Dr. Voytas discovered the yeast transposon Ty5 by sequence analysis of the S.c. genome. Because the yeast sequence was functionally defective, he cloned the homologous sequence from S. paradoxus and demonstrated that it would transpose in S. c. He went on to develop an elegant assay for transposition in which a HIS3 marker with an artificial intron insert is placed in the Ty5 transcription unit driven by the GAL promoter. Because HIS3 is oriented backward relative to the Ty5 unit, but the intron is in the forward orientation, Ty5 RNA but not HIS3-promoted RNA can be spliced to generate a HIS3+ gene. His+ clones arising from this construction have a Ty5 element flanked by a 5 base repeat at genomic sites. Surprisingly, Ty5 inserts preferentially into silenced regions of the genome, at telomeres and HML and HMR. This is quite a novel finding and was published in a notable paper. Dr. Voytas has adapted a system that was used to show a role of ini tRNA in transposition of Ty1. The difficulty showing a requirement of Ini tRNA is that the tRNA gene is essential for cell viability. However, using a plasmid shuffle scheme, mutations that do not destroy the activity of the tRNA in initiation can be tested for allowing Ty transposition. Dr. Voytas showed in this way that mutations in the tRNA anticodon stem can reduce the frequency of transposition 5-6 fold. This region of the tRNA was a candidate for the primer because it has a 13 base region complementary to a region in the 5'LTR of Ty5. The PI suggests that a half tRNA generated by processing may serve as primer, as has been shown for the copia element of Drosophila.
The specific aims i n this First proposal are: 1. Identify the primer used for minus-strand DNA synthesis by Ty5. 2. Identify initiator met tRNA sequences important for transposition. 3. Identify Ty and host mutations that suppress tRNA-induced transposition defects.
