Faculty Bibliography

We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.

Yeast Ty1 elements are retrotransposons that transpose via an RNA intermediate found in a virus-like particle (Ty-VLP). A Ty-encoded reverse transcriptase activity found inside the particles is capable of giving rise to full-length reverse transcripts. The predominant form of these reverse transcripts is a full-length linear duplex DNA. We have developed a cell-free system for transposition of Ty1 DNA molecules into a bacteriophage lambda target. Purified Ty-VLPs and target DNA are the only macromolecular components required for the transposition reaction. A TYB-encoded protein, p90-TYB, contains amino acid sequences that are similar to those of retroviral integrase proteins. Mutations in the integrase coding region abolish transposition both in vivo and in vitro.

Saccharomyces cerevisiae Ty elements are transposons closely related to retroviruses. The DNA sequence of a functional Ty element (TyH3) is presented. The long terminal repeat sequences are different, suggesting that TyH3 is a recombinant Ty element. A chromosomal Ty element near the LYS2 gene, Ty173, was found to be nonfunctional, even though it has no detectable insertions or deletions. The defect in Ty173 transposition is caused by a missense mutation giving rise to a Leu-to-Ile substitution in the TYB (pol) open reading frame. Several chromosomal Ty elements carry this lesion in their DNA, indicating that nonfunctional Ty elements are common in the yeast genome.

Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced. These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene. Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome. A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette. This may be because it does not allow the transcription run-through necessary for Ty1 transposition. Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.

The yeast retrotransposon Ty can be used to insert multiple copies of a gene at new sites in the genome. The gene of interest is inserted into a GALI-Ty fusion construct; the entire "amplification cassette" is then introduced into yeast on a high copy number plasmid vector. Transposition of the Ty element carrying the gene occurs at multiple sites in the genome. Two genes, a bacterial neomycin phosphotransferase gene and the yeast TRPl gene, were amplified in this way. Although the amplified genes were about 1 kilobase in length, they were amplified to about the same extent as a 40-base pair segment. The benefit of this "shotgun" approach is that amplification can be achieved in one set of manipulations.

By following the fates of genetically marked Ty elements, we observed a very high frequency (80-90%) of deletion between directly repeated marker sequences during transposition. From blot hybridization analyses of Ty RNA and DNA species found in the Ty virus-like particles, we determined that the deletion events occurred during or immediately after reverse transcription of Ty RNA but before integration of Ty DNA. The results suggest that the Ty reverse-transcription machinery can recognize homologous sequences in the template. This capacity may be utilized in the replication and recombination processes of retrotransposons and retroviruses.

We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars.

5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element. The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101. Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F. Winston, K. J. Durbin, and G. R. Fink, Cell 39:675-682, 1984). Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent. Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid. In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.