J. Anim Sci.
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Published online first on November 6, 2009
J. Anim Sci. 1910. doi:10.2527/jas.2009-1832
© 2009 American Society of Animal Science
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Molecular cloning and characterization of porcine calcinuerin-{alpha} subunit espression in skeletal muscle

F. F. S. Depreux*, J. M. Scheffler{dagger}, A. L. Grant*, C. A. Bidwell* and D. E. Gerrard{dagger}

* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 {dagger} Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306

dgerrard{at}vt.edu

Abstract

The calmodulin/Ca2+-dependent serine/threonine phophatase, calcineurin (CaN), has been implicated in controlling muscle fiber phenotype. However, little information is available concerning the expression of CaN in porcine skeletal muscle. Therefore, the porcine CaN{alpha} (CaN-A) was cloned by reverse transcriptase PCR and its expression characterized in selected porcine skeletal muscles. We successfully cloned porcine CaN gene using semitendinosus muscle (GenBank accession number AF193515). Sequence analysis showed both the full length and a 30-bp deletion splice variant in coding region of the gene reported in other species. The deduced amino acid sequence showed 99.4% homology with the rat CaN-A {delta} isoform gene. Real time PCR analysis showed CaN is present in all tissues. However, using primers targeting the region containing the 30-bp deletion, the full length sequence is only found in skeletal muscle and brain tissues. Using a CaN-A monoclonal antibody, we localized CaN-A in porcine longissimus and soleus muscles and the red and white portions of the semitendinosus muscle. The CaN-A protein was abundant in fast fibers and primarily localized in the cytoplasm, whereas slow fibers expressed low levels of CaN-A. Further studies are required to understand the functions of CaN-A isoform in skeletal muscle.

Key Words: calcineurin • fiber type • gene sequence • pig • protein phosphatase 2B






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