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J Abelson

Publications and source records attributed to J Abelson.

At least 73 records · Page 4Linked to original sources
PubMed1990

Domain structure in yeast tRNA ligase.

Yeast tRNA ligase is one of two proteins required for the splicing of precursor tRNA molecules containing introns. The 95-kDa tRNA ligase has been purified to homogeneity from a strain of Escherichia coli which overexpresses the protein. The ligation reaction requires three enzymatic activities: phosphodiesterase, polynucleotide kinase, and ligase. By partial proteolytic digestion, we have produced fragments of tRNA ligase which contain the constituent activities. These results provide evidence for a model in which the three constituent activities of ligase are located in three distinct domains separated by protease-sensitive regions. We have also located the active adenylylated site in the ligase domains. It is lysine-114. The tRNA ligase sequence in this region has limited homology to the active-site region of T4 RNA ligase.

Adenosine Triphosphate↗
PubMed1990

Construction of Escherichia coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency.

Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA. We have measured the levels of in vivo suppression resulting from introducing each tRNA gene into E. coli via a plasmid vector. The suppressors function at varying efficiencies. Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency. Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not. We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs. Both tRNA(CUAHis) and tRNA(CUAGlu) were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression. The tRNA(CUAPro) was inactive, but a hybrid suppressor tRNA consisting of the tRNA(CUAPhe) anticodon stem and loop together with the remainder of the tRNA(Pro) proved highly efficient at suppressing nonsense codons. Protein chemistry results reported in the accompanying paper show that the altered tRNA(CUAHis) and the hybrid tRNA(CUAPro) insert only histidine and proline, respectively, whereas the altered tRNA(CUAGlu) inserts principally glutamic acid but some glutamine. Also, a strain deficient in release factor I was employed to increase the efficiency of weak nonsense suppressors.

Anticodon↗
PubMed1990

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity.

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.

Amino Acid Sequence↗
PubMed1990

Do health service organizations and community health centres have higher disease prevention and health promotion levels than fee-for-service practices?

We interviewed health care providers representing 23 fee-for-service (FFS) practices, 19 health service organizations (HSOs) and 11 community health centres (CHCs) in Ontario to compare self-reported approaches to disease prevention and health promotion. Few significant differences were found across practice types in the presence of recall systems for screening or in knowledge of, compliance with or estimated coverage for selected preventive maneuvers recommended by the Canadian Task Force on the Periodic Health Examination. CHCs reported a significantly greater variety of formal health promotion programs and a greater tendency to use nonphysician health care personnel to carry out both prevention and health promotion activities. The results must be interpreted with caution because of the use of self-reported data, the low response rate for FFS practices and the use of a restrictive definition of disease prevention tied to evidence from the reports of the task force. Thus, the results cast some doubt on the common assumption that increasing the population served by alternative modes of delivery such as HSOs and CHCs necessarily increases the level of disease prevention and health promotion activity.

Aged↗
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

PRP5: a helicase-like protein required for mRNA splicing in yeast.

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein.

Amino Acid Sequence↗
PubMed1990

Identification of five putative yeast RNA helicase genes.

The RNA helicase gene family encodes a group of eight homologous proteins that share regions of sequence similarity. This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double-stranded RNA. Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mitochondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation. We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes. Northern and Southern blot analyses demonstrated that these genes are single copy and expressed in yeast. Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes.

Amino Acid Sequence↗
PubMed1990

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs.

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single-strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein.

Antibodies↗
PubMed1990

Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.

U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.

Base 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

In vitro reconstitution of functional yeast U2 snRNPs.

A system for the functional reconstitution of yeast U2 snRNPs using synthetic U2 RNAs is described. We use oligonucleotide-directed RNase H cleavage to specifically deplete yeast extracts of their endogenous full-length U2 snRNA and consequently inactivate pre-mRNA splicing activity. The subsequent addition of synthetic yeast U2 RNAs, derived by in vitro transcription (T7U2 RNAs), to these oligonucleotide-treated extracts efficiently reconstitutes their ability to splice pre-mRNA. The use of deletion derivatives of the T7U2 RNA has demonstrated that the region downstream from the conserved Sm-binding site sequence in the yeast U2 RNA is not absolutely required for pre-mRNA splicing activity in vitro. Furthermore, we found that both human and rat U2 RNAs can function in yeast extracts. We also show that point mutations in the yeast U2 RNA can be analyzed using the in vitro reconstitution system. Allele-specific suppression of mutations in pre-mRNA branch site sequence is observed when the appropriate compensatory mutations in the branch site recognition region of the T7U2 RNA are introduced. Finally, we present a model for the interaction of the U2 and U6 snRNAs during pre-mRNA splicing.

Base Sequence↗
PubMed1989

In vitro assembly of yeast U6 snRNP: a functional assay.

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.

Base Sequence↗
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↗
PubMed1989

Protein engineering with synthetic Escherichia coli amber suppressor genes.

We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.

Amino Acid Sequence↗
PubMed1989

tRNA identity.

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Amino Acids↗
PubMed1988

Electron microscopic identification of the yeast spliceosome.

We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.

Bacterial Proteins↗