Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.

Abstract

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GALl upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone aL-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.Retroviruses and long terminal repeat (LTR)-containing retrotransposons have similar structures and transposition cycles (58). The common steps of replication and integration for these elements, henceforth referred to collectively as retroviruslike elements, are (i) transcription of an integrated DNA copy of the element into RNA, (ii) translation of the RNA followed by assembly of particles composed of the RNA as well as the proteins encoded by the element, (iii) reverse transcription of the RNA into DNA, and (iv) integration of this DNA copy into the host cell genome. Retrotransposons are competent for transposition in the cell within which they reside, while retroviruses infect other cells.Retroviruslike elements have genomes of between 2 and 8 kbp which include LTRs of a few hundred base pairs and code for both major structural proteins of the viral particles and enzymes necessary for transposition. The LTRs contain information for initiating transcription of the element and for terminating and processing transcripts. The genes of retroviruses are generally classified as gag, pol, and env. The gag gene products include the capsid protein (CA), which is the main structural component of the retroviruslike element particle; the nucleocapsid protein (NC), which binds the genomic RNA of the element; and, in some cases, the protease (PR), which processes the polyprotein translation products of gag and pol. In other retroviruses, PR is encoded by the pol gene. The other poi gene products are the reverse transcriptase (...

RNA polymerase III interferes with Ty3 integration

“…1. PCR assay for Ty3 position specific integration [30]. VLPs shown in upper left function as the source of Ty3 DNA and integrase activity.…”

“…Integration was detected by PCR amplification of a diagnostic fragment using primers which annealed in Ty3 (primer 278) and upstream of the modified tRNA gene carried on pDLC374 (primer 279) as described previously [30] and as illustrated in Fig. 1.…”

“…It also carries a modified SUP2 tRNA Tyr gene. The positive control plasmid pTM842 was derived from pDLC374 via an in vivo integration of Ty3 at position -1, relative to the nt of tRNA gene transcription initiation in an orientation such that transcription from the tRNA gene and from the Ty3 promoter were divergent [30]. The plasmid pEGTy3-l has been described previously [31].…”

“…The pellets were washed with 70% ethanol, dried, and dissolved in 50 ul of buffer containing 10 mM TrisHCl, pH 8.0 and 1 mM EDTA. The products were analyzed by polymerase chain reaction (PCR) [30].…”

“…In order to normalize for the amount of target plasmid recovered from integration reactions in the 278/279 PCR reactions, PCRs were performed using primers which detect the HIS3 gene (primers 284 and 285) [30]. This gene is carried on the target plasmid.…”