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 connective tissue mast cells selectively occurring in some tissues, e.g., the tongue. Its amino acid sequence is known (Le Trong et al., Biochem. 1987, 26, 6988-6994) but not the corresponding nucleotide sequence. Amplification of mRNAs from rat tongue was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers corresponding to the translated region of rat mast cell protease 2 (RMCP2) 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 the N-terminus and 13 amino acids at the C-terminus. The amino acid identity was of 74% with RMCP2, and of 76%, 65% and 90% with the mouse proteases MMCP1, MMCP2 and MMCP4, respectively. Based on the sequence of RMCP1 or RMCP2 cDNAs, selective oligoprobes were designed and their specificity established by Northern blot analysis of mRNAs purified from tongue and jejunum, two tissues containing selectively type 1 and 2 protease, respectively. Single 1.2 and 1.0 kb transcripts were evidenced in tongue and jejunum, respectively. In addition, a RT-PCR method was developed to amplify selectively each transcript which may serve as reliable markers in the analysis of mast cell heterogeneity, differentiation and function.
Rouleau
A
A
Unité de Neurobiologie et Pharmacologie (U. 109) de l'INSERM, Centre Paul Broca, Paris, France.
Garbarg
M
M
Schwartz
J C
JC
Ruat
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D053818:Chymases; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D015870:Gene Expression; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007583:Jejunum / Q000201:enzymology; D008297:Male; D008407:Mast Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D014059:Tongue / Q000201:enzymology; D014158:Transcription, Genetic
D017931:DNA Primers; D007527:Isoenzymes; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053818:Chymases; C509230:Mcpt1 protein, mouse; C509231:Mcpt1 protein, rat