Yeast MSH2 project in an undergraduate course

I will teach a section of BIO125 molecular biology and genomics in Spring semester 2013. This course is based on Gammie and Erdeniz "Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory in molecular biology", Cell Biology Education, 2004.  BIO125 is a project based course. We expect  21 students,  working in 7 groups, in each section. Each group will work on one msh2 mutant. Each yeast msh2 mutant is designed based on equivalent mutations on human hMSH2.

Growth media with selection are designed to be -HIS-TRP-LEU-THR-URA, in which  -HIS for the pRS413 plasmid that can carry MSH2 genes and their mutants, -TRP for pSH44 reporter pladmids with dinucleotide-URA3, -LEU-THR to induce expression of the GT16-URE3 chimera, and -URA to "ensure no instabilities of dinucleotide repeats occur before the assays are inititated" (a statement that I do not grasp at the first reading).

To evaluate the dinucleotide instability, GE04 used synthetic media -HIS-TRP-LEU-THR+5FOA. 5FOA is purchased from Toronto Research Chemicals, Inc. To evaluate single bp mutations, the media is -HIS-TRP-ARG+CAN, canavanine (Sigma).

GE04 said that yeast strains were grown in liquid synthetic medium -HIS-TRP overnight at 30C. For the reporter strain AGY75 which does not have pRS413, I think it must have been grown in +HIS media, because -HIS is used to select for the HIS3 marker on pRS413 , a yeast centromere vector. The genotype of AGY75 is MATa ade2-1 trp1-1 ura3-1, leu2-3,112 his3-11,15, msh2::LEU2 RAD5.   GE04 indicated that AGY75 is derived from W303 (see footnote of Table 2).  Due to the ade2 mutation, most colonies should be pink. Interestingly, yeast strains different msh2 mutations have different frequencies for white colonies, based on the previous experience in Bio125.

I want to connect mismatch repair stress with cell cycle, and oxidative stress response. So, comparison of cell cycle status, DHE and DHR staining maybe an interesting questions. I should go back to review Burhans's paper on replication stress and (chronological?) life span.

GE illustra puReTag Ready-to-go PCR beads is used in this class.

Some major items that I think will be needed include 8 set of replica blocks and velveteen squares (7 for students groups and 1 for instructor) available from Corastyle.com;


Notes:
msh2 mutant grow very slow in SD-Trp-His. It can take 2.5 day to reach OD=0.6 for FOA assays.


Yeast media from Sunrise:

Qty	Item #	Description	Total Price
	1709-300	SD-Trp Powder, 10 x 0.5 liter pouches Price Each: $99.00	$99.00
	1709-010	SD-Trp Powder, 10 grams Price Each: $45.00	$45.00
	1705-100	SD-His Powder, 100 Grams Price Each: $76.00	$76.00
	1715-500	SD-His-Trp Powder, 500 Grams Price Each: $275.00	$275.00
	1715-300	SD-His-Trp Powder, 10 x 0.5 liter pouches Price Each: $99.00	$99.00

See also http://hongqinlab.blogspot.com/2013/05/msh2-project-problems-in-bio125-spring.html
MSH2 resources
Wikipedia entry