Cloning and expression of the dog mast cell alpha-chymase gene.
Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.
Caughey
G H
GH
Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco 94143-0911, USA. ghc@itsa.ucsf.edu
Blount
J L
JL
Koerber
K L
KL
Kitamura
M
M
Fang
K C
KC
D000818:Animals; D001483:Base Sequence; D053818:Chymases; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D006801:Humans; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053818:Chymases