Saccromyces cerevisiae is a budding yeast that has been widely used for study of the cell cycle due to its short life cycle and the ease with which is can be genetically manipulated.
| Saccromyces cerevisiae is a budding yeast that has been widely used for study of the cell cycle due to its short life cycle and the ease with which is can be genetically manipulated. |
doc6-, a mutation that causes budding yeast containing a Gal-CLN2 fusion to arrest or die (we are unsure which) when placed on galactose media, was first isolated by Maria Yuste-Rojas at The Rockefeller University. doc6- strains also behave as though they were Trp- (cannot make the amino acid Tryptophan), even though they are genotypically Trp+.In order to isolate the DOC6+ (wild-type) gene, we transformed a doc6- strain with a leucine yeast genomic DNA library and selected for transformants on plates lacking leucine. We then replica-plated the transformants onto galactose plates lacking leucine (Gal-Leu), to select for DOC6+ transformants, and dextrose plates lacking tryptophan (Dex-Trp), to select for transformants that overcame the Trp- phenotype. We isolated four transformants, named LL8, LL11, LL19 and LL26 after the plates from which they came, which grew on both Gal-leu and Dex-Trp plates.
We isolated the transforming, and therefore putative DOC6+, plasmids and transformed them into E. coli by electroporation to replicate them to high copy. Two plasmids, LL11 and LL26, produced E. coli transformants. Plasmid 26 was sequenced at The Rockefeller University by Fred Cross' lab. Using the yeast genome search engine on the sequence from plasmid 26a, we concluded that our gene is most likely contained on this region of chromosome IV. Through restriction mapping of plasmid 26 and 11, we concluded that the only overlapping region of the two plasmids is the ARO1 gene region, which encodes five proteins involved in the synthesis of aromatic amino acids.