Genomic cloning of human thioredoxin-encoding gene: mapping of the transcription start point and analysis of the promoter.

Thioredoxin (TR) is a small ubiquitous dithiol-reductase enzyme first identified in bacteria and plants. In recent years, this protein has been recognized as playing an important role in the growth control of eukaryotic cells, especially in lymphocytes. It was first cloned from a human Epstein-Barr virus-transformed lymphoblastoid B-cell line by our group in 1988 [Wollman et al., J. Biol. Chem. 263 (1988) 15506-15512] and localized on chromosome 3 p11-p12 by in situ hybridization [Lafage-Pochitaloff-Huvalé et al., FEBS Lett. 255 (1989) 89-91]. The present work was performed to study the genomic organization of the human thioredoxin (hTR)-encoding gene (hTR). The screening of a human genomic library in lambda EMBL4 phage led to the identification of two genomic clones which encompassed the entire gene, including the promoter region. The coding region of hTR spans over 13 kb and is organized into five exons separated by four introns which were 60% sequenced. We determined the transcription start point (tsp) by primer extension. This tsp located, in lymphocytes, 22-bp downstream from a TATA box (TATAA) defines a 5' untranslated region of 74 bp. We analyzed 2149-bp upstream from the promoter for sequence motifs which could bind regulatory proteins. This promoter contains many possible regulatory elements compatible with both a basal constitutive expression and a regulated inducible transcription, especially by cytokines such as interleukin-6 and interferons. Finally, Southern hybridization of genomic DNAs from several donors detected only one active gene encoding hTR.

The gene for human thioredoxin maps on the short arm of chromosome 3 at bands 3p11-p12.

Thioredoxin, a ubiquitous enzyme possessing an oxidoreductase activity, has recently been cloned in human. Using in situ chromosomal hybridization with a human thioredoxin cDNA probe, we have precisely localized the thioredoxin gene on chromosome 3 at bands 3p11-p12.

Cloning and expression of a cDNA for human thioredoxin.

Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine). This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin. Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase. Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.