Open AccessCloning and sequencing of mammalian glutathione reductase cDNA
Author(s) -
TUTIC Mevlida,
LU Xiang,
SCHIRMER R. Heiner,
WERNER Dieter
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15431.x
Subject(s) - complementary dna , microbiology and biotechnology , cdna library , ecori , biology , expression vector , molecular cloning , genomic library , escherichia coli , biochemistry , peptide sequence , plasmid , recombinant dna , dna , gene
The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full‐length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated λGRM‐B11 was isolated by plaque‐screening of an induced mouse cDNA expression library in the λgt11 vector with a rabbit antibody probe to human glutathione reductase. 125 Iodine‐labelled whole anti‐rabbit immunoglobulin was used as second antibody. Eco RI digestion of the λGRM‐B11 clone released a 720‐bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with λGRM‐B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti‐(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM‐B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM‐B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318. The insert of the pGRM‐B11 plasmid was used as a bona fide nucleic acid probe to screen mouse and human cDNA libraries prepared in the λgt11 or in the λgt10 vector. The first full‐length cDNA clone (λGRH‐Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three Eco RI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH‐B) is colinear with the mouse sequence cloned in the pGRM‐B11 plasmid. The fragment of intermediate size (pGRH‐CT) comprises the 3′ end of the mRNA and the poly(A) tail while the short fragment (pGRH‐NT) corresponds to the 5′ region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.