Abstract
Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.
Highlights
Nucleotide sequencing of a rat carboxypeptidase B rat CPA1, CPA2, CPB, and other pancreas-specific (CPB)cDNA and directsequencing of the CPB mRNA genes have been identified
Sequence Determination of theRat CPB mRNA-A rat cDNA clone, pcX4-73, which contains a 650-bp insert was isolated by random selection from a pancreaticcDNA library constructed in pBR322
This clone was identified as the rat CPB cDNA on the basis of 80%identity between its predicted amino acid sequence and thesequence of bovine CPB (Titani et al, 1975)
Summary
Sequence Determination of theRat CPB mRNA-A rat cDNA clone, pcX4-73, which contains a 650-bp insert was isolated by random selection from a pancreaticcDNA library constructed in pBR322. Hybrid- sequence of the 5’ flanking region of the CPAlgene (Quinto ization of this radiolabeled genomic fragment to a Southern et al, 1982)ascertained that themissing first nucleotide is an blot of rat genomicDNA revealed that therewere no repetitive adenosine. This assignment is in agreement with compiled sequences in the probe which would hamper our screening examples of transcription-initiation sequences The genomic inserts of two of these Nucleotide sequencing efforts have elucidated approxiclones, XgCPBlO and XgCPB12, contain an extensive stretch mately 80%of the sequence of the CPAlgene including that of genomic DNA immediately 5’ to exon 1 of the rat CPB of the exons and the exon/intron boundaries (Fig. 4).
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