Construction of a S. cerevisiae Ty1-less strain.

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Our initial observation that retrotransposition is self-limiting depending on element copy number, and subsequent discovery that this control is mediated by Ty1 antisense RNA, relied heavily on the use of an unusual Saccharomyces strain that lacked any Ty1 elements. Natural (wild) isolates of Saccharomyces vary in Ty1 copy number, although they usually contain fewer elements than the common laboratory strains of S. cerevisiae. The natural Ty1-less strain we used was initially found to have an unusually low Ty1 element load (one element) and that single element was deleted to derive a strain free of Ty1 elements. A genomic analysis to search for residual long terminal repeats (LTR), indicative of sites where elements had been lost due to LTR-LTR recombination, revealed that at least 11 elements had populated an ancestral strain, and that this strain is not strictly S. cerevisiae , but rather, appeared to be a hybrid between S. cerevisiae and S. paradoxus. This has hindered our analyses and comparisons to S. cerevisiae as the genomic sequence of S. paradoxus is incomplete and poorly annotated, and without sequencing the entire Ty1-less yeast genome, we cannot discern the speciation or extent of hybridization. To address this disparity, we have undertaken the systematic deletion of all 32 Ty1 elements in the laboratory strain of S. cerevisiae. by transformation with a PCR fragment containing the URA3 gene flanked by Ty1 sequence. The replacement of a Ty1 element by the URA3 gene and subsequent loss through recombination can be monitored by growth of the cells in the presence of 5-fluoro-orotic acid (FOA), a drug that is toxic to cells when URA3 is expressed. Unwanted gross chromosomal abnormalities or rearrangements generated by this process are detected by pulse field gel electrophoresis. Using this method, strains with as few as one element have been constructed.