Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.
Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.
Smith
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C
Sloan-Kettering Institute for Cancer Research, Molecular Biology Program, New York, New York.
Giordano
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H
DeLotto
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R
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology*; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000235:genetics; D010641:Phenotype; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism
D029721:Drosophila Proteins; D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C081508:snk protein, Drosophila