Lab 3: Linkage Testing, Backcrossing and Mapping
Confirm phenotype of the backcrossed strain; specifically, your backcrossed mutant should breed true meaning that it should produce again a homozygote for the mutation Genotype dpy/dpy; mutant phenotype (the crosses are the following (P: dpy/dpy x +/+, F1: dpy/+ self fertilizes to produce F2: +/+, dpy/+ and dpy/dpy) What percentage of the mutagenized chromosomes have you replaced with wild-type, unmutagenized chromosomes?
To determine in what chromosome or linkage group your dpy mutation is located record the number of dpy, unc and dpy unc mutants by examining, scoring the phenotype and removing that animal from the plate for each of your five crosses with reference unc mutations. If you see no, or significantly fewer than one in seven double mutants dpy unc, it could be taken as an indication that your dpy mutation is on the same chromosome or linkage group as that unc mutation. Remember that your mutation will only reside on a single chromosome; therefore, the ideal result is that your mutation segregates independently with respect to 4 of the 5 reference mutations.
Assuming that you have determined the linkage group on which your mutation resides, you will continue working with that strain only where you suspect that the two mutations are one the same chromosome. To calculate distance between the two markers pick 5 Dpy mutants to each of 5 plates. As described above, most of these are wild type for the reference mutation (Unc) r+/r+, but hopefully some are heterozygous for the reference mutation (Unc) rm/r+m.
3-4 days later
Mapping: screen your 5 plates for double mutants. Pick 3 such putative double mutants to separate plates.
It is not unusual to have series of mutations that confer similar phenotypes and also map to a identical or similar location on a chromosome. In such cases, the practicing geneticist performs a complementation test to determine if the mutations are allelic (that is, in the same gene) or non-allelic. The specifics of strain construction vary depending on the experimental organism. However, the basic strategy in all cases is to construct a trans double heterozygote and then examine the phenotype of this organism. As is apparent from consultation of your genetics textbook, a wild-type phenotype indicates that the two mutations complement one another and are therefore in different genes. Conversely, a mutant phenotype suggests the mutations are allelic to one another (that is, they fail to complement). We will construct double hets as follows:
Cross #1: unknown Dpy (dpy-u/dpy-u) x N2 males (+/+) yields dpy-u/+ progeny
Cross #2: dpy-u/+ males x known dpy hermaphrodites (dpy-k/dpy-k) yields dpy-u +/+ dpy-k progeny
The phenotype of the trans double heterozygote is then scored.
In this experiment, you will determine the identity of your “unknown” Dpy mutation and place these mutations in trans to five mutations in different Dpy genes that have been previously mapped (i.e., known mutations). We are attempting to determine if, our “unknown” Dpy mutations are allelic to any of the five mutations.
3-4 days after lab:
Cross your unknown Dpy times N2 males by placing three to five L4 Dpy's on a plate in the presence of 5 to 8 N2 males. It is essential that the only wild-type animals present on this plate are males (because you desire +/dpy males from this plate, and not the +/+ males that would result of wild-type hermaphrodites were included on the plate).
Incubate the worms at 23°C until next lab period.
C. elegans General Information
Tools and Techniques
Lab 1: Welcome to C. elegans and Mutant Hunt
Lab 2: Linkage Test and Backcross
Lab 4: Mapping Part 2
Lab 5: Score!