['318', 'present', 'more', 'sequence', 'divergence', 'in', 'the', '3’', 'noncoding', 'regions.', 'We', 'have', 'previously', 'shown', 'by', 'genomic', 'DNA', 'Southern', 'blot', 'analysis', 'that', 'the', 'rat', 'and', 'mouse', 'glutathione', 'S-transferase', 'Ya', 'subunit', 'is', 'encoded', 'by', 'a', 'multigene', 'family', 'that', 'is', 'located', 'in', 'mouse', 'on', 'a', 'single', 'chromosome', '(Czosnek', 'ef', 'a/.,', '1984).', 'In', 'this', 'report', 'we', 'present', 'the', 'structure', 'and', 'se-', 'quence', 'of', 'a', 'mouse', 'glutathione', 'S-transferase', 'Ya', 'subunit', 'gene,', 'The', 'isolation', 'of', 'a', 'complete', 'rat', 'Ya', 'cDNA', 'clone,', 're-', 'Jated', 'to', 'Ya', 'clone', 'pGTB38', 'described', 'by', 'Pickett', 'ef', 'a/.', '(1984)', 'but', 'containing', 'the', 'entire', "5'", 'untranslated', 'region', 'of', 'the', 'mRNA,', 'has', 'enabled', 'the', 'assignment', 'of', 'the', 'exons', 'in', 'the', 'mouse', 'Ya', 'gene.', 'A', 'genomic', 'clone', 'containing', 'part', 'of', 'the', 'rat', 'Ya', 'gene', 'was', 'also', 'isolated', 'and', 'sequenced.', 'A', 'comparison', 'of', 'the', 'DNA', 'sequences', 'of', 'the', 'mouse', 'and', 'rat', 'Ya', 'genes', 'showed', 'a', 'high', 'degree', 'of', 'homology', 'in', 'the', 'exons', 'and', 'the', 'flanking', 'in-', 'tron', 'regions.', 'MATERIALS', 'AND', 'METHODS', 'Materials', 'Restriction', 'enzymes', 'and', '&eo', 'RI', 'linkers', 'were', 'purchased', 'from', 'Bio', 'Labs', '(New', 'England)', 'T4', 'DNA', 'Ligase', 'from', 'P-L', 'Biochemicals,', 'T4', 'polynucleotide', 'kinase', 'and', 'calf', 'intestinal', 'alkaline', 'phosphatase', 'from', 'Boehringer,', 'A\\DNA', 'in', 'vitro', 'packaging', 'kit', 'and', 'radioactive', 'nucleotides', 'from', 'Amersham', 'Corporation.', 'Preparation', 'of', 'mRNA', 'and', 'cDNA', 'library', 'Rats', 'and', 'mice', 'were', 'injected', 'with', 'phenobarbital', '(10', 'mg/', '100', 'g', 'body', 'weight),', 'free', 'polysomes', 'were', 'prepared', 'from', 'livers,', 'and', 'poly(A)’', 'RNA', 'was', 'isolated', 'as', 'previously', 'de-', 'scribed', '(Daniel', 'ef', 'a/.,', '1983).', 'A', 'rat', 'liver', 'cDNA', 'library', 'was', 'constructed', 'in', '\\gt', '10', 'vector', 'following', 'the', 'procedure', 'of', 'Huynh', 'ef', 'af.', '(1985)', 'and', 'screened', 'for', 'Ya', 'clones', 'with', 'a', 'probe', 'from', 'a', 'partial', 'Ya', 'cDNA', '(Daniel', 'er', 'af,', '1983).', 'A', 'cDNA', 'clone,', 'Agt!O-rYal2,', 'was', 'isolated', 'and', 'found', 'to', 'con-', 'tain', 'the', 'entire', 'sequence', 'of', 'a', 'rat', 'liver', 'Ya', 'mRNA', 'species.', 'Isolation', 'of', 'Ya', 'genomic', 'clones', 'Ya', 'genomic', 'clones', 'were', 'isolated', 'from', 'mouse', '(Czosnek', 'er', 'ai.,', '1984)', 'and', 'rat', '(Lasser-Weiss', 'ef', 'a/.,', '1981)', 'genomic', 'li-', 'braries', 'constructed', 'by', 'partial', 'Eco', 'RI', 'digestion.', 'The', 'li-', 'braries', 'were', 'screened', 'and', 'genomic', 'DNA', 'inserts', 'of', 'positive', 'clones', 'were', 'mapped', 'with', 'probes', 'derived', 'from', 'Ya', 'cDNA', 'insert', 'of', '\\gt]€-r¥al2', 'clone.', 'Genomic', 'DNA', 'fragments', 'were', 'subcloned', 'into', 'the', 'appropriate', 'sites', 'of', 'pBR322', 'or', 'M13', 'for', 'further', 'propagation', 'and', 'sequence', 'analysis.', 'DNA', 'sequencing', 'DNA', 'sequence', 'detcrminations', 'were', 'performed', 'by', 'M13', 'cloning', 'and', 'dideoxynucleotide', 'sequencing', 'method', '(Sanger', 'et', 'af.,', '1977)', 'and', 'by', 'the', 'chemical', 'method', 'of', 'Maxam', 'and', 'Gilbert', '(1980).', 'For', 'the', 'chemical', 'method,', 'DNA', 'restriction', 'fragments', 'were', '5’-end', 'labeled', 'by', 'polynucleotide', 'kinase', 'and', '[y-?', 'P]ATP.', 'DANIEL', 'ET', 'AL.', 'S!', 'mapping', 'Location', 'of', 'the', '5', 'end', 'of', 'the', 'mouse', 'Ya', 'gene', 'was', 'deter-', 'mined', 'by', 'the', 'method', 'of', 'SI', 'nuclease', 'mapping', '(Berk', 'and', 'Sharp,', '1977).', 'A', '3’', 'Hinf', 'I', 'fragment', '(134', 'bp)', 'fram', 'the', 'mouse', 'gene', 'in', 'clorc', '\\mYal', 'was', 'used', 'for', 'SI', 'nuclease-', 'mouse', 'liver', 'Ya', 'mRNA', 'protection', 'experiments.', 'RESULTS', 'Identification', 'of', '«', 'complete', 'Ya', 'cDNA', 'clone', 'A', 'rat', 'liver', 'Ya', 'cDNA', 'clone,', 'Agt]0-rYal2,', 'containing', 'the', 'complete', 'sequence', 'of', 'Ya', 'mRNA', 'was', 'isolated', 'from', 'a', 'cDNA', 'library.', 'DN/\\', 'sequence', 'of', 'this', 'clone', 'showed', 'it', 'to', 'represent', 'a', 'Ya', 'mRNA', 'species', 'identical', 'with', 'that', 'of', 'the', 'Ya', 'cDNA', 'clone', 'pGT3&', 'described', 'by', 'Pickett', 'ef', 'af.', '(1984).', 'In', 'addition,', 'our', 'clone', 'contained', 'the', 'entire', '5’', 'noncoding', 'region', 'of', 'Ya', 'mRNA', '(Fig.', '1).', '10', '20', '30', 't', 't', '1', '5!', 'GAGTTGGGAGCTGAGTGGAGAAGAAGCCAC', '40', '50', '60', '1', '1', 'GACTCTCGCTAGACAGTGAAGCACAGTTGC', 'TCCTATG', 'FG.', '1.)', 'The', '5’', 'nootranslated', 'region', 'of', 'ral', 'Ya', 'mRNA', 'in', 'pr¥al2', 'cDNA', 'clone.', 'The', 'Ya', 'cDNA', 'in.ert', 'of', 'clone', 'A10-rYal2', 'was', 'subcloned', 'into', 'the', 'Eco', 'R1', 'site', 'of', 'pBR322', 'and', 'the', 'resulting', 'plasmid', 'pr¥al2', 'was', 'amplified', 'in', '£.', 'coli', 'HB101.', 'DNA', 'restriction', 'fragments', 'of', 'the', 'pral2', 'insert', 'were', 'used', 'to', 'locate', 'exons', 'in', 'the', 'Ya', 'genomic', 'clones.', 'Structure', 'and', 'sequence', 'of', 'the', 'mouse', 'Ya', 'gene', 'Recombinant', '\\Cl4A-mouse', 'DNA', 'clones', 'containing', 'the', 'Ya', 'sequence', 'were', 'isolated', 'from', 'a', 'mouse', 'genomic', 'library', 'using', 'radioactively', 'labeled', 'Ya', 'cDNA', 'probes.', 'A', 'clone,', 'AmYal', 'containing', 'a', 'single', 'Eco', 'RI', 'DNA', 'fragment', 'of', '15', 'kb', 'hybridized', 'with', 'both', '5’', 'and', '9', 'regions', 'of', 'the', 'Ya', 'cDNA.', 'The', 'mouse', 'DNA', 'insert', 'of', 'AmYal', 'was', 'cleaved', 'by', 'restric-', 'tion', 'endonucleases', 'and', 'the', 'fragments', 'subcloned', 'and', 'am-', 'plified', 'into', 'pBR322', 'to', 'determine', 'the', 'sequence', 'of', 'Ya', 'gene.', 'Figure', '2', 'shows', 'the', 'map', 'of', 'the', 'endonuclease', 'restriction', 'sites', 'used', 'for', 'sequencing', 'of', 'the', 'mouse', 'Ya', 'gene.', 'The', 'assignment', 'of', 'all', 'exon-intron', 'junctions,', 'with', 'the', 'exception', 'of', 'the', 'S’', 'end', 'of', 'exon', '1,', 'is', 'bused', 'on', 'Ya', 'cDNA', 'sequence.', 'The', 'pre-', 'dominant', 'site', 'of', 'transcription', 'initiation', 'was', 'determined', 'by', 'S1', 'mapping.', 'It', 'was', 'found', 'that', 'the', 'mouse', 'Ya', 'gene', 'spans', 'about', '11', 'kb', 'and', 'contains', 'seven', 'exons.', 'The', 'DNA', 'sequence', '(Fig.', '3)', 'shows', 'that', 'the', 'first', 'exon', 'is', 'noncoding', 'and', 'the', 'translation', 'start', 'codon', 'is', 'located', '23', 'bp', 'into', 'the', 'second', ' ']