MOLECULAR-CLONING OF RAT MAST-CELL PROTEASE-1 AND DEVELOPMENT OF SPECIFIC PROBES FOR ITS GENE TRANSCRIPT

Rat mast cell protease of type 1 (RMCP1) is a specific marker of conne ctive tissue mast cells selectively occuring in some tissues, e.g., th e tongue. Its amino acid sequence is known (Le Trong et al., Biochem. 1987, 26, 6988-6994) but not the corresponding nucleotide sequence. Am plification of mRNAs from rat tongue was performed by reverse transcri ptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers corresponding to the translated region of rat mast cell protease 2 (R MCP2) gene. The cDNA obtained was subcloned and sequenced, leading to an amino acid sequence which matched the known 227 amino acid sequence . In addition there was, however, two sequences of 11 amino acids at t he N-terminus and 13 amino acids at the C-terminus. The amino acid ide ntity was of 74% with RMCP2, and of 76%, 65% and 90% with the mouse pr oteases MMCP1, MMCP2 and MMCP4, respectively. Based on the sequence of RMCP1 or RMCP2 cDNAs, selective oligoprobes were designed and their s pecificity established by Northern blot analysis of mRNAs purified fro m tongue and jejunum, two tissues containing selectively type 1 and 2 protease, respectively. Single 1.2 and 1.0 kb transcripts were evidenc ed in tongue and jejunum, respectively. In addition, a RT-PCR method w as developed to amplify selectively each transcript which may serve as reliable markers in the analysis of mast cell heterogeneity, differen tiation and function. (C) 1994 Academic Press, Inc.