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

Abstract

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 (h TR). The screening of a human genomic library in λEMBL4 phage led to the identification of two genomic clones which encompassed the entire gene, including the promoter region. The coding region of h TR 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.