Thioredoxin Gene Research Articles

Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae.

Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.

Structures of the Human Gene for the Protein Disulfide Isomerase-Related Polypeptide ERp60 and a Processed Gene and Assignment of These Genes to 15q15 and 1q21

ERp60 (also known as ERp61 or GRP58) is an isoform of protein disulfide isomerase (PDI) that has two thioredoxin-like domainsaanda′ in positions corresponding to those of domainsaanda′ in the PDI polypeptide and shows a significant amino acid sequence similarity to PDI in almost all parts. We report here that the human ERp60 gene is about 18 kb in size and consists of 13 exons. No distinct correlation was found between its exon–intron organization and the modular structure of the ERp60 polypeptide, nor were any similarities in exon–intron organization found between the human ERp60, PDI, and thioredoxin genes. The 5′ flanking region of the ERp60 gene has no TATAA box or CCAAT motif but contains several potential binding sites for transcription factors. The highest levels of expression of the ERp60 mRNA were found by Northern blotting in the liver, placenta, lung, pancreas, and kidney, and the lowest in the heart, skeletal muscle, and brain. We also isolated an intronless ERp60 gene that probably represents a pseudogene. The ERp60 gene was mapped by fluorescencein situhybridization to 15q15 and the processed gene to 1q21, so that neither was located on the same chromosome as the human PDI and thioredoxin genes.

Detection of Membrane Associated Thioredoxin on Human Cell Lines

Thioredoxin (TRX) is a ubiquitous dithiol-oxidoreduction enzyme broadly expressed in cells from prokaryote to eukaryote organisms. Human thioredoxin (human TRX) gene, previously cloned in our laboratory, codes for a 12-kDa protein found in the culture supernatant of several hemopoı̈etic human cell lines. This protein is secreted by a nonclassical pathway. The role of the secreted enzyme as a signalling soluble mediator was demonstrated, but nothing is known about a membrane associated form of thioredoxin which could be involved in cell/cell contacts and accessory signal function. Thus, we performed experiments to determine if human TRX is also expressed at the cell surface. We report here positive results based upon indirect immunofluorescence flow cytometric analysis of different human cell lines (HeLa, U 937, Jurkat, 3B6, Daudi and Raji) using a cross reactive sheep antiE. coliTRX polyclonal antibody demonstrating a significant expression of human TRX at the surface of human cells.

A Gene for a Novel Zinc-finger Protein Expressed in Differentiated Epithelial Cells and Transiently in Certain Mesenchymal Cells

We have identified a novel zinc-finger protein whose mRNA is expressed at high levels in the epidermal layer of the skin and in epithelial cells in the tongue, palate, esophagus, stomach, and colon of newborn mice. Expression in epithelial cells is first detected at the time of their differentiation during embryonic development. In addition, during early embryonic development there is expression in mesenchymal cells of the skeletal primordia and the metanephric kidney which is later down-regulated. The expression pattern suggests that the protein could be involved in terminal differentiation of several epithelial cell types and could also be involved in early differentiation of the skeleton and kidney. The carboxyl terminus of the protein contains three zinc fingers with a high degree of homology to erythroid krüppel-like factor and binds to DNA fragments containing CACCC motifs. The amino-terminal portion of the protein is proline and serine-rich and can function as a transcriptional activator. The chromosomal location of the gene was mapped using mouse interspecific backcrosses and was shown to localize to mouse chromosome 4 and to cosegregate with the thioredoxin gene.

Transactivation of an inducible anti-oxidative stress protein, human thioredoxin by HTLV-I Tax

Adult T-cell leukemia derived factor (ADF)/human thioredoxin (TRX), which has thiol reducing and radical scavenging activities, plays an essential role on cellular protection against oxidative stress and cell death. TRX itself is induced by various oxidative stress as well as the Human T-cell lymphotropic virus type I (HTLV-I) Tax protein. To investigate the mechanism of this induction, the promoter region of the TRX gene was analyzed. Chloramphenicol acetyltransferase (CAT) reporter constructs containing the TRX promoter sequences responded to the overexpression of the Tax protein, whereas various oxidative agents activated the TRX promoter through a newly identified oxidative responsive element. Moreover, TRX was translocated from the cytoplasm into the nucleus by ultraviolet irradiation, suggesting its possible role on sensing and transducing oxidative signals.

Human Thioredoxin Reductase Gene Localization to Chromosomal Position 12q23–q24.1 and mRNA Distribution in Human Tissue

Thioredoxin reductase is a member of the pyridine nucleotide–disulfide oxidoreductase family of enzymes. By delivering reducing equivalents to thioredoxin, thioredoxin reductase exerts control over a number of redox-sensitive factors in the cell, including ribonucleotide reductase and several transcription factors. We have localized the human thioredoxin gene to chromosomal position 12q23–q24.1 byin situhybridization. We have also determined the relative tissue distribution of thioredoxin reductase mRNA as well as thioredoxin mRNA by probing a Northern blot of several human normal tissues.

Alternative forms of the human thioredoxin mRNA: identification and characterization

Thioredoxin (TRX) is an ubiquitous and relatively conserved oxidoreductant enzyme which is involved in a multitude of redox reactions through the formation of reversible disulfide bonds. A recent report indicates the presence of novel isoforms of TRX proteins isolated from MP6 cell lines [Rosén et al., Int. Immunol. 7 (1995) 625–633]. In these isoforms, as evidenced from amino acid sequencing, several Lys residues of the wild-type sequence were replaced by Arg. Although the human genome contains several (isoformic) copies of the TRX gene, only one appears to be transcriptionally active [Kaghad et al., Gene, 140 (1994) 273–278]. As we characterized the isoforms of TRX mRNAs, we found that several MP6 TRX cDNA clones were devoid of the characteristic poly(A) tail. In order to increase the efficiency of isolating mRNAs without the poly(A) tail, we developed a novel procedure for exclusive capturing of a specific mRNA by magnetic beads coated with biotinylated antisense oligodeoxyribonucleotide. Using this method on MP6 cell total RNA, we isolated an additional truncated version of the TRX mRNA. This latter form does not produce any variant TRX enzyme, as an inframe stop codon truncates the product. This isoform was also present in mRNAs isolated from human placenta, leucocyte cells and Molt4 cells, the latter two being the progenitors of MP6 cells. From a thorough analysis of the sequence of the truncated TRX mRNA, we conclude that this variant originated from an event of altered splicing, as consensus splice sites were present in the normal TRX cDNA at precise positions.

Early Embryonic Lethality Caused by Targeted Disruption of the Mouse Thioredoxin Gene

Thioredoxins belong to a widely distributed group of small proteins with strong reducing activities mediated by a consensus redox-active dithiol (Cys-Gly-Pro-Cys). Thioredoxin was first isolated as a hydrogen donor for enzymatic synthesis of deoxyribonucleotides by ribonucleotide reductase inEscherichia coli.Recent studies have revealed a variety of roles that thioredoxin plays in transcription, growth control, and immune function. In this report, we describe the phenotype of mice carrying a targeted disruption of the thioredoxin gene (Txn). Heterozygotes are viable, fertile, and appear normal. In contrast, homozygous mutants die shortly after implantation, and the concepti were resorbed prior to gastrulation. When preimplantation embryos were placed in culture, the inner cell mass cells of the homozygous embryos failed to proliferate. These results indicate thatTxnexpression is essential for early differentiation and morphogenesis of the mouse embryo.

The cyanobacterial thioredoxin gene is required for both photoautotrophic and heterotrophic growth.

The gene encoding thioredoxin in the facultative heterotrophic cyanobacterium Synechocytis sp. PCC 6803 (trxA) has been cloned by heterologous hybridization using the corresponding gene trxM from the cyanobacterium Anacystis nidulans as a probe. The deduced amino acid sequence of trxA predicts a protein of relative molecular weight of 11,750 and has strong identity with its cyanobacterial counterparts and other m-type thioredoxins of photo-synthetic eukaryotes. The trxA gene has been expressed Escherichia coli as a functional protein of 12 kD and has been shown by western blot analysis to be the same size as in Synechocystis. The trxA gene is transcribed in Synechocystis as a single transcript of 450 nucleotides and accumulates to the same level under photosynthetic and heterotrophic growth conditions. The trxA gene was inactivated with a kanamycin-resistant cassette, and total segregation of the disrupted trxA gene was obtained only when the trxM gene from A. nidulans (E.D.G. Muller, B.B. Buchanan [1989] J Biol Chem 264: 4008-4014) was simultaneously expressed in Synechocytis. Our results suggest an essential role of thioredoxin not only when cells grow photosynthetically but also under heterotrophic conditions.

THE CLONING, EXPRESSION AND PURIFICATION OF CERVINE INTERLEUKIN 2

The cloning, sequencing and expression of cervine interleukin 2 (IL–2) is described. Cervine IL–2 cDNA is 489 base pairs long and shows high homology to bovine and ovine IL–2 (94%) with lower homologies to human (50%) and mouse (53%). The predicted protein sequence is 162 amino acids long with a signal sequence containing 20 amino acids. A molecular weight of 16 273 Da was predicted for the mature protein. The expression plasmid pTRXFUS was redesigned to allow recombinant proteins to be expressed at high levels in a soluble form and subsequently affinity purified. This new plasmid, pTRXHIS, has been used to express the first cervine cytokine, IL–2. The fusion of the cervine IL–2 gene to the thioredoxin gene (TRX) stabilizes the recombinant product allowing the high expression of soluble IL–2. A polyHis (6 × Histidines) tag has been inserted between the two fusion partners which allows the fusion product to be affinity purified on a nickel—nitrilo-tri-acetic acid (Ni-NTA) column. The purified cervine IL–2 fusion protein was shown to be biologically active despite the presence of the TRX at the amino terminus. The TRX can be removed enzymatically with enterokinase releasing the biologically active IL–2 molecule. This expression system has several features that are useful in producing and purifying large quantities of biologically active cytokines.

A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene.

Adult T cell leukemia-derived factor (ADF) is a human thioredoxin (Trx) and is a disulfide reducing protein with various biological functions. We found that expression of the ADF/Trx gene was increased by oxidative agents such as hydrogen peroxide, diamide and menadione in Jurkat cells. Analysis using a CAT expression vector plasmid under the control of the ADF/Trx gene promoter revealed that CAT gene expression in Jurkat cells was increased after exposure to oxidative agents. A series of deletion analyses showed that a region from -976 to -890 of the 5' flanking sequence was required for enhancement of ADF/Trx promoter activity against the oxidative agents. Gel mobility shift assay revealed the specific DNA binding activities to the sequences from -953 to -930 in the nuclear extracts from the Jurkat cells. The sequences in this region showed no homology with any known consensus sequences for DNA binding factors. It is suggested that ADF/Trx gene expression is enhanced through a novel cis-acting regulatory element responsive for the oxidative stress and a new factor(s) is involved in this oxidative stress responsive element.

Identification of a third thioredoxin gene from Corynebacterium nephridii

We identified and sequenced a gene encoding a third thioredoxin (C3) from Corynebacterium nephridii. The determined nucleotide sequence encodes a thioredoxin of 145 amino acid residues, which is larger than most thioredoxins found in microbial cells and contains 6 cysteine residues. C. nephridii thioredoxin C3 is able to serve a · a subunit of T7 DNA polymerase. C. nephridii is the first nonphotosynthetic procaryotic organism know to carry three different thioredoxins.

Expression of soluble cloned porcine pepsinogen A in Escherichia coli.

A system for the production of soluble porcine pepsinogen A (EC 3.4.23.1) was developed by fusing the pepsinogen and thioredoxin genes and then expressing the fused product (Trx-PG) in Escherichia coli. The expressed fusion protein was purified using a combination of ion-exchange and hydrophobic chromatography. Trypsin digestion of the fusion protein yielded pepsinogen which was one residue longer than the intrinsic length. Acidification of either the fusion protein or pepsinogen (tryptic digestion of Trx-PG) yielded recombinant pepsin A (r-pepsin). When compared with commercial porcine pepsin A, r-pepsin had similar milk-clotting and proteolytic activities, kinetic parameters and pH dependency. The above results indicate that an expression system was developed which yielded fully active soluble pepsin(ogen) from Escherichia coli.

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae.

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a "segregation structure") into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one "low molecular weight activity" (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.

Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen.

The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.

15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1–24)

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

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Expression of the thioredoxin gene (

Molecular genetic analysis of a thioredoxin gene from Thiobacillus ferrooxidans

The Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71% identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E. coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.

The role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae.

The YAP1 and YAP2 genes encode yeast transcription factors of the c-jun family. We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae. They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary-phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes. However, resistance towards H2O2 under these conditions is significantly reduced. We show that expression of the yeast GSH1 gene (encoding gamma-glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants. Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion. However, we show that Yap1 is not required for the superoxide anion-mediated induction of GSH1 gene expression. Furthermore, while the H2O2-mediated induction of SSA1 expression is shown to by YAP1 dependent, the heat-shock-mediated induction of the SSA1 gene does not require YAP1. We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes.

A Redox-Dependent Function of Thioredoxin Is Necessary to Sustain a Rapid Rate of DNA Synthesis in Yeast

DNA replication is impaired in mutants of Saccharomyces cerevisiae which lack the two thioredoxin genes TRX1 and TRX2. Trx1p supports a normal rate of DNA replication only if the active site contains the redox active cysteines. Two mutant forms of Trx1p, one containing a Cys30Ser mutation and a second containing the Cys30Ser mutation in combination with a Cys33Ser mutation, were unable to sustain normal rates of DNA synthesis. The thioredoxin active-site mutants completed a round of replication in 66 min as opposed to 18 min observed for an isogenic wild type culture. Western blot analysis, using antibody generated against purified 6× His-tagged Trx1p, showed that both mutant forms of Trx1p were present at the same levels as the wild-type protein. Thus the inability of the mutant proteins to promote DNA synthesis is not caused by degradation or poor expression, but rather by the loss of their reductive capacity. The results show that an optimal rate of DNA synthesis requires a redox function of thioredoxin. Since the measured levels of deoxyribonucleotides are normal in the thioredoxin mutants, thioredoxin either participates with ribonucleotide reductase in channeling a small subset of deoxyribonucleotides to sites of replication, or thioredoxin reduces and thereby activates an unidentified component of the replication machinery.