This study involves the previously identified temperature sensitive (ts) cdc K. lactis mutants RCY246 and RCY435. In order to determine its defective gene, RCY246 was transformed with a K. lactis genomic DNA library and screened for transformants that grew at the restrictive temperature. A complementing transformant for RCY246 was found and designated RCY946. To amplify the complementing plasmid, the plasmid DNA from RCY946 was isolated and transformed into E. coli. Retransformation of RCY246 with the proposed complementary plasmid indicated partial complementation of RCY 246; however, the plasmid was unstable. In an attempt to amplify the insert region of the plasmid, a polymerase chain reaction was used and the major products were sequenced. To determine the genes of interest, the sequence was input into a BLAST program that revealed the presence of E. coli genomic DNA. Information about the complementing plasmid has yet to be discovered. In addition, RCY436 has a previously identified complemented transformant, RCY947. The complementing plasmid was isolated from RCY947 and amplified in E. coli. To verify complementation, the plasmid DNA could be retransformed into RCY435 and transformants could grow at the restrictive temperature.
Carnagey, E. (2009). Investigation of Complemented Transformants of "Kluyveromyces lactis" Temperature Sensitive Cell Division Cycle Mutants RCY426 and RCY435 (Undergraduate honors thesis, University of Redlands). Retrieved from http://inspire.redlands.edu/cas_honors/121