Biochem. J. (1999) 339
(685–693) (Printed in Great Britain)
Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene
Nasser FOTOUHI-ARDAKANI*† and Gerald BATIST*†1
*McGill Center for Translational Research in Cancer, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis–Jewish General Hospital, 3755 Cote-Ste-Catherine, Montreal, Quebec, Canada H3T IE2, and †Division of Experimental Medicine, McGill University, 3655 Drummond St. West, Montreal, Quebec, Canada H3G IY6
The rat glutathione S-transferase-A3-subunit (GSTA3) gene
is a member of the class Alpha GSTs, which we have previously reported
to be overexpressed in anti-cancer-drug-resistant cells. In this study,
we report the isolation and characterization of the entire rat GSTA3
(rGST Yc1) subunit gene. The rat GSTA3 subunit gene is
approximately 15 kb in length and consists of seven exons interrupted
by introns of different lengths. Exon 1, with a length of 219 bp,
contains only the 5´-untranslated region of the gene. Each exon–intron
splicing junction exhibited the consensus sequence for a mammalian splice
site. The transcription start site and exon 1 of rat GSTA3 were
characterized by a combination of primer extension and rapid amplification
of the cDNA ends. Position +1 was identified 219 bp upstream of the
first exon–intron splicing junction. The proximal promoter region
of the rat GSTA3 subunit gene does not contain typical TATA or CAAT
boxes. A computer-based search for potential transcription-factor binding
sites revealed the existence of a number of motifs such as anti-oxidant-responsive
element, ras-response element, activator protein-1, nuclear factor-
B, cAMP-response-element-binding protein, Barbie
box and E box. The functional activity of the regulatory region of the
rat GSTA3 subunit gene was shown by its ability to drive the expression
of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma
cells, and its activity was greater in melphalan-resistant cells known
to have transcriptional activation of this gene by previous studies. The
structure of the gene, with a large intron upstream of the translation-initiation
site, may explain why the isolation of this promoter has been so elusive.
This information will provide the opportunity to examine the involvement
of the rat GSTA3 subunit gene in drug resistance and carcinogenesis.
Abbreviations used: GST, glutathione S-transferase; r, rat; ARE, anti-oxidant-responsive
element; Arnt, aryl hydrocarbon-receptor nuclear transporter; WT, wild-type;
MLNr, melphalan-resistant; CAT, chloramphenicol acetyltransferase; UTR,
untranslated region; RRE,
ras-response element; AP-1,
activator protein-1; NF-
kB, nuclear factor-
B; CREB, cAMP-response-element-binding protein;
5´-RACE, rapid amplification of cDNA 5´ ends.
1
To whom correspondence should be addressed (e-mail gbatist@onc.jgh.mcgill.ca).
The nucleotide sequences reported in this paper have been submitted
to the DDBJ/EMBL/GenBank Nucleotide Sequence Databases with accession numbers
AF067442 and AF111160.
Key words: drug resistance, expression, rGSTA3 isolation, structural organization.
Received 29 October 1998/7 January 1999; accepted 10 February 1999
The Biochemical Society, London © 1999
Full Editorial Board
RSS feeds
Table of contents by email
My BJ toolbox
