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Inducible expression of bovine calpastatin in C(2)C(12) mouse myoblasts and the relationship of restriction fragment length polymorphisms at the bovine calpastatin locus to postmortem calpastatin activity and Warner-Bratzler shear force in beef longissimus steaks
Abstract
Two separate studies to define further the role of calpastatin in muscle growth and development and postmortem tenderization of meat were conducted. The objective of the first study was to determine if restriction fragment length polymorphisms (RFLP) at the bovine calpastatin locus are related to variations in calpastatin activity or beef tenderness in unrelated animals. A sample of 83 crossbred steers from sires representing eight different breeds was examined to determine this relationship. No associations between EcoRI and BamHI RFLP and 24-h calpastatin activity or Warner-Bratzler shear force at 14 d postmortmen were detected. Therefore, these polymorphisms cannot be used to predict tenderness of aged beef from unrelated animals of mixed breeding. These results do not exclude the possibility that other DNA sequences in or near the bovine calpastatin gene are responsible for variation in calpastatin activity or meat tenderness. Further development of calpastatin-based methods for predicting beef tenderness in unrelated animals should focus on basic factors influencing calpastatin activity at the molecular and cellular level. The objective of the second study was to develop a model of inducible calpastatin expression in C$\sb2$C$\sb{12}$ mouse myoblast. A 2.2-kb cDNA corresponding to bovine skeletal muscle calpastatin domains 2, 3, 4, and a 3$\sp\prime$ untranslated region was subcloned to an eukaryotic expression vector. The inducible expression vector (pOPRSVI) is under the control of the lac repressor. This expression vector and a lac repressor vector (p3$\sp\prime$SS) were transfected to C$\sb2$C$\sb{12}$ mouse myoblasts. Bovine calpastatin fragment expression was induced with 5 mM isopropyl-$\beta$- scD-thiogalactopyranos ide and was detected on immunoblots and activity assays after 2 h of incubation and was still detected at 48 h of incubation This model of inducible expression of calpastatin in C$\sb2$C$\sb{12}$ mouse myoblasts provides a means to study the role of the calpains and calpastatin in myoblast development and protein metabolism.
Subject Area
Food science|Molecular biology
Recommended Citation
Lonergan, Steven Michael, "Inducible expression of bovine calpastatin in C(2)C(12) mouse myoblasts and the relationship of restriction fragment length polymorphisms at the bovine calpastatin locus to postmortem calpastatin activity and Warner-Bratzler shear force in beef longissimus steaks" (1995). ETD collection for University of Nebraska-Lincoln. AAI9600744.
https://digitalcommons.unl.edu/dissertations/AAI9600744