cDNA cloning, sequence analysis of the porcine LIM and cysteine-rich domain 1 gene.
LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.
Wang
Jun
J
Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China.
Deng
Chang-Yan
CY
Xiong
Yuan-Zhu
YZ
Zuo
Bo
B
Xing
Lei
L
Li
Feng-E
FE
Lei
Ming-Gang
MG
Zheng
Rong
R
Jiang
Si-Wen
SW
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D010802:Phylogeny; D011110:Polymorphism, Genetic; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000235:genetics*
D002352:Carrier Proteins