Abstract
We have isolated, cloned, and characterized cDNA and genomic DNA corresponding to the mRNA and gene for the rat mast cell protease, RMCP II. The amino acid sequence deduced from the cDNA provides evidence that this protease is synthesized as a precursor, with a signal peptide and additional residues at the N and C termini. RNA homologous to the cDNA is expressed only in mast cells. Analysis of RNA from the two subclasses of mast cell, mucosal and serosal, indicates subclass specific expression of the different proteases found in each of these two subclasses. S1 protection analysis and the sequence of the genomic clone indicate that the serosal mast cell protease, RMCP I, is likely to be coded for by a separate, highly homologous gene. A comparison of the exon/intron structure of the RMCP II gene with genes of related serine proteases further indicates that RMCP II is a member of a family of proteases distinct from those found in the pancreas. We have also isolated a third gene, highly homologous to RMCP II but different from it and from the gene for RMCP I. Analysis of the 5' transcriptional control region of both genes showed striking homology to the TATA box and enhancer regions of the pancreatic proteases.
Highlights
We have isolated, cloned, and characterized cDNA and genomic DNA corresponding tothe mRNA and gene for the rat mast cell protease, RMCP 11
Preparation and Analysis of RNA-For the preparation of RNA for the cDNA library in pBR322 the rat basophilic leukemia (RBL)’ cell line (Eccleston et al, 1973; received from Dr Henry Metzger), was grown in roller bottles in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 5% fetal calf serum to confluence at the MIT cell culture center.Frozen cell pellets were ground in liquid nitrogen and lyophilized
After addition of homopolymericdC tails (Roychoudhuryet al., 1976)t,he cDNA was inserted into pBR322 cut at the PstI site and modified by the (Seldin et al.,1985; Kid0 et al, 1986)), was probed with synthetic oligonucleotides derived from two regionsof the amino acid sequence of RMCP I1 (Woodbury et al, 1978b).One clone, pcMCP1, with an insert of approximately addition of homopolymeric dG tails (New England Nuclear)
Summary
From the Departmentof Genetics, Harvard Medical School, and the Howard Hughes Medical Institute, Boston, Massachusetts 02115. We have isolated, cloned, and characterized cDNA and genomic DNA corresponding tothe mRNA and gene for the rat mast cell protease, RMCP 11. Analysis of RNA from the two subclasses of mast cell, mucosal and serosal, indicates subclass specific expression of the different proteases found in each of these twsoubclasses. Total RNA was prepared by lysis in a buffer of 6 M urea, 2% SDS, 35 mM NaCI, 0.1 mM EDTA, and 1 mM Tris, pH 8.0, followed by phenol/chloroform extraction and centrifugation through CsCl as previously described (Ross, 1976; Glisin et al, 1974; Holmes and Bonner, 1973) This method was used on one occasion to isolate RNA from rat serosal mast cells (kindly provided by Drs Robert Lewis and Richard Stevens). Hybridization to 5 r g of total RNA was as described (Berk and Sharp, 1977) with modifications as described in Taub et al (1984)
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