Cloning And Functional Characterization Of Human Genes Pank, Rab2b Of Nm23-h1b Vrk3 The Study

From our large cDNA cloning and sequencing plan, by cDNA microarray analysis together with bioinformatics analysis, we selected 4 genes as follows for further research with intention for human disease related gene.Pantothenate Kinase (PanK) is a key regulatory enzyme in the CoA biosynthetic pathway in bacteria and mammalian cells. It catalyzes the first committed step in the universal biosynthetic pathway leading to CoA. We report here the cloning and characterization of a novel human PanK gene. The gene encoded a protein of 314 amino acids residues, which shared high homology to mouse Pantothenate Kinase (mPanK) 1beta. Northern blot analysis revealed a transcript of the gene of 2.6kb in human liver and kidney. The human PanK gene was located to human chromosome between 10q23.1-10q23.2 by bioinformatics analysis. cDNA microarray analysis revealed its expression level is move down in liver cancer and lung cancer.Rab proteins are small molecular weight GTPases that control vesicular traffic in eukaryotic cells. The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex. We identified a novel human Rab (Rab2B) gene that was 2312bp of length and encoded a protein of 216 amino acids residues. The protein shared high homology to mouse Rab2 (identity 83%, similarity 91%). The expression pattern of human Rab2B gene showed that there is a transcript in kidney, prostate, lung, liver, thymus, colon, placenta, pancreas and skeletal muscle, whereas in heart, brain, spleen, testis, ovary, small intestine and leukocyte specific bands of the transcript could not be detected. An over-expression has been observed in colon adenocarcinoma CX-1. Rab2B gene consists of nine exons and eight introns and is mapped to14q11.1-14q11.2 in chromosome. cDNA microarray analysis indicated its expression level is move up in liver cancer and stomach cancer. NM23 gene is a conspicuous metastasis-suppressor gene and related to cell growth, differentiation and tumor pathogenesis. We cloned a different transcript (NM23-H1B) of human NM23-H1. The cDNA is 987 base pairs of length and encodes a protein of 177 amino acid residues. Compared with NM23H1, the cDNA contained an additional NH2-terminal region (25 aminoacid residues). It is mapped to 17q21.3 in chromosome and shows that the second exon is not exit in NM23-H1. The expression pattern of NM23-H1B showed that it was ubiquitously expressed in normal tissues (15 tissues except colon) in different levels. Our data also indicated that the expression of transcript in tumor related to tumor differentiation. We also detected the expression of NM23-H1A, the results revealed different with NM23-H1B. cDNA microarray analysis indicated its expression level is move up in liver cancer. Also, treated with drugs in 0.5hr or 1hr, the expression level of NM23-H1 was move down in different cancer cells.Vaccinia virus B1R gene product acts as protein kinase with an essential role in viral DNA replication. The human vaccinia virus B1R kinase related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein. We identified a novel human VRK (VRK3) gene that was 1927bp of length and encoded a protein of 474 amino acids residues. The protein shared high homology to mouse VRK1 (identity 38%, similarity 54%). VRK3 gene consists of fifteen exons and fourteen introns and is mapped to 19q13.3-13.4 in chromosome. The expression pattern of human VRK3 gene showed that it expressed ubiquitously. cDNA microarray analysis indicated its expression level may move down in liver cancer. GFP location of VRK3 gene product shows this protein located in the cell nucleus. The ORF of VRK3 was subcloned into reconstructed pQE30 plasmid and the fusion protein was expressed in E.coli. and then purified. No kinase activity of VRK3 has been detected in kinase activity assay. We have done yeast-two-hybrid analysis of VRK3 to identify other proteins with which it interacts. The intera...