Lab 4

Goals of the lab:

• Score the test cross to confirm linkage between goi-1 and the unc reference gene, and to determine the recombination frequency between these two genes
• Begin complementation testing

Test Cross and Map Distance: Scoring phenotypes

We are now ready to analyze our data from our test cross.

TO DO:

• Look over all of your test crosses.
• Discard any crosses that do not display all four of the expected phenotypes. Note that single mutants may be rare.
• Discard any crosses that do not contain males. Since this generation should be the result of a mating, there should be plenty of male progeny. Lack of male progeny indicates that your hermaphrodites may have self-fertilized instead of mating.
• Score all of the ADULT worms from each test cross the same way you did in Lab 4. It is important to score only adult worms as larval worms may not display the phenotype despite being homozygous for the mutant allele. Remember to move randomly around the plate, and to flame each worm as you count it so as not to double count. Move some mutants to a separate plate and have your partner and lab instructor double check your identification early on!
• Record your counts in your notebook, and transfer your data into the group spreadsheet.
• Once all of the data are entered into the spreadsheet, complete the Chi-square analysis to prove whether goi-1 and the unc reference gene are linked. If so, calculate the recombination frequency (RF). RF is directly proportional to map units (mu, also measured as centimorgans or cM). Note that it is difficult to translate map distance in cM to actual distance in bases. The conversion factor varies among species and changes as distance between genes changes. Therefore, we will limit our mapping to calculating distances in map units.

You will determine map distance of the dpy gene of interest from an unc gene using the formula: RF (recombinant frequency) =the number of single mutants (both dpy and unc single mutants) divided by the total number of worms counted * 100 (to obtain RF in % recombinants and thus in map units).

Complementation

In order to determine whether a mutation is in a novel gene, or is in a previously characterized gene, we can perform a complementation analysis. It is not unusual to have series of mutations that confer similar phenotypes and also map to the same chromosome. Complementation testing can determine if two mutations are allelic (that is, in the same gene) or non-allelic (in different genes but both causing the same phenotype). This is done by crossing a mutant with a series of reference strains so that the progeny contain one experimental mutant allele and one reference allele. If the two mutations are allelic there will be no complementation, meaning that the mutations can not "rescue" each other because you do not have a wild type copy of the gene causing the mutant phenotype; thus, you observe the mutant phenotype when the two strains combine. If the mutations are non-allelic there should be complementation, meaning they "rescue" each other: because there is one wild-type copy of each gene, the progeny can show a wild type phenotype.

Complementation analysis can be evaluated this way only when you are working with recessive mutations. For more information about complementation, see your Genetics textbook.

If goi-1 has been previously characterized, this complementation analysis might tell us the published name of our gene of interest. If it has not been previously characterized, we will be able to name it.

In our case, we will use several different reference mutant strains. All have similar phenotypes, but in each strain the gene responsible for the phenotype is located to a different known region of a chromosome.

We have the following strains available, all maintained as homozygous hermaphrodites:

Chromosome I:

• dpy-5
• dpy-14
• dpy-24

Chromosome II:

• dpy-2
• dpy-10
• dpy-25

Chromosome III:

• dpy-1
• dpy-17
• dpy-18

Chromosome IV:

• dpy-4
• dpy-13
• dpy-20

Chromosome V:

• dpy-11
• dpy-21
• dpy-30

To Do Today

• With your partner, design a cross using the available worms listed above that will enable you to determine whether goi-1 is a novel gene or a previously characterized one.
  • NOTE: This will require more than one generation of worms! Remember that homozygous mutant males are unable to mate. In your design, indicate the genotypes of the worms you will mate, the genotypes of the gametes they can produce, and the genotypes and phenotypes of the resulting progeny. In designing your crosses, consider the following: What chromosome do you think goi-1 is on? Which reference strains do you need to use? What would you see in the progeny if goi-1 is allelic to a reference strain? What if it is non-allelic? How many matings would show complementation? How many would not?
• Have your instructor check your strategy before moving on.
• Pick 3 single mutant L4 hermaphrodites to each of 2 separate mating plates. The genotype and phenotype of these worms match those of the worms you started with in Lab 2.
• Next, transfer 3-4 wild-type males onto a transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to each of the mating plates.
• Label your crosses with your ORANGE Sharpie. Be sure you can differentiate between these plates and the ones you set up for the mapping and test cross! Incubate your worms at 23C for 3 days.

To Do In 3-4 Days

In the last lab, you set up a cross to generate heterozygous male worms carrying our mutation in goi-1. Today you will set up the actual complementation crosses to determine whether this mutation is allelic or non-allelic to any of several reference alleles.

TO DO:

• Obtain plates with worms from the three strains you will use for complementation testing.
• Observe the reference strains carefully, making note of the phenotype. Do you see the phenotype in adult worms? Larval worms? Both? How does the phenotype compare to that of our mutant? Make careful notes in your lab notebook about the similarities and differences.
• For each of the reference strains, pick 3-5 single mutant L4 hermaphrodites to a mating plate.
• Next, transfer 3-4 heterozygous male progeny onto a transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to the mating plate. Be careful not to get these mixed up with the double heterozygotes you used to set up the mapping crosses!
• Label your crosses with your ORANGE Sharpie. Incubate your worms at 23C.