Author
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Ernst, Catherine |
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Koohmaraie, Mohammad |
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Submitted to: Journal of Animal Science Supplement
Publication Type: Abstract Only Publication Acceptance Date: 3/18/1996 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: The calpain proteolytic system has been shown to be responsible for postmortem meat tenderization. Calpastatin is an endogenous inhibitory protein specific for the calpains. However, little is known about the mechanisms regulating calpastatin gene expression. Therefore, a project was initiated to clone the gene for bovine calpastatin. A bovine genomic library (Stratagene) was used to obtain clones containing portions of the calpastatin gene. Library screening was performed using a cDNA probe corresponding to domains L and 1 of bovine skeletal muscle calpastatin. Eight clones were obtained and those containing unique sequences were identified by digestion with six restriction enzymes, followed by Southern analysis using the same calpastatin cDNA probe used for library screening. Seven clones contained unique sequences. The seven clones were digested with EcoRI and either BamHI or XbaI. Fragments were subcloned into the vector pBluescript II SK+ (Stratagene) and ligation mixtures were used to transform the host strain, XL1 Blue (Stratagene). Transformed colonies were screened for plasmids containing the desired fragments. Ten fragments from 4 of the 7 clones have been aligned. These clones span approximately 27-kb of sequence corresponding to domains L, 1, and a portion of domain 2, as well as an approximately 4-kb portion in the 5-prime untranscribed region of the gene. Two fragments have been completely sequenced and the remaining fragments have been partially sequenced. The availability of genomic clones for bovine calpastatin makes it possible to characterize the promoter region of the gene in order to identify mechanisms regulating calpastatin expression. |
