Faculty Bibliography

The Saccharomyces cerevisiae dbr1 mutation has been mapped on the left arm of chromosome XI. XIL is a chromosome arm that was until now rather sparsely populated with accurately mapped markers. On the basis of physical data, the overall order of markers is inverted relative to the existing genetic map of XI. We present tetrad analyses using a variety of markers on XI that indicate that the existing genetic map of XIL should be inverted, at least for the strains in which our mapping was carried out, and probably for other S. cerevisiae strains.

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.

The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments demonstrated that mutations at one histone locus, HTA1-HTB1, do cause lethality when in conjunction with mutations in the SPT10 gene. SPT10 has been shown to be required for normal levels of transcription of several genes in S. cerevisiae. Motivated by this double-mutant lethality, we have now investigated the interactions of mutations in SPT10 and in a functionally related gene, SPT21, with mutations at each of the four histone loci. These experiments have demonstrated that both SPT10 and SPT21 are required for transcription at two particular histone loci, HTA2-HTB2 and HHF2-HHT2, but not at the other two histone loci. These results suggest that under some conditions, S. cerevisiae may control the level of histone proteins by differential expression of its histone genes.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus.

A plasmid bearing a GAL1::Ty1 fusion that is competent to transpose was mutagenized by insertion of oligodeoxyribonucleotides that precisely introduce four or five codons semirandomly throughout the plasmid. Approximately one quarter of these resulted in inactivation of transposition; these include inactivating insertions in both the TYA and TYB genes, corresponding to retroviral gag and pol genes. Examples of transposition-inactivating mutations map within each of the known or proposed functional domains of TYB, suggesting that these are all required for retrotransposition. All of the transposition-inactivating mutations were found to be recessive with the exception of a single mutation in TYA. The remaining mutations have slightly deleterious to no effect on Ty1 transposition.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.

Mutations in the SPT10 and SPT21 genes were originally isolated as suppressors of Ty and LTR (delta) insertion mutations in Saccharomyces cerevisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPT10 and SPT21. Since the spt10 mutation used to clone SPT10 resulted in very poor transformation efficiency, a novel method making use of the kar1-1 mutation was used. Neither SPT gene is essential for growth, and constructed null alleles cause phenotypes similar to those caused by spontaneous mutations in the genes. spt10 null alleles are strong suppressor mutations and cause extremely slow growth. Certain spt10 spontaneous alleles are good suppressors but have a normal growth rate, suggesting that the SPT10 protein may have two distinct functions. An amino acid sequence motif that is similar to the Zn-finger motif was found in SPT10. Mutation of the second Cys residue in this motif resulted in loss of complementation of the suppression phenotype but a normal growth rate. Thus, this motif may reside in a part of the SPT10 protein that is important for transcriptional regulation but not for normal growth. Both the SPT10 and SPT21 proteins are relatively tolerant of large deletions; in both cases deletions of the C-terminus resulted in at least partially functional proteins; also, a large internal deletion in SPT21 was phenotypically wild type.

Yeast RNA lariat debranching enzyme has been purified to near homogeneity using a bacterial overproducer of the enzyme. The enzyme is capable of digesting a variety of branched nucleic acid substrates, including group II intron lariats, multicopy single-stranded DNAs (msDNAs), and a variety of synthetic branched RNAs. A trinucleotide release assay using radiolabeled msDNA substrates was developed and used to determine the basic biochemical parameters for the enzyme. The debranching enzyme shows a strong preference for purines at the 2'-position in both msDNA and synthetic branched RNA substrates, in accord with the structure of its native substrate, which always has a 2'-G residue. The use of small synthetic branched RNA substrates will allow systematic mechanistic and structural studies of this unique enzyme.