hisT Gene Research Articles

The diagnostic value of long non-coding RNA MIR31HG and its role in esophageal squamous cell carcinoma

AimsThis study aimed to assess plasma lncRNA microRNA-31 hist gene (MIR31HG) as a novel diagnostic and therapeutic biomarker for esophageal squamous cell carcinoma (ESCC) and to investigate its role in ESCC. Main methodsThe expression of MIR31HG, Furin and MMP1 was examined via quantitative real-time polymerase chain reaction. MIR31HG expression between plasma and ESCC tissues was compared using Pearson correlation analysis; furthermore, the association between Furin/MMP1 levels and MIR31HG levels in ESCC tissues was analyzed. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of plasma MIR31HG. A WST-1 assay was performed to assess cell proliferation. The migratability and invasiveness of cells was determined via Transwell assays. Key findingsMIR31HG was significantly upregulated in ESCC tissues and plasma (P < 0.01). A significant positive association was obtained between plasma and tissue MIR31HG expression in ESCC (r = 0.78, P < 0.01). Furthermore, MIR31HG displayed high diagnostic sensitivity and specificity for predicting ESCC occurance. Furthermore, knockdown of MIR31HG suppressed the capacity for proliferation, migration, and invasion of ESCC cells (P < 0.01). In addition, silencing of MIR31HG inhibited the expression of Furin and MMP1 in EC9706 and EC1 and the level of Furin/MMP1 in ESCC tissues displayed a significant positive correlation with MIR31HG (P < 0.01). SignificanceMIR31HG can be used as a novel potential diagnostic biomarker and a potential therapeutic target for ESCC.

Comparative analysis of expression of histone H2a genes in mouse

BackgroundAt least 18 replication-dependent histone H2a genes are distributed in 3 Hist gene clusters on different chromosomes of the mouse genome. In this analysis we designed specific PCR primers for each histone H2a transcript and studied the expression levels and patterns using quantitative RT-PCR (qRT-PCR). In addition, we compared histone H3 K9 acetylation levels in the promoter regions of H2a genes by ChIP (chromatin immunoprecipitation) – quantitative PCR (qPCR) analysis.ResultsRT-PCR analysis indicated that all 20 histone H2a genes assessed in this study are expressed. The replication-dependent histone H2a genes have different expression levels but similar expression patterns. Among the 20 histone H2a genes, the expression-level of H2afz, a replication-independent gene, was highest, and that of Hist1h2aa, a replication-dependent gene, was lowest. Among 18 replication-dependent H2a genes, the expression level of Hist3h2a was highest. The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in promoter regions of both H2afz and Hist3h2a are clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa is similar to that in the γ-satellite region.ConclusionThese results strongly suggest that histone H3 K9 acetylation plays a role in the expression of histone genes.

Characterization of Yeast Protein Deg1 as Pseudouridine Synthase (Pus3) Catalyzing the Formation of Ψ38 and Ψ39 in tRNA Anticodon Loop

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).

Proton-nitrogen-15 NMR studies of Escherichia coli tRNAPhe from HisT mutants: a structural role for pseudouridine

Escherichia coli tRNA(Phe)U39 was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(Phe)U39. The double mutant permitted efficient incorporation of [3-15N]uracil, resulting in greater than 95% 15N enrichment of uracil-derived bases. 1H and 1H-15N NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. 1H NMR assignments indicate that tRNA(Phe)U39 has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (psi) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U----psi modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.

Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I.

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.

HisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.

Structural features of the hisT operon of Escherichia coli K-12.

The DNA sequence of a 2,3-kilobase segment of the E. coli hisT operon was determined. Analysis of the sequence indicated that the upstream gene in the operon encodes a 36,364-dalton polypeptide, which runs aberrantly on SDS-polyacrylamide gels. The distal hisT gene encodes the tRNA modification enzyme, pseudouridine synthase I, which was shown to have a polypeptide molecular mass of 30,399 daltons. The DNA sequence was consistent with the phenotypes and hisT expression of mutant operons. Analysis of the sequence and genetic complementation experiments demonstrated that the upstream and hisT genes are evolutionarily, structurally, and functionally unrelated; however, translation signals for the two genes overlap, which is consistent with genetic evidence suggesting translational coupling. Codon usage in the upstream gene is radically different from the hisT gene and may underlie the differential expression observed from the operon. Gene-inactivation experiments and S1-mapping of in vivo transcripts indicated that the operon contains an additional upstream gene. S1-mapping experiments also confirmed the presence of an internal promoter, which might be stringently controlled. Taken together, these results show that the structure of the hisT operon is complex and suggest that the operon might be regulated at several levels.

Specific mistranslation in hisT mutants of Escherichia coli.

Certain strains of Escherichia coli mistranslate at very high frequencies when starved for asparagine or histidine. This mistranslation is the result of misreading events on the ribosome. The introduction of a hisT mutation into such a strain decreases the frequency of mistranslation during histidine starvation but not during asparagine starvation. The most likely explanation is that the replacement of the pseudouridine residue in the anticodon loop of glutamine specific transfer ribonucleic acid by uridine in hisT mutants leads to an increase in fidelity of transfer ribonucleic acid function. The hisT gene in Escherichia coli has also been more accurately mapped, giving the gene order purF-hisT-aroC-fadL-dsdA.

Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium.

Mutations in the hisT gene of Salmonella typhimurium alter pseudouridine synthetase I, the enzyme that modifies two uridines in the anticodon loop of numerous transfer ribonucleic acid species. We have examined two strains carrying different hisT mutations for their ability to grow on a variety of nitrogen sources. The hisT mutants grew more rapidly than did hisT+ strains with either arginine or proline as the nitrogen source and glucose as the carbon source. The hisT mutations were transduced into new strains to show that these growth properties were due to the hisT mutations. The hisT mutations did not influence the growth of mutants having altered glutamine synthetase regulation. Assays of the three primary ammonia-assimilatory enzymes, glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, showed that glutamate synthase activities were lower in hisT mutants than in isogenic hisT+ controls; however, the glutamate dehydrogenase activity was about threefold higher in the hisT strains grown in glucose-arginine medium. The results suggest that the controls for enzyme synthesis for nitrogen utilization respond either directly or indirectly to transfer ribonucleic acid species affected by the hisT mutation.

Biosynthesis of Pseudouridine in Transfer Ribonucleic Acid

An enzyme that catalyzes the formation of specific pseudouridine residues in the anticodon region of tRNA has been characterized in extracts of Salmonella typhimurium and Escherichia coli. It has previously been shown that hisT regulatory mutants of S. typhimurium lack the pseudouridine modifications in the anticodon region of histidine and several other tRNA species. This has permitted the development of a simple radiochemical assay for the modification enzyme, based on the release of tritium from hisT tRNA isolated from cells grown in [5-3H]uridine. This enzyme activity is absent from hisT mutants and is relatively heat-labile in extracts from temperature-sensitive hisT mutants, which suggests that the modification enzyme itself is the primary gene product of the hisT gene.

Histidine regulation in Salmonella typhimurium: XI. The percentage of transfer RNA His charged in vivo and its relation to the repression of the histidine operon

The cellular concentration of charged transfer RNAHis is shown to be directly correlated with the repression of the histidine operon in Salmonella typhimurium. This conclusion is based on determinations of the amounts of charged and total tRNAHis, tRNAVal and tRNAArg present in vivo in exponentially growing cells of the wild type and of the six classes of regulatory mutants of the histidine biosynthetic operon. The derepression observed in the regulatory mutants hisS (histidyl-tRNA synthetase gene), hisR (tRNAHis structural gene), and hisU and hisW (genes presumably involved in tRNA maturation) may be explained by the lesser amount of charged tRNAHis which these mutants contain. However, mutants in the hisT gene (coding for a dispensable tRNA modification enzyme: uridine to pseudouridine) contain wild-type levels of charged and total tRNAHis, yet are derepressed. HisT tRNAHis then, is a tRNA functional in the charging reaction and protein synthesis, but defective in repression. Mutants in hisO (the operator gene), as expected, contain wild-type levels of charged tRNAHis. Exponentially growing cells of wild type bacteria have about 77% of their tRNAHis charged and 23% uncharged. Adding external histidine increases the percentage of charged tRNAHis to 88%, which is consistent with the known increase in the pool of histidine in the cell and the Km of the histidyl-tRNA synthetase for histidine. Bacteria derepressed physiologically, due to a partial defect in histidine biosynthesis, contain 12% charged and 88% uncharged tRNAHis. Addition of histidine or triazolealanine (a histidine analogue effective in causing repression) to this strain restores charging to the wild-type level. It is shown that a modification of the Folk & Berg (1970) method for the determination of the tRNA charging in vivo gives precise and accurate results.

Histidine Regulation in Salmonella typhimurium: IX. HISTIDINE TRANSFER RIBONUCLEIC ACID OF THE REGULATORY MUTANTS

Abstract Four classes of mutants constitutive for the histidine operon of Salmonella typhimurium, hisR, hisT, hisU, and hisW, were examined for a possible role in the production of histidine tRNA. The content of tRNAhis in tRNA isolated from each of the mutant types was compared to that of the wild type, and the chromatographic behavior of the tRNAhis obtained from the mutants and the wild type was examined. Chromatography of the tRNAhis of the wild type gave no evidence for multiple species. Two factors suggest that hisR is a structural gene for histidine tRNA. First, hisR strains were found to have 25 to 45% less tRNAhis than the wild type; and second, introduction of an episome carrying the hisR gene into the wild type resulted in a 2.5-fold increase in the quantity of tRNAhis. The tRNAhis of a hisT mutant chromatographed differently from that of the wild type. Since the hisT gene has previously been shown to code for a dispensible protein, we conclude that this protein is involved in tRNAhis maturation. The tRNAhis from hisT mutants is present in normal amounts, is charged normally, and appears to behave normally in protein synthesis, although not in repression control. The tRNA from a cold-sensitive hisW mutant was found to have an altered acceptance for several amino acids. This indicates that hisW also codes for a tRNA maturation enzyme. No increase in tRNAhis levels was obtained when an episome carrying the hisW gene was inserted into the wild type, and the tRNAhis from a hisW mutant behaved as the tRNA from the wild type on chromatography. No difference was found between the tRNAhis of hisU mutants and that of the wild type.

Histidine regulation in Salmonella typhimurium. 8. Mutations of the hisT gene.

The hisT gene, one of six genes in which mutation causes derepression of the histidine operon in Salmonella typhimurium, is shown to code for a protein that is not essential for the growth of the bacteria. This is indicated by the characterization of particular classes of mutations in the hisT gene: amber mutations, frame-shift mutations, and temperature-sensitive mutations that affect repression but not growth. In addition, the class of semilethal mutations was selected for but not found.