Lab 7: Series2 Forward Genetics Project- SCORE!
Mapping: If you haven't already done so, form a hypothesis about your expected recombination frequency (RF) for the test cross you will score today. RF is directly proportional to map units (in centimorgans cM) a measurement of the distance that linked genes are apart. You can check previous research (Wormbase is a good place to look) for the number of map units between the your linkage group (gene of interest and reference gene). How many recombinants (total) do you expect in each 100 worms scored? Note that it is difficult to translate map distance in cM to distance between genes in bases. The conversion factor varies among species and changes as distance between genes changes. Roughly, in humans, a cM is equal to approximately a million bases but that conversion factor does not apply to map units in C. elegans. Therefore, we will limit our mapping to calculating distances in map units.
Scoring the Test Cross: Each partner should count all the adult progeny from one test cross plate, scoring the phenotype as either wild type , Dpy , Unc or Dpy Unc. Remember to remove each animal after you have determined its phenotype. Flame the WT and UncDpy progeny but transfer any single mutants to a new plate. When you have finished scoring your plate, ask both your partner and your lab instructor to confirm that the worm progeny that you scored as either Dpy or Unc are,in fact, single mutants. Record your totals on the google spreadsheet provided for course data. This spreadsheet will update as each lab section enters its data. You can find this spreadsheet in the DATA file in Resources in your lab section's Sakai site and on a computer in the back of the lab. You can calculate RF and map distance from your own group's and class' data before we have the course data completed, but, of course, the evidence used in your paper will be the RF calculated from the full data set.
Review the crosses that you diagrammed for this mapping project and make sure you understand how the test cross we set up differentiates, phenotypically, progeny of parental gametes from the progeny of recombinant gametes. Hint: how did you end up with either of the single mutant classes ( Unc or Dpy) from these parental genotypes d u/+ + (genotype of the male) and d u/ d u (genotype of the hermaphrodite parent)?
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).
Congratulations! You now have calculated the location of the dpy mutation in map unit distance away from your reference linked unc gene on the a particular autosome. To check your location (and the accuracy of your recombination frequency relationship to map units), enter the linked unc gene name into Wormbase. Scroll down and click on Location and Mapping Data and find the gene that is your calculated number of map units away from the linked unc gene. (Remember that you will have to look in both directions on the chromosome). Is the dpy gene that you found allelic in complementation analysis at one of these map locations? Yes? Terrific! If not, your next step would be to see what is known about the gene or ORF at those locations on the chromosome and see if what's known fits in at all with your observations. If it does, great and, if not, you have some thinking to do about the identity of your dpy gene of interest. (You will NOT write a paper about "sources of error" in your experimental design or your execution of the experiment!!!!)
If you think you know the identity of your dpy gene, enter this name into the box at the top of the page in Wormbase and click Search. It will either bring you directly to that page or it will bring you to a page with multiple hits - click on the link that provides a definition for what the gene does.
On this new page should be all the known information about this particular gene. Its name, who named it, what the gene encodes - if that is known, and much more. At the bottom will be a list of references - or a link to a list of references. Find out the function of this gene.
Spend some time with Wormbase and marvel at all the hard work and years of research that went into discovering all this information about this tiny little soil nematode that causes us no harm (non-parasitic). Why do you think so many smart people have devoted so much of their time and energy to working out the genetics of "appearance or movement challenged" little worms? We will talk more about model organisms and the power of functional and comparative genomics in our next series.
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Planner section of the Weekly Calendar for other work to accomplish before the next lab. Direct link to information about writing up your series 2 work in classical genetics as a scientific research report due at the beginning of Lab 9 is found at BISC219/F12: Assignment_ Series2_Classical Genetics Paper.
Links to Labs& Project Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Continued
Lab 6: DNA sequence analysis; Mapping Continued
Lab 7: Complete Mapping: Score
Background Information on Project 3: Investigating Gene Regulation Using RNAi
Lab 7: Identifying a bacterial colony containing our plasmid of interest
Lab 8: Creating the feeding strain of bacteria for RNAi
Lab 9: Induction of feeding strain to produce dsRNA and feeding worms
Lab 10: Phenotypic analysis of treated vs untreated worms
Lab 11: Writing Workshop
Lab 12: Writing Conferences