Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.1.1.4 (leucyl-tRNA synthetase)
297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.
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PMID:Isolation and characterization of revertants of the mammalian temperature sensitive leucyl-tRNA synthetase mutant tsHl. 42 61

The genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl-tRNA synthetase mutant, which under non-permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L-73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1-like state with maintenance of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR-73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.
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PMID:Characterization of cell lines showing growth control isolated from both the wild type and a leucyl-tRNA synthetase mutant of Chinese hamster ovary cells. 43 1

Three forms (E1, E2 and E3) of leucyl-tRNA synthetase (LeuRS) were separated by DEAE-cellulose chromatography of total aminoacyl-tRNA synthetases from cow lactating mammary gland. The method of purification of all three components is described. E1 is a dimeric molecule (alpha 2) of molecular weight 182 000. Two other forms of molecular weight 67 000 and 64,000 consist of a single polypeptide chain as determined by polyacrylamide gel electrophoresis. Optimum conditions and kinetic parameters of leucyl-tRNA formation were studied for every enzyme form. The low values of Vmax and thermostability are characteristic of E3. All forms of LeuRS interact with 6 isoaccepting tRNA(Leu) from lactating mammary gland and can activate leucine in the absence of tRNA. E2 and E3 are supposed to derive from the native enzyme by endogenous proteolysis. The physico-chemical properties of native LeuRS from lactating mammary gland are compared with those of LeuRS's from other sources.
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PMID:[Purification and properties of leucyl-tRNA synthetase from the cow mammary gland]. 57 75

Forty-three temperature-sensitive mutants were isolated in the CHO cell line by selecting for noncycling cells using [3H]TdR and cytosine arabinoside. Cell division was extremely temperature sensitive in eight of the mutants, and these were studied in more detail. In seven of these eight mutants, the in vitro specific activity of a single aminoacyl-tRNA synthetase was greatly reduced; four had reduced levels of histidyl-tRNA synthetase, two of valyl-tRNA synthetase, and one of leucyl-tRNA synthetase. Cell hybridization studies showed that the mutants formed three complementation groups. In six of the seven mutants the aminoacyl-tRNA synthetase which had reduced activity was also more thermolabile than the wild-type enzyme. The spontaneous reversion frequency was low for these mutants, and in most cases could be increased by treatment with a chemical mutagen. The isolation of the valyl-tRNA synthetase mutant reported here brings to eight the number of different aminoacyl-tRNA synthetase mutants isolated in the CHO cell line.
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PMID:Mutations in the structural genes of CHO cell histidyl-, valyl-, and leucyl-tRNA synthetases. 69 22

The regulation of the transport of leucine, isoleucine, and valine in Escherichia coli B/r was studied in a mutant with a complete deletion of the leucine biosynthetic operon and a temperature-sensitive leucyl-tRNA synthetase [L-leucine:tRNALeu ligase (AMP-forming), EC 6.1.1.4]. Under conditions of excess leucine and a functional leucyl-tRNA synthetase transport activity was repressed. Shifting the culture to a temperature at which the activation of leucine to an appropriate tRNA species became growth-rate-limiting led to a large increase in the high-affinity transport of leucine, isoleucine, and valine (system LIV-I) while the uptake of histidine and proline was unchanged. A similar increase was observed for branched-chain amino-acid binding protein activity. The temperature change did not alter the transport activity for any of these substrates or the level of the binding proteins in an isogenic strain with a normal leucyl-tRNA synthetase. The increase in transport activity observed in the mutant was prevented by inhibitors of protein and RNA synthesis and probably represents an increase in the differential rate of synthesis of the protein(s) required for transport. These experiments demonstrate that the repression of branched-chain amino-acid transport involves the interaction of leucine with its aminoacyl-tRNA synthetase and its cognate leucyl-tRNA species.
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PMID:Role of leucyl-tRNA synthetase in regulation of branched-chain amino-acid transport. 110 69

Efficient selection procedures, using [3H]amino acids as the selecting agent, were developed for isolating temperature-sensitive (TS) mutations in CHO cells affecting protein synthesis. After chemical mutagenesis, leucyl-tRNA synthetase mutants were obtained when [3H]leucine was used as the selecting agent in two independent experiments. These mutations seem to involve the same genetic locus as the TSH1 mutant described previously (1). A selection with [3H]valine, in which all amino acids except leucine were at low concentration in the selective medium, resulted in a new class of mutants with reduced asparagyl-tRNA synthetase activity. These results were consistent with the finding that all mutants were phenotypically dependent on the concentration of amino acid, specific to the altered synthetase, in the medium. Our observations suggest that although leucyl synthetase mutations are a relatively common class of TS mutations in CHO cells, the spectrum of mutants obtained can be at least partially manipulated through concentrations of amino acids in selective media. The asparagyl-synthetase mutation was shown to be recessive and to complement the leucyl-synthetase mutation in cell-cell hybrids.
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PMID:Selection by [3H] amino acids of CHO-cell mutants with altered leucyl- and asparagyl-transfer RNA synthetases. 123 6

The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced. A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles. The leuS1 mutation was mapped to 270 degrees on the chromosome. Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon. The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B. subtilis ilv-leu operon. The DNA of the B. subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E. coli.
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PMID:Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis. 131 42

Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation. Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity. Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport. Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor. Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA. Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA. Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor. These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.
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PMID:Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli. 137 12

Streptomyces bldA gene, which encodes a tRNA corresponding to a very minor leucine codon, UUA, regulates pleiotropic gene expression which is involved in sporulation and secondary metabolism. The unique structural feature of this tRNA is the lack of GG sequence in dihydrouridine loop (D-loop) that generally is conserved in tRNAs involved in cytoplasmic protein biosynthesis. In order to investigate the relationship between the D-loop structure and the stability and leucine accepting activity of this tRNA, the wild and D-loop mutant tRNA transcripts were constructed with T7 RNA polymerase in vitro. The wild type tRNA(UUALeu) showed the structural stability and leucine accepting activity at physiological temperature for Streptomyces. The E.coli type D-loop mutant, which has a larger loop size and contains a GG doublet, exhibited increased thermostability. The kinetical analyses of the aminoacylation reaction of tRNA(UUALeu) with S.lividans and E.coli leucyl-tRNA synthetase (LeuRS) suggest there is a unique recognition mechanism of Streptomyces LeuRS toward tRNA(UUALeu).
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PMID:The effects of a unique D-loop structure of a minor tRNA(UUALeu) from Streptomyces on its structural stability and amino acid accepting activity. 138 Jun 90

The cdc60 mutation (for cell division cycle) of the yeast, Saccharomyces cerevisiae, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature [Bedard et al., Curr. Genet. 4 (1981) 205-214]. We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype. Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurospora crassa, implying that CDC60 encodes the corresponding yeast protein. Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle. Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA. The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.
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PMID:The cell division cycle gene CDC60 encodes cytosolic leucyl-tRNA synthetase in Saccharomyces cerevisiae. 139 22


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