric rings of sensory organs arranged around the mouth. These organs include chemoreceptors and mechanoreceptors. These receptors allow the nematode to detect certain chemicals and physical stimuli and will turn and go in the opposite direction upon exposure to these various agitations. (Hartman & Caudle, 2003)
C. elegans has two different sexual types: a self-fertilizing hermaphrodite and male. The wild-type phenotype of the nematode is the self-fertilizing hermaphrodite. The two different sexes can be easily distinguished between one another. The hermaphrodites are typical larger and symmetrical on both ends of the nematode. The males are usually smaller and have a “fan” like structure on the posterior end of the nematode. Males also occur at a much lower frequency than the self-fertilizing hermaphrodites. Males are produced by hermaphrodites due to meiotic nondisjunction at a rate of .01%. Hermaphrodites and males can mate together to produce a progeny of half males and half hermaphrodites. As C. elegans matures, it will undergo four different molts (L1, L2, L3, and L4). The four different larval molts help identify how old an individual may be. L4 larvae can be distinguished by a crescent shaped structure near the middle of the body, by the vulva. (Hartman & Caudle, 2003)
This experiment looked at three different mutations of C. elegans: dumpy (dpy), uncoordinated (unc), and dumpy and uncoordinated (dpyunc). The dumpy mutation was described as an individual that is much shorter and wider than the average wild-type hermaphrodite. Also, the dumpy mutation could be on two loci of the gene and is described as either dpy-1 or dpy-11. The uncoordinated mutation was found in individuals who do not move or move very slowly. This is tested by disturbing the petri dish and examining if an individual reacts to the disturbance. The uncoordinated mutation is expressed at one locus and described as unc- 32. The dumpy and uncoordinated mutation was a combination of both the dumpy and the uncoordinated mutations. This can be described as either dpy-1 unc-32 or dpy-11 unc-32.
The reason for this experiment was to determine whether if the dumpy, uncoordinated, and dumpy and uncoordinated mutations were linked or unlinked autosomal genes. The null hypothesis was that the dumpy and uncoordinated mutations are unlinked. In the F1 generation, all of the progeny is expected to be wildtype. The F2 generation would yield a phenotypic ration of 9:3:3:1 (wild-type, dmy, unc, dmyunc). The alternative hypothesis would be that the dumpy and uncoordinated mutations were linked on an autosomal gene. The F1 generation would all be 100% wild-type. The F2 generation would yield a progeny that made the parental phenotype being more prominent than the recombinant.
Methods:
Before any specific animal was captured and transferred to a new petri dish, the transferring technique had to be perfected. This technique involved scraping Escherichia coli (E. coli, the food in the petri dish for C. elegans) and then pressing on a nematode with a small pick. This caused an individual to adhere to the bottom of the pick. Once adhered, the pick was then scraped on the ager in a new petri dish, thus effectively transferring the nematode. The pick was then sterilized using an open flame after every attempt to transfer an individual.
On the first day, five, young, wild-type males located from a male only petri dish and transferred (as the technique described above) to a new host petri. Next, three mutant L4 larval stage hermaphrodites (dpy-1, unc-32) were placed in the same dish as the original. Then, another petri dish was filled with another five wild-type males and 3 mutant L4 larval stage hermaphrodites. This time the mutation was dpy-11, unc-32. After the eight nematodes were placed in their respective new agar- and-E. coli-filled petri dish, a lid was placed on it and was wrapped in a flexible sheet in order to seal the dish to prevent contamination. The dish was also labeled appropriately in order to ensure a mix-up would not transpire. There were two crosses in order to determine if the different gene locus would have an affect on the subsequent progeny. Each cross had it’s own petri dish and was labeled “Cross 1A” and “Cross 1B” respectively.
On the third day, both petri dishes were examined to see if the first day crosses were successful. The individuals that made were in the two petri dishes on that day constituted the F1 generation. Both crosses were seen to have wild-type male and dumpy/uncoordinated hermaphrodite individuals. After observing all of the individuals in the petri dish labeled “Cross 1A,” one wild-type L4 hermaphrodite was placed in a new dish, labeled “Cross 2A.” The same was done in the second dish labeled “Cross 2B.” The wild-type L4 hermaphrodite was placed in the new petri dish labeled “Cross 2B.” The two new dishes were wrapped in the sheet similar to that of the first day in order to prevent contamination.
On the seventh and last day, all of the petri dishes that housed the crossed were observed and individuals were accounted for. Cross 1A and 2A were deemed unsuccessful due to a lack of C. elegans in the dish. A spare sample F2 dish was provided. Cross 1B and 2B were successful due to the abundance of individuals on the dish. The individuals on the sample dishes and Crosses 1B and 2B were accounted for by using a grid that was drawn on the underside of the dish. The lines of the grid could be seen through the agar in the petri dish and made counting and determining the phenotype of the individuals in the dish very easy.
Once note had been taken of the phenotype in every petri dish, a chi-squared test would be ran. This was the test used to determine the expected values of the various phenotypes (wild-type, dmy, unc, and dmyunc) and compare those numbers to the numbers that were observed as a result of the previous. The chi-squared equation also determined whether or not the null hypothesis would be accepted or rejected.
Results:
For the first cross, a wild-type male was crossed with a dmy-1 unc-32 mutant hermaphrodite. It was expected that the F1 generation would yield all wild-type hermaphrodites and males. It was observed from that cross that dmyunc and wild-type hermaphrodites, as well as, wild-type males were yielded in the F1 generation (Figure 1.). From the F1 generation, a wild-type hermaphrodite was taken and it was expected to yield a progeny with a ratio of 9 wild-type:3 dumpy:3 uncoordinated:1 dumpy and uncoordinated. There was a total of 74 individuals observed in the progeny, which translated to 41.625 wild-type, 13.875 dumpy, 13.875 uncoordinated, and 4.625 dumpy and uncoordinated nematodes (Table 1.). When the F2 generation was checked, there were no nematodes to be counted. In this event, another sample of the same cross was examined. That F2 generation yielded 46 wild-type, 17 dumpy, 9 uncoordinated, and 2 dumpy and uncoordinated (Table 1.). The chi-squared test for this cross gave a χ^2-value of 4.637 and 3 degrees of freedom (Table 1.). The critical value for the cross was .5<p-value<.2 (Table 1.).
For the second cross, a wild-type male was crossed with dmy-11 unc-32 mutant hermaphrodite. It was expected that the F1 generation would yield a progeny of all wild-type hermaphrodites and males. Mutant and wild-type hermaphrodites and wild-type males were observed in the F1 generation (Figure 2.). From this F1 generation, a wild-type hermaphrodite was taken and its expected progeny in the F2 generation was 9 wild-type:3 dumpy:3 uncoordinated: 1 dumpy and uncoordinated. There were 65 total nematodes in the F2 generation of the second cross. This translated to 36.56 wild-type, 12.188 dumpy, 12.188 uncoordinated, and 4.0625 dumpy and uncoordinated individuals (Table 2.). The asexual reproduction of the hermaphrodite placed in the dish yielded a progeny; a sample dish was unnecessary for the second cross, unlike cross 1. The F2 generation consisted of 25 wild-type, 23 dumpy, 13 uncoordinated, and 4 dumpy and uncoordinated individuals (Table 1.). The chi-squared test gave a χ^2-value of 13.3031, with 3 degrees of freedom. The critical value for this cross was <.01.
Because of the subsequent p-values, the cross 1 null hypothesis was not rejected, but the cross 2 null hypothesis was rejected.
Figure 1. Expected and Observed Phenotypes when Mating dmy-1 unc-32 with wild-type male (given as sample, not own data)
Shown above the cross of the wild-type male and the dumpy uncoordinated hermaphrodite. The expected and observed phenotypes are labeled on the F1 generation. The observed F1 phenotypes had a branch shown from it. This indicated that an F1 wild-type hermaphrodite was self-reproduced in order to create the F2. The expected and observed results are shown branching from the wild-type hermaphrodite from the F1 generation. The results from cross between the wild-type male and the dumpy uncoordinated hermaphrodite constituted Cross 1A and the self-reproducing F1 wild-type hermaphrodite constituted Cross 2A.
Figure 2. Expected and Observed Phenotypes when Mating dmy-11 unc-32 with wild-type male
Shown above the cross of the wild-type male and the dumpy-11 uncoordinated-32 hermaphrodite. The expected and observed phenotypes are labeled on the F1 generation. The observed F1 phenotypes had a branch shown from it. This indicated that an F1 wild-type hermaphrodite was self-reproduced in order to create the F2. The expected and observed results are shown branching from the wild-type hermaphrodite from the F1 generation. The results from cross between the wild-type male and the dumpy uncoordinated hermaphrodite constituted Cross 1B and the self-reproducing F1 wild-type hermaphrodite constituted Cross 2B.
Table 1. Observed Phenotypes in F2 Generation of Wild-type male X dumpy-1 uncoordinated-32 hermaphrodite
The “Observed Number of Offspring” column showed the number of each phenotype observed in the F2 generation. The “Expected Number of Offspring” column showed the number of each phenotype to be expected given the total number of individuals in the progeny, which was 74 total individuals. The expected number was a simple 9 wild-type:3 dmy:3 unc:1 dmyunc ratio.
Phenotype Observed Number of Offspring Expected Number of Offspring
Wild-type 46 41.625
Dumpy 17 13.875
Uncoordinated 9 13.875
Dumpy and Uncoordinated 2 4.625
χ^2=∑▒(observed-expected)^2/observed
χ^2=(46-41.625)^2/41.625+(17-13.875)^2/13.875+(9-13.875)^2/13.875+(2-4.625)^2/4.625=.4598+.7038+1.713+1.490=4.367
χ^2=4.367;Degrees of Freedom=3;critical value: .5<p-value<.2
Table 2. Observed Phenotypes in F2 Generation of Wild-type male X dumpy-11 uncoordinated-32 hermaphrodite
The “Observed Number of Offspring” column showed the number of each phenotype observed in the F2 generation. The “Expected Number of Offspring” column showed the number of each phenotype to be expected given the number of individuals in the progeny, which was 65. The expected number was a simple 9 wild-type:3 dmy:3 unc:1 dmyunc ratio.
Phenotype Observed Number of Offsrping Expected Number of Offsrping
Wild-type 25 36.563
Dumpy 23 12.188
Uncoordinated 13 12.188
Dumpy and Uncoordinated 4 4.0625
χ^2=∑▒(observed-expected)^2/observed
χ^2=(25-36.563)^2/36.563+(23-12.188)^2/12.188+(13-12.188)^2/12.188+(4-4.0625)^2/4.0625=3.657+9.591+.0541+.0010
χ^2=13.3031;Degrees of Freedom=3;critical value:<.01
Table 3. Genotypes and Phenotypes of the Parents, F1 Offspring, and F2 Offsrping
The parent, F1 offspring, and F2 offspring genotype and phenotype showed below. ++ indicated wild-type and +dpy or +unc indicated heterozygous.
Cross 1 Cross 2
Parent Genotype (++)/(++) x dpyunc/dpyunc (++)/(++) x dpyunc/dpyunc
Parent Phenotype Wild-type x dumpy and uncoordinated Wild-type x dumpy and uncoordinated
F1 Offspring Genotype (+dpy)/(+unc);(+dpy)/(+unc);dpyunc/dpyunc (+dpy)/(+unc);(+dpy)/(+unc);dpyunc/dpyunc
F1 Offspring Phenotype Wild-type male, wild-type hermaphrodite, and dumpy and uncoordinated mutant Wild-type male, wild-type hermaphrodite, and dumpy and uncoordinated mutant
F2 Offspring Genotype (+dpy)/(+unc);dpydpy/(+unc);(+dpy)/uncunc;dpydpy/uncunc (+dpy)/(+unc);dpydpy/(+unc);(+dpy)/uncunc;dpydpy/uncunc
F2 Offspring Phenotype Wild-type male and hermaphrodite, dumpy, uncoordinated, and dumpy and uncoordinated Wild-type male and hermaphrodite, dumpy, uncoordinated, and dumpy and uncoordinated
Discussion:
The purpose of this experiment was to determine if the dumpy-1 and uncoordinated-32 genes and the dumpy-11 and the uncoordinated-32 genes were located on the same chromosome. If those genes were located on the same chromosome, that would indicate and instance of gene linkage. The null hypotheses for both of these crosses were that the dumpy (1 and 11) and the uncoordinated-32 gene were not linked. Because cross 1’s null hypothesis was not rejected, it is concluded that there is no linkage between dumpy-1 and uncoordinated-32 genes. This was determined by finding where the critical value falls on a chi-square chart. Since it was between .5 and .2, the hypothesis was accepted (Table 1.). Inversely, because cross 2’s null hypothesis was rejected, it is concluded that there is linkage between dumpy-11 and uncoordinated-32 genes. This means that those genes are located on the same chromosome. As in cross 1, the critical value for cross 2 was placed on a chi square chart. Because it was not on chart (<.01, Table 2.), the null hypothesis was rejected.
Although the numbers reflected the data accurately, the dumpy-1 and uncoordinated-32 genes are located on the same chromosome (Brenner, 1973). Dumpy-1 and uncoordinated-32 are located on the third linkage group. Dumpy-11 is not located on the same linkage group. Since there are more than one gene that code for the same phenotype, it would be possible for dumpy-11 to be paired with a different uncoordinated that would lead to gene linkage (Brenner, 1973).
Because the data did suggested that dumpy-1 and uncoordinated-32 genes had no gene linkage and dumpy-11 and uncoordinated-32 genes, an error must have occurred. Errors could have happened at a number points throughout the experiment. When nematodes were being transferred from one petri dish to another, the correct sex or larval stage might not have been transferred. For example, if a male was transported to a new petri dish from the F1 generation, it would not be able to reproduce asexually and would lead to a lack of progeny in the F2 generation (potentially the error made during cross 1). An error could have also occurred while counting the different phenotypes of C. elegans. That would mean that the correct number of individuals were present, but were not appropriately accounted for. For this experiment to be executed effectively, these errors would need to be limited or not happen at all.
Literature Cited:
Works Cited
Brenner, S. (1973). The Genetics of Caenorhabditis Elegans. Medical Research Council Laboratory of Molecular Biology , 72-94.
Hartman, & Caudle. (2003). TCU Genetics lab manual.