Purpose: The object of this lab is to introduce the concept of genetic linkage and to demonstrate the ways in which it can be examined experimentally. Genes are said to be linked if they lie physically close to one another on the same chromosome. Such genetic linkage between genes can be observed experimentally by determining how frequently the allelles in question are inherited in the same products of meiosis. Genes that are unlinked (for example genes that are on different chromosomes) are segregated randomly. Linked genes tend to be inherited together. In this experiment, we will cross (mate) two haploid yeast strains to make a diploid strain. This diploid strain will then be coaxed into meiosis and the resulting products of meiosis (tetrads) will be dissected. The haploid progeny will then be tested to determine which set of genes it has inherited from the diploid parent.
Strains: YMM28 Mat a ade2 his4 ura3-52
YMM54 Mat a his4 lys2 cdc44-1.URA3 pol30-37 ura3-52
Day -1 Streak Mat a and Mat a strain across YPD plate and put at 30oC.
Day 1 Replica plate cross to selective plate where only diploid will grow (i.e. SD + His) and put at 30oC. Meet for lecture about meiosis, dissections, linkage. Pairs of students should book a time this week to try and dissect tetrads. This is best done before next week's class (day 8).
Day 3 Patch diploid cells to YPD plate and incubate at 30oC.
Day 4 Replica plate diploid patch to sporulation plate, put at 25oC.
Day 8 Take a swab of cells from the spo plate, disperse in water and look for tetrads under microscope. If you see tetrads, take a toothpickfull of sporulated cells and swirl into 50 ul of zymolyase (0.5 mg/ml in 1 M sorbitol) in an eppi tube. Incubate for 10 minutes at 30oC and then add 500 ul of water and put on ice. This treatment will free the spores from the cell wall and they can now be dissected. To do so, spread about 50 ul of the cells in a straight line across a thin YPD plate with a 1 ml glass pipet. Dissect out spores from 5-8 tetrads with a tetrad dissection microscope. Put dissection plate into 30oC incubator for 2-3 days to let the spores grow.
Day 8 continued Take a sterile frog pond and add 50 ul sterile water to each well. Use a sterile toothpick to innoculate cells from colonies into the appropriate wells. In all, frog haploid parent strains, diploid, and spores from 5-7 tetrads onto synthetic (SC) plates (30oC) to test for auxotrophies, and YPD plates (18, 24 and 30oC) to test for temperature sensitivities. Score 30oC plates after 2 days, 24oC plates after 3 days and 18oC plates after 5 days. Test cells on SC, SC - His, SC - Ura, SC - Ade, and SC - Lys plates for auxotrophies.
Day 10 -13 Score plates for growth, determine the genotype of the resulting haploid strains, and determine which, if any, genes were linked.
Questions to condsider for lab report: How did the various auxotrophies segregate and why? How did the cs- phenotype segregate and why? Are any of the phenotypes/genes linked?
Suggested Tables
Prepare a table listing the genotypes of the parents and all of the spores.
For the 6 combinations of genes, prepare a table listing the kinds of tetrads observed and whether the genes appear to be linked or not. For example:
TYPE OF TETRAD
Gene pairs
Parental D.T.
Non-Parental D.T.
Tetratypes
Unknown
Conclusion
ADE2 LYS2
1
2
4
0
UNLINKED
POL30 CDC44
ETC.
Since the POL30-containing combinations will have some undefined tetrads, make another table listing the genotypes of all of the known individual spores. For example:
GENOTYPE OF SPORES
GENE PAIRS
PARENTAL
NON-PARENTAL
CONCLUSIONS
POL30 ADE2
12
10
UNLINKED
POL30 LYS2
ETC.