tryptophanyl-tRNA Synthetase Gene Research Articles

Astragerm — activator of gene expression peroxisoms coactivator

The effect of the biologically active additive «Astragerm» on the activity of PGC-1-alpha was studied. The activating effect of astragerm on the expression of PGC-1-alpha in the «in vivo» system in rat muscle tissues was shown. Methods for quantitative assessment of mitochondria in tissues and analysis of the expression of genes included in the cascades of regulatory processes of energy metabolism were applied. Coordinated stimulation of tryptophanyl-tRNA synthetase gene expression, its non-canonical activity in the synthesis of the second messenger Ap3A, and expression of the PGC-1 alpha gene in «in vivo» systems under the action of astragerm was found, which indicates the unity of the mechanisms of energy regulation with the functions of protein synthesis and nonspecific immunity.

Properties of the drug Trekrezan to modulate protein synthesis in cells

New factors contributing to the processes of hormesis involving the peroxisome activator receptor coactivator 1 alpha gene (PGC-1α) and linking the mechanisms of energy regulation with the functions of protein synthesis were studied. Coordinated stimulation of the expression of the tryptophanyl-tRNA synthetase gene, its activity in the synthesis of the tertiary messenger aP3A and the expression of the PGC-1α gene in in vivo and in vitro systems, under the action of the immunostimulating and adaptogenic drug Trekrezan, was shown. It has been suggested that PGC—1α is a promising target for the therapeutic effect of drugs stimulating gene activity.

Regulation of an Auxiliary, Antibiotic-Resistant Tryptophanyl-tRNA Synthetase Gene via Ribosome-Mediated Transcriptional Attenuation

cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5' leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs of trpRS1 that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of a trpRS1 ortholog (SAV4725) in Streptomyces avermitilis. We conclude that the trpRS1 locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyl-tRNA synthetase genes are regulated in bacteria.

A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations.

Listeria monocytogenes as novel carrier system for the development of live vaccines

Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis (“zipper mechanism”) and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene ( trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.

Tryptophanyl-tRNA synthetase gene polymorphisms and risk of incident myocardial infarction

Tryptophanyl-tRNA synthetase (WARS) gene polymorphisms have been associated with the patho-physiology of vascular angiogenesis and homeostasis. Data from a recent genome-wide linkage analysis suggested a potential role of WARS in the risk of myocardial infarction. However, no genetic-epidemiological data are available. From a prospective cohort of 14,916 initially healthy American men, we evaluated five common polymorphisms within or close to the WARS locus, all with a minor allele frequency >0.10, amongst 386 individuals who subsequently developed myocardial infarction and 386 matched individuals who remained free of reported cardiovascular events during follow-up. The polymorphisms were: a G > C substitution in the 5′-flanking region (dbSNP rs2273804), an A > G substitution in intron 1 (dbSNP rs941931), a 335T > C substitution in exon 10 (dbSNP rs9453), a C > T substitution in intron 10 (dbSNP rs1570305), and a C > T substitution in the C14orf68 region (dbSNP rs3736951). The observed genotypes were in Hardy–Weinberg equilibrium in the control group. Genotype- and haplotype-frequency distributions were similar between cases and controls. Further investigation using a haplotype-based matched logistic regression analysis, adjusting for age, smoking, randomized treatment-assignment (likelihood ratio test: χ 3 2 = 3.20 , p = 0.36) or with additional adjustment for BMI, hypertension, and diabetes (likelihood ratio test: χ 3 2 = 2.38 , p = 0.50) yielded similar null findings. An alternative haplotype analysis based on evolutionary arguments again yielded null results. In conclusion, we found no evidence for an association between the common polymorphisms or haplotypes of the tryptophanyl-tRNA synthetase gene tested and risk of myocardial infarction.

Phorbol ester stimulates expression of the human tryptophanyl-tRNA synthetase gene.

The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on expression of the human interferon (IFN)-inducible tryptophanyl-tRNA synthetase (WRS) gene was studied. PMA caused an increase in the basal and IFNgamma-induced WRS protein content in HeLa and HEK293 cultured cells. Besides, PMA upregulated WRS mRNA level in HeLa cells. Since PMA is known as a selective activator of protein kinase C (PKC) and is widely used to study the PKC-related pathways, these results show possible PKC involvement in regulation of the WRS gene expression. PKC inhibition by staurosporine (10 and 100 nM) had no effect on either basal or IFNgamma-induced expression of WRS in either cell line. Consequently, PKC is not an indispensable element in WRS induction by IFNgamma. Rather, PKC may activate WRS gene expression only by a distinct pathway.

WRS-85D: A tryptophanyl-tRNA synthetase expressed to high levels in the developing Drosophila salivary gland.

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.

Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression.

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon-gamma (IFN-gamma). Transforming growth factor-beta (TGF-beta) antagonizes many cellular responses to IFN-gamma. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF-beta caused time- and dose-dependent abrogation of IFN-gamma-stimulated expression of IDO and tryptophanyl-tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down-regulation of IFN-gamma signaling by TGF-beta. In parallel with its effect on IDO mRNA expression, TGF-beta caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN-gamma. IFN-gamma caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre-mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF-beta partially reversed this stimulation. IFN regulatory factor (IRF)-1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN-gamma. The stimulation by IFN-gamma of IRF-1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF-beta. Furthermore, gel mobility shift assays indicated that TGF-beta did not inhibit the induction of stat1 and IRF-1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF-beta, as shown by determination of mRNA half-lives following blockade of transcription with 5,6-dichlorobenzimidazole riboside. The findings indicate that TGF-beta prevents the induction of IDO and tryptophanyl-tRNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF-beta is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF-beta in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control.

Identification and expression of the Saccharomyces cerevisiae cytoplasmic tryptophanyl-tRNA synthetase gene.

The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site. The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiae enzyme. The HRE342 ORF plus about 250 bp of 5' and 3' flanking sequence was amplified by polymerase chain reaction, cloned into a 2 mu based vector, and transformed into a host strain, S. cerevisiae JG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert. Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1. A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetase suggests there is sufficient similarity to identify both class I and class II genes.

Isolation and characterization of the Bacillus subtilis tryptophanyl-tRNA synthetase gene( trpS) conferring 5-fluorotryptophan resistance and temperature sensitivity

The mutant trpS gene of Bacillus subtilis encoding a thermosensitive tryptophanyl-tRNA synthetase that also confers resistance to growth inhibition by 5-fluorotryptophan at permissive temperature has been isolated and characterized. A point mutation at codon 82 of the gene from a wild-type TCA codon for Ser to a TTA codon for Leu has been identified.

Assignment of the human tryptophanyl-tRNA synthetase gene (WARS) to chromosome 14q32.2 --> q32.32.

Tryptophanyl-tRNA synthetase catalyzes the aminoacylation of tRNAtrp with tryptophan, an essential function in the cell's protein synthesis machinery. It has been shown that tryptophanyl-tRNA synthetase is induced by interferon, and this has led to hypotheses about other possible functions of tryptophanyl-tRNA synthetase. We have mapped a cDNA probe of the tryptophanyl-tRNA synthetase gene (WARS) by a combination of somatic cell hybrid analysis, fluorescence in situ hybridization (FISH), and linkage analysis. Both FISH and linkage analysis independently supported a more distal position of WARS than had been previously reported. FISH mapping indicated a most likely location at 14q32.31. Linkage analysis was based on the 40 reference families from the CEPH collaboration and resulted in a 13-point map, placing WARS, with odds of more than 1,000:1, within an area of approximately 10 cM and, with odds of 198:1, in an approximately 6 cM interval between pCMM101 and D14S27. The study provides additional integration of the physical and genetic maps of the distal part of 14q, as well as genetic tools enabling a more complete understanding of the function of tryptophanyl-tRNA synthetase, especially with regard to the cryptic inducibility of interferon.

Localization of the tryptophanyl tRNA synthetase gene (WARS) on human and bovine chromosomes by in situ hybridization

Bovine tryptophanyl-tRNA synthetase (ECC 6.1.1.2.; WARS) is one of the best studied synthetases from multicellular organisms. Previous assignment of the WARS gene to the long arm of human Chromosome (Chr) 14 has been made by somatic cell hybrid techniques (Denney and Craig 1976; Shimizu et al. 1976). Francke and co-workers (1977) mapped it on 14q21q32. We describe here the localization of the WARS gene to the bovine and human chromosomes with specific cDNA probes (Frolova et al. 1991) for in situ hybridization. Detailed protocols for the in situ hybridization, chromosome staining, and silver grain distribution analysis have been given previously (Graphodatsky et al. 1992). Standard nomenclatures of the human and cattle chromosomes were used (ISCN 1985; ISCNDA 1989). Our results confirm the previously reported assignment of the tryptophanyl-tRNA synthetase gene to Chr 14 (Denney and Craig 1976; Shimizu et al. 1976; Francke et al. 1977). Twenty-four percent of all grains (202 grains from 104 total metaphases) were mapped on Chr 14 (• = 185.0; P < 0.001). Sixty-three percent of the grains over Chr 14 were localized to the 14q23q31 region; 33% to the 14q24 band. These results demonstrate the location of the WARS gene in the 14q23Lq31 region and presumably more precisely in the 14q24 band. Unexpected results were obtained with bovine chromosomes probed with cloned human cDNA. Thirteen percent of all the grains (353 grains per 103 metaphases) were mapped on Chr 3 (• = 53.6; P < 0.001). However, these grains were not condensed in one locus but were concentrated in two separate loci: 38% of grains on BTA3 were found over the qll--q21 region and 36% over the q42-45 region. Figure 1 shows the Gand RBG-banding patterns of cattle 3 (BTA3) Chr and human Chr 14 (HSA14).

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Intrachromosomal gene mapping in man: the gene for tryptophyl-tRNA synthetase maps in region q21 leads to qter of chromosome 14.

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter leads to Xp22::14q21 leads to 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 leads to 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.

Assignment of a human gene for tryptophanyl-tRNA synthetase to chromosome 14 using human-mouse somatic cell hybrids.

Human tryptophanyl-tRNA SYNTHETASE (Trp-RS, EC 6.1.1.2) can be separated from its mouse counterpart by Cellogel electrophoresis. Analysis of the presence or absence of human Trp-RS and other human enzyme markers in eleven independently dervied cell lines of human-mouse somatic cell hybrids revealed that the expression of Trp-RS is correlated with the expression of human nucleoside phosphorylase (NP, EC 2.4.2.1). The syntenic relationship between Trp-RS and NP permits the assignment of the structural gene for Trp-RS to human chromosome 14. Karyotype and isozyme analysis of these hybrid clones rules out other linkage assignments.

Assignment of the human gene for tryptophanyl-tRNA synthetase using human-mouse somatic cell hybrids.

Assignment of the human gene for tryptophanyl-tRNA synthetase using human-mouse somatic cell hybrids.

Regulatory mechanism of the tryptophan operon in Escherichia coli: Possible interaction between trpR and trpS gene products

The trpS5 mutation (a mutation in the structural gene for tryptophanyl-tRNA synthetase (TRSase) in E. coli), when present in the genetic background of strain KY913 (HfrH), results in the failure to grow at high temperature (42° C) in a complete medium. The rel (RC relaxed) marker present in this strain was found to be partly responsible for this temperature sensitivity. TRSase in such a strain was rapidly inactivated during growth at 42° C in rich media, but not in minimal media or in the presence of chloramphenicol. A partial derepression of anthranilate synthetase formation took place in the presence of excess tryptophan at growth-restricting temperatures. When some of the trpR mutations (including amber mutations) were combined with trpS5, the resulting double mutants (trpR trpS5) were temperature-insensitive, and TRSase was not inactivated at high temperature, in contrast to the trpR+trpS5 strain. This effect of trpR mutations on temperature sensivity was shown not to be a secondary consequence of the constitutive expression of the trp operon. These findings suggest that the trpR+ product interacts with the TRSase of the trpS5 mutant so as to bring about the growth-dependent inactivation of the enzyme. Furthermore, a special class of trpR mutants was obtained whose constitutivity with respect to the trp operon is manifested only in strains carrying trpS5 (but not trpS+) grown at high temperatures. It is proposed that TRSase participates in repression trrough direct interaction with the product of the trpR gene.