Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.     

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.     

Chromatin structure in a region of a yeast transposable element regulating adjacent gene expression.

The chromatin structure of a portion of yeast transposable elements known to be responsible for regulation of the expression of the adjacent HIS4 gene has been investigated, using the nuclease probe micrococcal nuclease. Yeast strains containing Ty917 or derivatives of this element that possess either a His-, weak His+, or strong His+ phenotype were examined. The chromatin at the Ty/HIS4 junction region was accessible to micrococcal nuclease. A partial nucleosome ladder was observed upon digestion with micrococcal nuclease indicating the presence of three phased nucleosomes located in Ty sequences upstream of the HIS4 gene. Phased nucleosomes could not be detected upstream of the HIS4 gene in wild-type cells. These data suggest that nucleosomal structure is not a major contributor to Ty917-regulated adjacent gene expression at HIS4.     

A genetic and structural study of genome rearrangements mediated by high copy repeat Ty1 elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.     

Deletions extending from a single Ty1 element in Saccharomyces cerevisiae.

Chromosomal rearrangements associated with one Ty1 element in the iso-1-cytochrome c (CYC1) region of Saccharomyces cerevisiae yeast cells were examined. Most of the rearrangements were deletions of the three linked genes, CYC1, OSM1, and RAD7, and resulted from recombination involving the single Ty1 element and a solo delta in the same orientation. These deletions differed by the number of Ty1 elements (zero, one, or two) remaining after deletion and by restriction site heterogeneities associated with these elements. A single Ty1 element remained at the deletion junction point much more frequently than no Ty1. Apparently the Ty1-associated delta element nearer to the solo delta was involved more often in recombination than the more distal Ty1-associated delta element. The restriction site data implicate gene conversion and suggest that site-specific recombination within the deltas, if occurring, is not the only mechanism of delta-delta recombination. Three other rearrangements bore deletions which began at the end of the Ty1 element and extended into regions not bearing Ty1 or delta sequences. Two of these deletions eliminated 7 kilobases of DNA, although they differed by an associated reciprocal translocation. The third involved a deletion of 14.7 kilobases of DNA associated with an overlapping inversion.     

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.     

Mutations Affecting Ty-Mediated Expression of the HIS4 Gene of SACCHAROMYCES CEREVISIAE

We have identified mutations in seven unlinked genes (SPT genes) that affect the phenotypes of Ty and δ insertion mutations in the 5' noncoding region of the HIS4 gene of S. cerevisiae. Spt mutants were selected for suppression of his4-912δ, a solo δ derivative of Ty912. Other Ty and δ insertions at HIS4 are suppressed by mutations in some but not all of the SPT genes. Only spt4 suppresses a non-Ty insertion at HIS4. In addition to their effects on Ty and δ insertions, mutations in several SPT genes show defects in general cellular functions—mating. DNA repair and growth.