Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho⁻) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O⁻₂) into the cells. Accumulation of O⁻₂ and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho⁻, Δsco1), or in cells pretreated with O⁻₂ scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.     

Selective autophagy regulates insertional mutagenesis by the Ty1 retrotransposon in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic components and is ubiquitously found in eukaryotic cells. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia superfamily, is the most abundant retrotransposon in the yeast Saccharomyces cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like particles (VLPs) localized in the cytoplasm. Here we show that selective autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to autophagosomes by an interaction with Atg19. We propose that selective autophagy safeguards genome integrity against excessive insertional mutagenesis caused during nutrient starvation by transposable elements in eukaryotic cells.     

Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty

Summary Distribution of a delta () sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis. More than 100 copies of the sequence were non-randomly distributed on the chromosome. Using the sequence as a recombination site, mouse -amylase and human -endorphin genes were introduced into the chromosomal DNA. The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five. It was also found that single- or multi-copy integration occured at a single or multiple sites on the particular chromosome. The integrants secreted -amylase and -endorphin by three-to fivefold compared with single-copy integrants. This type of integration was mitotically stable over a period of 50 generations under non-selective conditions. Offprint requests to: A. Sakai     

Genetic mapping of Ty elements in Saccharomyces cerevisiae.

We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetically sites of several Ty elements. The 24 transformants recovered at Ty sites define 19 distinct loci. Seven of these were placed on the genetic map. Two classes of Ty elements were identified in these experiments: a Ty1-17 class and Ty elements different from Ty1-17. Statistical analysis of the number of transformants at each class of Ty elements shows that there is preferential integration of the CV9 plasmid into the Ty1-17 class.     

Integration of an aberrant retrotransposon in Saccharomyces cerevisiae.

We describe an atypical composite Ty1 element that apparently resulted from the concurrent integration of two complete elements. A portion of the central region of one of these elements was inverted between two long terminal repeats. Inversions of this type have been detected among unintegrated retroviral circles. It now appears that such intermediates can be incorporated into the genome.     

The Saccharomyces retrotransposon Ty5 influences the organization of chromosome ends.

Retrotransposons are ubiquitous components of eukaryotic genomes suggesting that they have played a significant role in genome organization. In Saccharomyces cerevisiae, eight of 10 endogenous insertions of the Ty5 retrotransposon family are located within 15 kb of chromosome ends, and two are located near the subtelomeric HMR locus. This genomic organization is the consequence of targeted transposition, as 14 of 15 newly transposed Ty5 elements map to telomeric regions on 10 different chromosomes. Nine of these insertions are within 0.8 kb and three are within 1.5 kb of the autonomously replicating consensus sequence in the subtelomeric X repeat. This suggests that the X repeat plays an important role in directing Ty5 integration. Analysis of endogenous insertions from S.cerevisiae and its close relative S.paradoxus revealed that only one of 12 insertions has target site duplications, indicating that recombination occurs between elements. This is further supported by the observation that Ty5 insertions mark boundaries of sequence duplications and rearrangements in these species. These data suggest that transposable elements like Ty5 can shape the organization of chromosome ends through both transposition and recombination.     

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.     

Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements.

The structural analysis of aberrant chromosomes is important for our understanding of the molecular mechanisms underlying chromosomal rearrangements. We have identified a number of diploid Saccharomyces cerevisiae clones that have undergone loss of heterozygosity (LOH) leading to functional inactivation of the hemizygous URA3 marker placed on the right arm of chromosome III. Aberrant-sized chromosomes derived from chromosome III were detected in approximately 8% of LOH clones. Here, we have analyzed the structure of the aberrant chromosomes in 45 LOH clones with a PCR-based method that determines the ploidy of a series of loci on chromosome III. The alterations included various deletions and amplifications. Sequencing of the junctions revealed that all the breakpoints had been made within repeat sequences in the yeast genome, namely, MAT-HMR, which resulted in intrachromosomal deletion, and retrotransposon Ty1 elements, which were involved in various translocations. Although the translocations involved different breakpoints on different chromosomes, all breakpoints were exclusively within Ty1 elements. Some of the resulting Ty1 elements left at the breakpoints had a complex construction that indicated the involvement of other Ty1 elements not present at the parental breakpoints. These indicate that Ty1 elements are crucially involved in the generation of chromosomal rearrangements in diploid yeast cells.