Regulation of retrotransposition in Saccharomyces cerevisiae

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at a rate of 10(-5) to 10(-7) per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.     

Construction of yeast strains containing genetically marked transposons

Haploid yeast cells contain approximately 35 Ty (transposon yeast) elements. To facilitate the study of these elements, we have constructed yeast strains in which one of these transposons carries a genetic marker. The elements that we have modified are Ty912 and Ty917, elements originally detected at the HIS4 locus in spontaneously occurring his4- mutants. The strain constructions took place in three stages: 1) cloning of the mutant HIS4 genes containing the Ty elements; 2) introduction of a HindIII restriction fragment containing the yeast URA3 gene into the cloned transposons; and 3) replacement of the chromosomal HIS4 gene with the modified genes constructed in vitro. These strains will be extremely useful in the study of Ty transposition and other Ty-promoted DNA rearrangements.     

Multimeric arrays of the yeast retrotransposon Ty.

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.     

Polymorphisms on the right arm of yeast chromosome III associated with Ty transposition and recombination events.

The region of Saccharomyces cerevisiae chromosome III centromere-distal to the PGK gene is the site of frequent chromosome polymorphisms. We have sequenced this region from fragments of chromosome III isolated from three different yeast strains, GRF88, CN31C and CF4-16B. The sequence analysis demonstrates that these polymorphisms are associated with the presence of Ty and delta elements and defines a region of the chromosome which is a hot-spot for transposition events (the RAHS). The three strains can be arranged into a logical evolutionary series in which successive transposition and recombination events insert Ty elements and fuse them with consequent deletions of chromosome and of transposon sequences. The influence of such events on yeast genome evolution is discussed.     

Transposon Tagging Using Ty Elements in Yeast

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.     

Analysis of Yeast Retrotransposon Ty Insertions at the Can1 Locus

The target site distribution for 55 independent Ty insertions that inactivate the function of the Saccharomyces cerevisiae CAN1 gene is reported. Under some selection conditions Ty elements inserted preferentially into the promoter and exhibited an orientation bias. In contrast, under other conditions no insertions were detected in the promoter region and transposition appeared to occur randomly throughout the CAN1 coding sequence. These results show that the target site distribution for Ty insertions may be a function of the selection conditions.