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.     

Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae

The yeast, Saccharomyces cerevisiae is currently used for the production of recombinant DNA-generated proteins derived from a variety of eukaryotic organisms. The applications of a yeast-based technology in the production of proteins for pharmaceutical and industrial purposes is discussed including current methods for introducing recombinant genes into yeast and strategies for maximizing their expression.     

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.     

Phosphorylation regulates integration of the yeast Ty5 retrotransposon into heterochromatin

The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5's mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock's hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge.     

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.     

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.     

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Ty1-copia group retrotransposon sequences in amphibia and reptilia

We have isolated sequences belonging to Tyl-copia group retrotransposons from the genomes of an amphibian (Pyxicephalus adspersa) and three reptiles (Conolophus subscristatus, Amblyrynchus cristatus and Pytas mucosus). Two different seqences were found in the amphibian (Tpa1 and Tpa2). Each is present in several copies per genome and absent from the genomes of two other amphibian species. The C. subcristatus sequence Tcs1 is present in multiple copies in both its host genome (Galapagos land iguana) and the genome of the related Galapagos marine iguana (A. cristatus). There is little or no polymorphism in Tcs1 insertions between different individual animals, suggesting that this sequence is not transposing rapidly in either iguana genome. The P. mucosus sequence Tpm1 shows a discontinuous distribution in snake species, suggesting that it has either been lost from many lineages during vertical germline transmission or has been transferred horizontally in some snake species. Phylogenetic comparisons of all these sequences with each other and with other members of this retrotransposon group from other animals and plants show that sequences within a particular vertebrate species are most closely related to each other, consistent with a vertical transmission model for their evolution.     

Genome evolution mediated by Ty elements in Saccharomyces

How mobile genetic elements molded eukaryotic genomes is a key evolutionary question that gained wider popularity when mobile DNA sequences were shown to comprise about half of the human genome. Although Saccharomyces cerevisiae does not suffer such "genome obesity", five families of LTR-retrotransposons, Ty1, Ty2, Ty3, Ty4, and Ty5 elements, comprise about 3% of its genome. The availability of complete genome sequences from several Saccharomyces species, including members of the closely related sensu stricto group, present new opportunities for analyzing molecular mechanisms for chromosome evolution, speciation, and reproductive isolation. In this review I present key experiments from both the pre- and current genomic sequencing eras suggesting how Ty elements mediate genome evolution.