To identify host cell factors involved in the transposition process, a genetic screen for host cell mutations which allow transposition at elevated temperatures has been initiated. A yeast strain (genotype MATa ura3-167 leu2D1 his3 D 200 trp1 D 1) carrying a TRP1-marked Ty1 element on a high-copy URA3 plasmid was chemically mutagenized. After plating on glucose medium, approximately 2000 colonies were replica-plated to galactose plates pre-warmed to 37°C. After two days of induction, the colonies were replica-plated to rich media (YPD), then to 5-fluorooratic acid (5-FOA) to induce plasmid loss, and finally to medium lacking tryptophan to select for transposition. Colony growth indicated that a mutation was acquired which allowed transposition at high temperature. To distinguish plasmid mutations (the plasmid was present during mutagenesis) from genomic mutations, promising mutant colonies were grown from the master plates on rich media to lose the Ty plasmid, and fresh plasmid then introduced. After this screening step, three mutant strains were identified which had some level of transposition above wild-type at high temperature. The mutants have been given the designation htt for High Temperature Transposition. The screening process and one of the mutants is shown in Figure 1.

The screening process proved to be difficult due to variability in the results of the transposition assays. After careful analysis, the optimum temperature (where the difference in transposition between the isogenic wild-type and mutant strains is greatest), was found to be ~35-35.5° C.  We have focused on the mutant with the strongest phenotype, htt19.  Genetic analysis has been complicated by the presence of the trp1D1 mutation in the original strain.   The trp1D1 mutation present in the mutant strain removes a portion of the UAS of the GAL3 gene, and inducer of the gal induction pathway. As a result, the level of galactose-induced transposition in trp1D1 strains is slightly reduced. At high temperature, the difference in transposition between TRP1 and trp1D1 strains is significantly greater. Taking the TRP1 genotype of the spores into account, the phenotype of transposition at 35°C segregates 2:2 in 5 of the 6 tetrads, indicating that trt19 maps to a single locus. (Figure 2) For subsequent analysis, we selected a TRP1 HTT19 spore and a TRP1 htt19 spore, 5B and 5D respectively. Analysis of diploids made from various TRP1 spores indicates that the htt19 mutation is recessive.

Each of the strains carrying htt mutations have been made TRP+ by transformation of the missing sequence and selection on SC-Trp medium.  These strains are being used in all further studies.

We originally attempted to clone the gene by transforming a wild type library into the htt19 mutant strain and looking for a reduction in transposition.  This strategy failed to yield any clones.  We are currently attempting to clone the gene by transforming a htt19/HTT19 diploid with the Snyder Tn-lacZ disruption library.  Since the gene is recessive, the diploid strain does not transpose at high temperature.  Disruption of the HTT19 allele should allow for transposition at high temperature.  Unfortunately, we have gotten very poor transformation efficiencies with this strain and have been unable to get enough colonies for a screen.