Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Properties of asparagine synthetase in asparagine-independent variants of Jensen rat sarcoma cells induced by 5-azacytidine.

Jensen rat sarcoma cells in culture require L-asparagine for growth and lack detectable levels of asparagine synthetase. Cultures exposed for 24 h to graded concentrations of 5-azacytidine give rise to asparagine-independent variants in high frequency. These prototrophs are stable phenotypically whether maintained in the presence or absence of L-asparagine. Asparagine synthetase activity in several variant clones was uniform in thermolability and several kinetic parameters, as well as in immunological properties. Parental Jensen rat sarcoma cells contained no detectable immunologically cross-reacting material. Our data suggest that transitions between asparagine dependence and independence in these cells are mediated by stable shifts in gene expression rather than by structural gene mutations.

Histidyl-tRNA synthetase of Chinese hamster ovary cells contains phosphoserine.

The Chinese hamster ovary cell line 2H2-5, which expresses elevated levels of histidyl-tRNA synthetase, was used to demonstrate that histidyl-tRNA synthetase contains phosphoserine. After growth of the cells in medium containing [32P]orthophosphate, histidyl-tRNA synthetase was isolated by partial purification and immunoprecipitation and shown to be a phosphoprotein. Phosphoamino acid analysis showed that histidyl-tRNA synthetase is phosphorylated on serine, as has previously been shown for threonyl-tRNA synthetase of CHO cells.

Nucleotide and deduced amino acid sequence of human cysteinyl-tRNA synthetase.

We have determined the nucleotide sequence of a cDNA coding human cysteinyl-tRNA synthetase. The predicted protein sequence of 638 amino acids has substantial sequence similarity to the E. coli cysteinyl-tRNA synthetase in the nucleotide-binding fold region that constitutes the potential active site. The results are consistent with the hypothesis that evolution of the full complement of aminoacyl-tRNA synthetases occurred prior to the divergence of prokaryotes and eukaryotes.

Bouveret's syndrome: successful endoscopic treatment of gastric outlet obstruction caused by an impacted gallstone.

Bouveret's syndrome is defined as gastric outlet obstruction caused by duodenal impaction of a large gallstone which passes into the duodenal bulb through a cholecystogastric or cholecystoduodenal fistula. This is a report of a 62 years old female who presented with complaint of persistent vomiting and upper abdominal pain for the last 5 days. Ultrasound abdomen was suggestive of pneumobilia. CT scan of upper abdomen showed cholecystoduodenal fistula and complete obstruction of third part of duodenum by a large stone, which was reported as Bouveret's syndrome. She underwent emergency gastroscopy. The stone was retrieved by Dormia basket, crushed with lithotripter and extracted endoscopically.Complete intestinal obstruction was relieved endoscopically.

Threonyl-tRNA synthetase from Chinese hamster ovary cells is phosphorylated on serine.

A Chinese hamster ovary cell line, 2000A, in which threonyl-tRNA synthetase accounts for 1.5% of the total soluble protein, was used to demonstrate that this enzyme is a phosphoprotein. Threonyl-tRNA synthetase was isolated by immunoprecipitation from cells labeled with 32Pi for 18 h. Phosphoamino acid analysis of radiolabeled threonyl-tRNA synthetase showed that phosphorylation occurs on serine.

Chinese hamster ovary cells resistant to borrelidin overproduce threonyl-tRNA synthetase.

Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase.

Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis.

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.

Genetic differences inthe te of histidase synthesis in inbred mice.

Inbred strains of mice fall into two groups with respect to their liver histidase activity levels, high strains having approximately twice as much activity as low strains. Analysis of the F1, F2, and backcross progeny of the mating of a high activity strain (C57BL/6J) and a low activity strain (C3H/HeJ) indicates that the difference between the strains is determined by a single genetic locus with two alleles exhibiting additive inheritance. No differences with respect to various physical and kinetic parameters were found in studies of partially purified histidase from both strains. Quantitation of the amount of enzyme present by immunotitration showed that the amount of enzyme antigen is proportional to the level of enzyme activity in the two strains. Measurements of the relative rates of histidase synthesis by combined radiochemical and immunological techniques showed that the relative rate of synthesis was closely correlated with the amount of enzyme present. Rates of enzyme degradation in the two strains, measured by recovery of activity after irreversible inhibition with nitromethane, were the same.

Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes.

The nucleotide sequence of a cDNA coding human threonyl-tRNA synthetase has been determined. The predicted protein sequence is highly homologous to that of the yeast cytoplasmic, yeast mitochondria and Escherichia coli threonyl-tRNA synthetases. In particular, the three structural motifs recently shown to be common to class II aminoacyl-tRNA synthetases are present in the threonyl-tRNA synthetases from all sources. Primer extension and S1 nuclease analyses indicate that transcription initiates approximately 220-230 nucleotides upstream of the putative initiator methionine codon. This region contains a 10-nucleotide interrupted inverted repeat flanked by a 13-nucleotide interrupted direct repeat.

Inhibition of growth of Salmonella typhimurium and of threonine deaminase and transaminase B by beta-chloroalanine.

beta-Chloro-l-alanine (CA) was found to inhibit the growth of Salmonella typhimurium. The inhibition was overcome by isoleucine plus valine. CA inhibited the activity of threonine deaminase and transaminase B in vitro. The inhibition of threonine deaminase was reversible and was not affected by desensitization to isoleucine inhibition. Transaminase B was irreversibly inactivated. Derepression of some of the isoleucine and valine biosynthetic enzymes was achieved by growth of wild-type cells on low levels of CA.

Aminoacyl-tRNA synthetase mutants degrade protein at a normal rate.

The stability of both rapidly and slowly degraded proteins in wild type CHO cells is similar to that in three ts aminoacyl-tRNA synthetase mutants at both permissive and non-permissive temperatures, although the degree of tRNA charging in the synthetase mutants differs considerably with temperature. These results indicate that the altered rate of protein breakdown seen under a variety of physiological conditions in eukaryotic systems is not mediated by uncharged tRNA.

Assignment of histidase-regulating locus to chromosome 10 of the mouse.

Data from four sets of recombinant inbred strains confirm that variation at a single genetic locus is responsible for the previously observed differences in the rate of histidase synthesis in inbred mice. Linkage testing stocks were used to demonstrate linkage with steel (Sl) on chromosome 10.

Increased levels of threonyl-tRNA synthetase in a borrelidin-resistant Chinese hamster ovary cell line.

The growth of Chinese hamster ovary cells in medium containing reduced concentrations of threonine is inhibited by borrelidin, a macrolide antibiotic. Borrelidin-resistant clones have been isolated after ethyl methanesulfonate mutagenesis. One clone, 1C-1, has a 3-fold increased level of threonyl-tRNA synthetase [L-threonine:tRNAThr ligase (AMP-forming), EC 6.1.1.3] as determined by both activity measurements and antiserum titrations. The levels of four other aminoacyl-tRNA synthetases and of tRNAThr are the same in strain 1C-1 and in the wild-type parent. The phenotype of increased threonyl-tRNA synthetase activity is recessive to wild type in cell hybrids.

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family.

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.

Threonyl-tRNA synthetase gene maps close to leucyl-tRNA synthetase gene on human chromosome 5.

The structural gene for threonyl-tRNA synthetase was mapped to human chromosome 5 by an analysis of the isoelectric focusing patterns of this enzyme from human X Chinese hamster interspecific somatic cell hybrids. The threonyl-tRNA synthetase gene is the fourth of seven aminoacyl-tRNA synthetase genes mapped in humans to be assigned to this chromosome. Regional mapping studies showed that the threonyl-tRNA synthetase gene is on the short arm of chromosome 5, p13-cen, and is close to, but separable from, the gene for leucyl-tRNA synthetase which maps to 5cen-5q11.