Nucleic Acids Research, 1982, Vol. 10, No. 18 5407-5419
© 1982
MOLECULAR BIOLOGY |
Closing and sequence analysis of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits
Department of Biochemistry, Microbiology, Molecular and Cell Biology
Center for Air Environment Studies, The Pennsylvania State University, University Park PA 16802, USA
*To whom correspondence should be addressed
Received July 2, 1982. Revised August 30, 1982. Accepted August 30, 1982.
We describe the construction and characterization of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits. Poly(A)-RNA isolated from rat livers was enriched for glutathione S-transferase mRNA activity and used as templates to synthesize double stranded cDNA. The double stranded cDNAs were annealed to pBR322 through terminal deoxynucleotidyl transferase generated GC-tails followed by transformation into E. coll. Several candidate clones were selected by colony hybridization using polynucleotide kinase labeled liver and testis poly(A)-RNA probes. These candidate clones were further characterized by hybrid-selected translation of mRNA followed by immunoprecipitation and SDS gel electrophoresis. The positive clone, pGTR112 was mapped with restriction endonuclease analysis and sequenced by the chemical method of Maxam and Gilbert. The largest open reading frame contains 142 amino acids very rich in Arg and Lys residues. The C-terminal residue phenylalanine of this open reading frame is consistent with what was reported for one of the ligandin subunits by Bhargava et al., (J. Bio1. Chem. 253, 4116–4119, 1978). Among the 352 nucleotides covered by both pGTK112 and pGST94 described by Kalinyak and Taylor (J. Biol. Chem. 257, 523–530, 1982), there are only 9 nucleotide differences resulting in four changes of amino acid sequences.