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T Stearns

Publications and source records attributed to T Stearns.

12 recordsLinked to original sources
PubMed1991

Gamma-tubulin is a highly conserved component of the centrosome.

We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.

Amino Acid Sequence↗
PubMed1990

Manipulating yeast genome using plasmid vectors.

The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

Cloning, Molecular↗
PubMed1990

ADP-ribosylation factor is functionally and physically associated with the Golgi complex.

ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.

ADP-Ribosylation Factor 1↗
PubMed1990

Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function.

Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.

Benomyl↗
PubMed1990

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes.

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

Benomyl↗
PubMed1990

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes.

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

ADP-Ribosylation Factor 1↗
PubMed1989

DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage.

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

Aneuploidy↗
PubMed1988

Unlinked noncomplementation: isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae.

Mutations in genes of Saccharomyces cerevisiae that code for proteins that interact with beta-tubulin were sought by screening for unlinked mutations that fail to complement mutations in the single beta-tubulin-encoding gene (TUB2). Among the first three noncomplementing mutations examined, two are linked to TUB2 while one is unlinked. The unlinked mutation was shown to be a conditional-lethal allele of the major alpha-tubulin-encoding gene (TUB1) and represents the first such mutation in that gene. The tub1-1 mutation itself causes a cold-sensitive cell-cycle arrest, and confers supersensitivity to the antimicrotubule drug benomyl. These phenotypes occur in the presence of a wild-type copy of the minor alpha-tubulin-encoding gene, TUB3; the combination of tub1-1 and a tub3 null mutation is inviable in haploids. Through further application of this method, new mutations in TUB2 and TUB3 were isolated as unlinked noncomplementers of tub1-1. The noncomplementation between tub1 and tub2 mutations is gene specific and allele specific, suggesting that the phenotype is due to an interaction at the protein level. We conclude that isolation of unlinked noncomplementing mutations is likely to be a generally useful method for isolating mutations in interacting gene products.

Crosses, Genetic↗