The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA i Met , the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T⌿C loop and the acceptor stem regions of the tRNA i Met affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.The Ty1 element of the yeast Saccharomyces cerevisiae is a 6-kb retrotransposon with long terminal repeats (LTRs) at each end (10). Transcription of the element produces an RNA molecule from which the element-encoded proteins TYA and TYB are translated. These proteins are functional homologs of retroviral Gag and Pol proteins, respectively. The TYA and TYB proteins assemble into structures termed virus-like particles (VLPs) because of their similarity in structure to retrovirus core particles. These VLPs are intermediates in transposition within which reverse transcription occurs (17). During the assembly process, the Ty1 RNA is packaged within the VLPs and subsequently reverse transcribed into a full-length cDNA. In the final step of transposition, the cDNA is integrated into a new site in the host genome, and the cycle begins again with transcription of the element.Retroviruses and LTR retrotransposons share the problem of having to generate a full-length cDNA from a message that initiates and ends within the LTRs of the element (diagrammed in Fig.

Homologous integration into the fission yeast Schizosaccharomyces pombe has not been well characterized. In this study, we have examined integration of plasmids carrying the leu1+ and ura4+ genes into their chromosomal loci. Genomic DNA blot analysis demonstrated that the majority of transformants have one or more copies of the plasmid vector integrated via homologous recombination with a much smaller fraction of gene conversion to leu1+ or ura4+. Non-homologous recombination events were not observed for either gene. We describe the construction of generally useful leu1+ and ura4+ plasmids for targeted integration at the leu1-32 and ura4-294 loci of S. pombe.

Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in Escherichia coli shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite ؉1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.