Goal: To identify yeast gene products important for accurate chromosome transmission in mitosis. Importance: Errors during chromosome transmission in humans can lead to cell death, genetic disorders (e.g., Down Syndrome), and cancer. Experimental Strategy: Plasmids containing yeast genes that suppress YAC loss in ysm83 and ysm84 mutant strains were identified in previous studies. The multiple genes present in each suppressor plasmid are being subcloned and introduced into yeast cells to determine their abilities to suppress YAC loss. Visual Assay For YAC Loss Analysis Of Candidate Genes That Suppress Chromosome Loss In Saccharomyces cerevisiae Mutants With Defects In Chromosome Transmission Naomi Adjei, Alyssa Ellis, Lanie Feigenbutz, Traci Gwinn, James Kolnik, Lindsey Miller, Neel Patel, Kevin Peterson, Alexandra Richardson, Christine Setsodi, Whitney Michaels, and Heidi Sleister. Biology Department, Drake University Results Experimental Question & Methods Blast search to determine which candidate yeast genes are present in each suppressor plasmid. Clone each candidate suppressor gene into vector (YEplac181, YEp13 or p366). Isolate vector and suppressor plasmid DNAs Linearize vector (restriction enzyme) and dephosphorylate Isolate yeast genes by PCR or restriction digest Ligate vector + insert (candidate suppressor gene) Transform ligation mixture into E. coli Screen transformants for correct constructs Assay subcloned yeast genes for suppression of YAC loss in ysm mutant strains. Transform correct constructs into ysm mutant strains Screen transformed cells for suppression of YAC loss Background Figure 3. Restriction digests and gel electrophoresis to isolate candidate suppressor genes. Digestion of suppressor plasmid ysm83-p26 with BamHI-PstI (lane 2), Sau3A (lane 4), and EcoRI (lane6) resulted in DNA bands containing TIM22 (1337bp), DTD1 (1903bp), and YDL218W (1794bp), respectively (marked with yellow arrows). DNA extracted from these bands was ligated to vector YEplac181 cut with BamHI- PstI, Sau3A, and EcoRI, respectively. Lanes 1 and 8 contain a 100bp plus ladder Many of the candidate suppressor genes have known roles related to chromosome transmission. For example, CAK1 and OCA1 are involved in cell cycle regulation. Suppressor plasmid ysm83-p26 contains full-length genes DTD1, YDL218W, and TIM22 (Figure 2). These genes were isolated from ysm83-p26 using restriction digests (Figure 3) and ligated to vector YEplac181 (Figure 1). Correct vector + insert constructs for TIM22 and YDL218W were created (Figure 5), transformed into yeast ysm84 cells (similar to ysm83), and assayed for suppression of chromosome loss. Preliminary results suggest YDL218W at least partially suppresses YAC loss in ysm84 cells. Suppressor plasmid ysm83-p41 contains full-length genes OCA1, RAS2, and PHO23. All three genes were isolated by PCR, ligated to BamHI- linearized YEp13, and transformed into E. coli. Transformants are now being screened for presence of the insert. Suppressor plasmid ysm83-p71 contains full-length genes SSF1, RRP3 and HTD2. Two of these genes were isolated by PCR (Figure 4) and ligated to BamHI-linearized YEp13. Transformants are now being screened for presence of the insert. Suppressor plasmid ysm84-p11 contains full-length genes AGX1 and CAK1. These genes were isolated by PCR, separately ligated to BamHI- linearized vector YEp13, and transformed into E. coli. Transformants are now being screened for presence of the insert. Suppressor plasmid ysm84-p57 contains a single gene, SMF1. To determine if SMF1 can suppress YAC loss in a single copy plasmid, the gene was isolated by PCR, ligated to BamHI-linearized single copy vector p366, and transformed into E. coli. Transformants are currently being screened. The BLAST analysis and restriction digests revealed that suppressor plasmid ysm76-p152 is rearranged. A primer walking approach is underway to determine the genes present in this plasmid. Figure 4. PCR amplification to isolate candidate genes. Candidate suppressor plasmid ysm83-p71 was used as a template for amplifying candidate suppressor genes HTD2 (1635bp, lane 2) and SSFI (1942bp, lane 3). PCR products were digested with BamHI and ligated to BamHI- linearized vector YEplac181 (lane 1). Lanes 4 and 5 contain markers: 100bp plus ladder and Lambda-HindIII. Wildtype (YSM+) yeast cells have low rates of YAC loss, resulting in white colonies with little to no red sectoring. Results 3.0 kb 2.0 kb 1.5 kb 1.0 kb Figure 5. Screening E. coli cells transformed with ligation of vector YEplac181 + insert candidate suppressor gene (TIM22 or YDL218W). Transformants were “cracked,” and resulting plasmid DNA was run on a 0.6% agarose gel. Plasmids containing an insert are larger than the vector alone (YEplac181, lane 1). Lanes 2-8: YEplac181 + TIM22 transformants; Lanes 9-16: YEplac181 + YDL218W transformants. Plasmids larger than the vector (*) were screened further. ****** Figure 1. Vector YEplac181. YEplac181 is a high copy yeast shuttle vector with selectable markers LEU2 and amp R. Cloning sites are BamHI, PstI, and EcoRI. Figure 2. Yeast DNA insert from suppressor plasmid ysm83-p26. The ysm83-p26 insert contains three full-length yeast genes: DTD1, YDL218W, and TIM22. Table 1. Summary of molecular cloning. Which candidate yeast genes are responsible for suppressing YAC loss in mutants with increased YAC loss? Conclusions & Current Work We thank previous Drake BIO106L and BIO124L (Genetics Research) students for isolation of the candidate suppressor plasmids described here. This work was supported, in part, by a Drake research grant. ysm83ysm84ysm76 ysm83 [p26] YSM+ Mutant strains ysm76, ysm83, and ysm84 have increased rates of YAC loss, resulting in white colonies with red sectors. Mutant ysm strains containing plasmid suppressors of YAC loss (e.g., ysm83 [p26]) have few red sectors.