Molecular cloning and characterization of porcine calcineurin-alpha subunit expression in skeletal muscle.
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 transcription-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 AA 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 LM and soleus muscle 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 reduced abundance of CaN-A. Further studies are required to understand the functions of CaN-A isoform in skeletal muscle.
Depreux
F F S
FF
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA.
Scheffler
J M
JM
Grant
A L
AL
Bidwell
C A
CA
Gerrard
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019703:Calcineurin / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D005796:Genes / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000096:biosynthesis; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013552:Swine / Q000235:genetics / Q000378:metabolism
D020033:Protein Isoforms; D019703:Calcineurin