Faculty Bibliography

The role of tRNAs in protein synthesis seems routine when compared with the novel ways in which the Ty retrotransposons of Saccharomyces cerevisiae use these interpreters of the genetic code. tRNAs and tRNA genes control essential steps in the retrotransposon life cycle by regulating protein expression, priming DNA synthesis and specifying integration target sites.

A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determined. The sequenced insertions all fell within a few very limited regions of chromosome III. Most of these regions contained tRNA coding regions and/or LTRs of preexisting transposable elements. Open reading frames were disrupted at a far lower frequency than expected for random transposition. The results suggest that the Ty1 integration machinery can detect regions of the genome that may represent "safe havens" for insertion. These regions of the genome do not contain any special DNA sequences, nor do they behave as particularly good targets for Ty1 integration in vitro, suggesting that the targeted regions have special properties allowing specific recognition in vivo.

Two families of retrotransposons, Tf1 and Tf2, have been isolated from the fission yeast, Schizosaccharomyces pombe. We report here the nucleotide (nt) sequence of a Tf2 element, the only retrotransposon family known from the commonly used laboratory strains, 972 and 975, and their derivatives. The total nt sequence of Tf2 was derived from the complete sequence of the coding region and 3' long terminal repeat (LTR) of randomly cloned element Tf2-1, and from a full 5' LTR and approximately one-third of the open reading frame (ORF) of Tf2-43, a Tf2 element found in the head-to-head orientation adjacent to the Sz. pombe rpb6 gene. The two Tf2 sequences are nearly identical and both of them contain a single ORF encoding a protein with regions of sequence similar to protease, reverse transcriptase, RNase H (RH) and integrase from other retrotransposons and retroviruses. Sequence comparisons between Tf1 and Tf2 indicate an extreme divergence of the putative capsid protein-encoding regions of these two elements, as well as divergence of a segment of the LTR, but otherwise virtually identical sequence.

Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol proteins. The different ORFs are thought to allow for overexpression of the Gag protein relative to Pol protein presumed necessary for the assembly of functional retrovirus particles and virus-like particles (VLPs). The results of in vivo experiments designed to detect the transposition of Tf1 show that Tf1 is indeed active and can insert itself into the host genome via a true retrotransposition process. Thus, a paradox emerged between the lack of any obvious means of overexpressing Tf1 Gag protein and the demonstrated functionality of the element. Epitope tagging experiments described here confirm that the Tf1 large ORF is intact and that there is no translational or transcriptional mechanism used to overexpress the Tf1 Gag protein. In addition, we used sucrose gradients and antisera specific for Tf1 capsid (CA) and integrase (IN) to show that the Tf1 proteins do assemble into uniform populations of macromolecular particles that also cosediment with Tf1 reverse transcription products. This evidence suggests that Tf1 proteins form VLPs without using the previously described mechanisms that retroviruses and retrotransposons require to overexpress Gag proteins.

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

Tf1, a retrotransposon from fission yeast, has LTRs and coding sequences resembling the protease, reverse transcriptase and integrase domains of retroviral pol genes. A unique aspect of Tf1 is that it contains a single open reading frame whereas other retroviruses and retrotransposons usually possess two or more open reading frames. To determine whether Tf1 can transpose, we overproduced Tf1 transcripts encoded by a plasmid copy of the element marked with a neo gene. Approximately 0.1-4.0% of the cell population acquired chromosomally inherited resistance to G418. DNA blot analysis demonstrated that such strains had acquired both Tf1 and neo specific sequences within a restriction fragment of the same size; the size of this restriction fragment varied between different isolates. Structural analysis of the cloned DNA flanking the Tf1-neo element of two transposition candidates with the same regions in the parent strain showed that the ability to grow on G418 was due to transposition of Tf1-neo and not other types of recombination events.

We describe the isolation, sequence and construction of a disruption of the yeast SDH1 gene, encoding the flavoprotein subunit of succinate dehydrogenase. This is the first eukaryotic flavoprotein subunit-encoding gene to be fully sequenced. The deduced amino acid (aa) sequence is 50% identical to the Escherichia coli enzyme sequence. The yeast gene encodes an N-terminal extension of 45 aa relative to the E. coli sequence which may act as a mitochondrial targeting signal. Disruption of the gene results in the inability to respire, assayed as the inability to utilize the nonfermentable carbon source, glycerol. This is the expected phenotype for disruption of an essential component of the yeast citric acid cycle.