biology, temperature sensitive mutant strains, kluyveromyces lactis, mutations, cloning by complementation, genes
Biology | Cell and Developmental Biology | Genetics and Genomics
Gaining a better understanding of genes, that when mutated, cause the cell-division cycle to malfunction will allow for better and more specialized cancer treatments. This project screened strains with potential mutations in cell-division cycle (cdc) genes in the budding yeast Kluyveromyces lactis. The goal was to confirm a Cdc phenotype in several temperature sensitive (ts) mutants in the manner introduced by Hartwell and colleagues in the 1970's. Although initially identified as a cdc mutant, RCY1125 was found not to be a cdc mutant upon rescreening. Screening for a cdc mutation in RCY11126 was inconclusive, as there were insufficient cells to be scored; however it showed interesting enlarged cell and elongated cell phenotypes. Four haploid mutant strains, RCY1110, RCY1120, RCY1123, and RCY1124 did show a Cdc phenotype so further analysis was performed to confirm that the cdc mutation was in a single gene and recessive. Having a recessive mutation in a single gene i necessary to allow for cloning by compmentation in later stages of the project. Each haploid was mated with LSY25, a wild type strain, yielding diploid strains that were used for recessiveness testing and tetrad dissection. Through recessiveness testing we were able to confirm recessive mutations in RCY1110 and RCY1124. Analysis on RCY1120 and RCY1123 was discontinued during recessiveness testing due to the haploids no longer showing a temperature sensitive phenotype, possibly due to osmotic effects. Tetrad dissections so far have shown a 2:2 segregation of the temperature sensitive phenotype for RCY110 and RCY1124, indicating a mutation in a single gene; however more tetrad analysis is still needed on both RCY110 and RCY1124. Preliminary tetrad analysis indicates linkage of the ts locus in RCY110 to ura3 and the ts locus of RCY1124 to ade1. Currently, RCY110 and RCY1124 appear to be good candidates for cloning by complementation and we hope they will fall into complementation groups not yet found by this lab.
Department 1 Awarding Honors Status
King, M. (2013). Investigation of Temperature Sensitive Mutant Stains RCY1110, RCY1120, RCY1123, RCY1124, RCY1125, and RCY1126 in Kluyveronmyces lactis for cdc Mutations and Potential for Cloning by Complementation (Undergraduate honors thesis, University of Redlands). Retrieved from https://inspire.redlands.edu/cas_honors/475
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