Abstract
RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a “living fossil”, are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first report on the RPS25 gene from the Giant Panda. The data will enrich and supplement the information about RPS25, which will contribute to the protection for gene resources and the discussion of the genetic polymorphism.
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References
Wool IG, Endo Y, Chan YL, Glück A (1990) Studies of the structure, function and evolution of mammalian ribosomes. In: Hill W, Dahlberg A, Garrett R, Moore P, Schlessinger D, Warner J (eds) Ribosome structure, function and evolution. Society for Microbiology, Washington DC, pp 203–214
Wool IG (1979) The structure and function of eukaryotic ribosomes. Annu Rev Biochem 48:719–754. doi:10.1146/annurev.bi.48.070179.003443
Wool IG, Chan YL, Glück A (1995) Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol 73:933–947
Mager WH (1988) Control of ribosomal protein gene expression. Biochem Biophys Acta 949(1):1–15
Laine RO, Hutson RG, Kilberg MS (1996) Eukaryotic gene expression: metabolite control by amino acids. Prog Nucleic Acid Res Mol Biol 53:219–248. doi:10.1016/S0079-6603(08)60146-4
Laine RO, Shay NF, Kilberg MS (1994) Nuclear retention of the induced mRNA following amino acid-dependent transcriptional regulation of mammalian ribosomal proteins L17 and S25. J Biol Chem 269:9693–9697
Sun D, Kar S, Carr BI (1995) Differentially expressed genes in TGF-β1 sensitive and resistant human hepatoma cells. Cancer Lett 89:73–79
Wang W, Chapin RE (2000) Differential gene expression detected by suppression subtractive hybridization in the ethylene glycol monomethyl ether-induced testicular lesion. Toxicol Sci 56(1):165–174. doi:10.1093/toxsci/56.1.165
Woods DB, Vousden KH (2001) Regulation of p53 function. J Exp Cell Res 264:56–66. doi:10.1006/excr.2000.5141
Nishiyama T, Yamamoto H, Uchiumi T, Nakashima N (2007) Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site. Nucleic Acids Res 35(5):1514–1521. doi:10.1093/nar/gkl1121
Kubota S, Copeland TD, Pomerantz RJ (1999) Nuclear and nucleolar targeting of human ribosomal protein S25: common features shared with HIV-1 regulatory proteins. Oncogene 18:1503–1514. doi:10.1038/sj.onc.1202429
Yu-jie D, Xiao-yan L, Yan-zhe H, Zhang T, Wan-ru H (2007) Cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the Giant Panda. Int J Biol Sci 3(7):428–433
Wan-ru H, Yu-jie D, Chen Y, Wu X, Zheng-song P, Yang J, Zhou C-Q (2007) Nucleotide sequence of cDNA encoding the mitochondrial precursor protein of the ATPase inhibitor from the Giant Panda (Ailuropoda melanoleuca). DNA Cell Biol 26(11):799–802. doi:10.1089/dna.2007.0626
Wan-ru H, Xiao-yan L, Yu-jie D, Chen Y, Wu X, Zheng-song P, Yang J, Zhou C-Q (2008) cDNA cloning and sequences analysis of RPS15 from the Giant Panda. Recent Pat DNA Seq 2(2):16–19
Wan-ru H, Yu C, Zhengsong P et al (2007) cDNA cloning and sequences analysis of ubiquinol-cytochrome c reductase complex ubiquinone-binding protein (QP-C) from Giant Panda. Acta Theriologica Sin 27(2):190–194
Peng R, Zeng B, Meng X, Yue B, Zhang Z, Zou F (2007) The complete mitochondrial genome and phylogenetic analysis of the Giant Panda (Ailuropoda melanoleuca). Gene 397:76–83. doi:10.1016/j.gene.2007.04.009
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Towbin H, Staehelin T, Gordon J (1979) Eletrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76(9):4350–4354
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This work is supported by the Key Chinese National Natural Science Foundation (30470261), Application Technology Project in Sichuan Province (2006J13-057), Key Scientific Research Foundation of Educational Committee of SiChuan Province (07ZA120) and Key Discipline Construction Project in Sichuan Province (SZD0420).
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Hao, Yz., Hou, Wr., Hou, Yl. et al. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda. Mol Biol Rep 36, 2139–2145 (2009). https://doi.org/10.1007/s11033-008-9427-9
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DOI: https://doi.org/10.1007/s11033-008-9427-9