Research Article

cDNA, genomic sequence cloning and overexpression of the ribosomal protein S13 gene in the giant panda (Ailuropoda melanoleuca)

Published: January 25, 2011
Genet. Mol. Res. 10 (1) : 121-132 DOI: 10.4238/vol10-1gmr1000

Abstract

The cDNA and the genomic sequence of ribosomal protein S13 (RPS13) of the giant panda (Ailuropoda melanoleuca) was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR, respectively. These two sequences were sequenced and analyzed, and the cDNA of the RPS13 gene was overexpressed in Escherichia coli BL21. We compared the nucleotide sequences of the coding region and the amino acid sequences with those of seven other mammalian species retrieved from GenBank. The cDNA fragment of the RPS13 cloned from the giant panda is 496 bp in size, containing an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 2277 bp, with five exons and four introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Canis lupus familiaris, Macaca mulatta, Mus musculus, Rattus norvegicus, and Pan troglodytes; the degree of homology was 91.23, 94.30, 94.74, 92.11, 87.94, 87.72, and 91.45%, respectively. The homologies for the deduced amino acid sequences reached as high as 99%. Primary structure analysis revealed that the molecular weight of the putative RPS13 protein is 17.22325 kDa, with a theoretical pI of 10.42. Based on topology prediction, there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, two N-myristoylation sites, and one ribosomal protein S15 signature in the RPS13 protein of the giant panda. The RPS13 gene can be expressed in E. coli and the RPS13 protein fused with the N-terminally GST-tagged form, which gave rise to the addition of an expected 43-kDa polypeptide.

The cDNA and the genomic sequence of ribosomal protein S13 (RPS13) of the giant panda (Ailuropoda melanoleuca) was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR, respectively. These two sequences were sequenced and analyzed, and the cDNA of the RPS13 gene was overexpressed in Escherichia coli BL21. We compared the nucleotide sequences of the coding region and the amino acid sequences with those of seven other mammalian species retrieved from GenBank. The cDNA fragment of the RPS13 cloned from the giant panda is 496 bp in size, containing an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 2277 bp, with five exons and four introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Canis lupus familiaris, Macaca mulatta, Mus musculus, Rattus norvegicus, and Pan troglodytes; the degree of homology was 91.23, 94.30, 94.74, 92.11, 87.94, 87.72, and 91.45%, respectively. The homologies for the deduced amino acid sequences reached as high as 99%. Primary structure analysis revealed that the molecular weight of the putative RPS13 protein is 17.22325 kDa, with a theoretical pI of 10.42. Based on topology prediction, there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, two N-myristoylation sites, and one ribosomal protein S15 signature in the RPS13 protein of the giant panda. The RPS13 gene can be expressed in E. coli and the RPS13 protein fused with the N-terminally GST-tagged form, which gave rise to the addition of an expected 43-kDa polypeptide.