Biomedical subjects

U Vijayraghavan

Publications and source records attributed to U Vijayraghavan.

6 recordsLinked to original sources

PubMed1990

Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae.

We have isolated a yeast mutant, ts352, that is temperature-sensitive for growth. The mutation has a general effect on mRNA metabolism and a specific effect on tRNA biosynthesis. Cells shifted to the nonpermissive temperature accumulate tRNAs that are shorter than mature tRNAs. The increased ability of these tRNAs to accept ATP demonstrates that growth of the ts352 mutant at the nonpermissive temperature results in accumulation of tRNA with defective 3' ends. The activity of ATP (CTP):tRNA-specific tRNA nucleotidyltransferase can readily be measured in extracts from wild type but not mutant cells. We have cloned and sequenced the wild type allele of the ts352 gene and find significant similarity between the yeast protein sequence predicted from the DNA sequence and the protein predicted from the sequence of the Escherichi coli tRNA nucleotidyltransferase gene. Expression of the yeast gene on a multicopy plasmid increases the activity of the tRNA nucleotidyltransferase in extracts. We conclude that the defect in the ts352 mutant is in the gene coding for yeast tRNA nucleotidyltransferase and that we have isolated the yeast gene that codes for this enzyme.

Amino Acid Sequence↗

PubMed1990

Accumulation of pre-tRNA splicing '2/3' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.

Base Sequence↗

PubMed1990

A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation.

We report on the characterization of the yeast prp20-1 mutant. In this temperature-sensitive mutant, multiple steps of mRNA metabolism are affected. The prp20-1 mutant strain showed alterations in mRNA steady-state levels, defective mRNA splicing and changes in transcription initiation or termination when shifted from the permissive to the non-permissive temperature. In addition, a change in the structure of the nucleus in these cells became apparent. Electron microscopy revealed an altered structure of the nucleoplasm of prp20-1 mutant cells when grown at the non-permissive temperature that was not observed in cells grown at the permissive temperature or in wild-type cells. The wild-type PRP20 gene was isolated and sequenced. The putative PRP20 protein has a molecular weight of 52 kDa. We found that the PRP20 gene is identical to the yeast SRM1 gene (Clark and Sprague 1989). In addition, the PRP20 protein sequence shows significant sequence similarity to the human RCC1 protein (Ohtsubo et al. 1987). This protein has been implicated in the control of chromosome condensation. Based on the phenotype of the prp20-1 mutant and the observed sequence similarity to the human RCC1 protein, we postulate that the yeast PRP20 protein is involved in the control of nuclear organization.

Amino Acid Sequence↗

PubMed1990

PRP18, a protein required for the second reaction in pre-mRNA splicing.

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

Cloning, Molecular↗

PubMed1989

Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae.

In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of approximately 1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(rna)17-prp(rna)27 with four phenotypic classes of mutants and 21 mutants in the prp2-prp11 complementation groups (formerly rna2-rna11). The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(rna)2-prp(rna)11 mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate of splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(rna)2-prp(rna)11 groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing.

Alleles↗

PubMed1986

Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome.

Yeast introns contain three highly conserved sequences which are known to be required for splicing of pre-mRNA. Using in vitro mutagenesis, we have synthesized seven point mutations at five different sites in these signals in the yeast actin intron. The mutant introns were then inserted into each of three constructs, which allowed us to assess the consequences both in vivo and in vitro. In virtually every case, we found the efficiency of splicing to be significantly depressed; mature mRNA levels in vivo ranged from 0 to 47% of wild-type. Surprisingly, the tightest mutations were not necessarily at the sites of nucleolytic cleavage and branch formation; these nucleotides are thus highly preferred, but are not absolutely necessary. Moreover, while particular nucleotides are specifically required for the final step in splicing, i.e. 3' cleavage and exon ligation, the predominant consequence of mutation within the conserved signals appears to be the inhibition of assembly of the splicing complex.

Actins↗