BISC 219/2009: Mod 1 Lab 3

Lab 3: Linkage Testing and Mapping

Linkage Testing
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 four crosses with reference unc mutations. If you see no double mutants (d u/d u), 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 3 of the 4 reference mutations. If you didn't get 4 successful reference crosses, score those you have and see your instructor for help. Can you determine the linkage group of your dpy mutation?

Mapping
Assuming that you have determined the linkage group on which your mutation resides, you will continue working with that strain only. You now suspect that the two mutations are on the same chromosome. To begin to calculate distance between the two markers separate 5 dpy mutants to 5 individual plates. As described above, most of these individuals are wild type for the Unc mutation (+ d/+ d), but hopefully some are heterozygous for the Unc mutation (u d/+ d). How does this happen?

1. Separate 5 dpy mutants to 5 individual plates
2. Label each of these plates with "Mapping 1" and your initials and the date with your PURPLE Sharpie.
   
3. Incubate the plates at 23°C for 3 days
   

3 days later

1. 'Screen your 5 plates for double mutants.
2. Pick 3 such putative double mutants to separate plates to allow them to self fertilize. If they are truly double mutants then all of their progeny should be double mutant as well and the progeny will be used for the next cross.
   
3. Label each of these plates with "Mapping 2", the genotype d u/d u and your initials and the date with your PURPLE Sharpie.
   
4. Incubate the plates at 23°C until next lab period.
   

Complementation

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 (different genes with the same phenotype). If the mutations are allelic there should be no complementation, whereas you could recover the wild type phenotype (though complementation) if the two mutations are in different genes. The specifics of strain construction vary depending on the experimental organism; however, the basic strategy in all cases is to construct a double heterozygote and then to examine the phenotype of this organism. Remember that a wild-type phenotype indicates that the two mutations complement one another (cancel each other out) and are therefore in different genes. Conversely, a mutant phenotype suggests the mutations are allelic to one another (that is, they fail to complement).

Over the next two weeks we will construct four different double heterozygotes containing your dumpy mutation of unknown location (dpy-u) and four different dumpy mutations of known location (dpy-k) as follows:

First obtain heterozygotes for your dpy mutation of unknown location through
Cross #1: unknown Dpy hermaphodites (dpy-u/dpy-u) x N2 males (+/+) yields dpy-u/+ progeny

Then use those heterozygous males resulting from Cross #1 in the next cross:
Cross #2: dpy-u/+ males x known dpy hermaphrodites (dpy-k/dpy-k) yields some dpy-u +/+ dpy-k progeny. [Note: we need to use heterozygote males for our unknown dpy mutation (dpy-u/+) because homozygote dumpy males do not mate properly]

The phenotype of the double heterozygote is then scored.

Keep in mind that, in this experiment, you will determine the allelic counterparts of your “unknown” Dpy mutation by placing it (your unknown mutation) with four mutations in different Dpy genes that have been previously mapped (i.e., known mutations). Our goal is to determine whether or not our “unknown” Dpy mutations are allelic to any of the four mutations.


3 days after lab: You will set up Cross #1: Cross your unknown Dpy hermaphrodites with N2 males [(dpy-u/dpy-u) x N2 males (+/+)] by placing three to five L4 Dpy's on a new plate with 3-4 N2 males. You will be picking your own wild type males from a mixed population of worms so be sure to transfer your males to a transfer plate before adding to the crosses. It is essential that the ONLY wild-type animals present on this plate are males.(There should be no wild type hermaphrodites because you desire +/dpy males from this plate for the next cross, and not the +/+ males that would result of wild-type hermaphrodites were included on the plate). Do this in duplicate.

Label your plates with your initials and the date and the genotype dpy/dpy (H) X +/+ (M) with your ORANGE Sharpie.

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!