Characterizations Of Two Differentially Expressed Genes Related To Human Gliomas

The Human Genome Project (HGP) is the first 'big' biology project that has ever been undertaken, it aims to sequence the 3×09 bp nucleotide sequence of human genome and identify all the estimated 70,000-100,000 genes. Although sequencing of the human genome had been completed, gene identification and annotation remains a challenge. Identifying and sequencing a set of full-length cDNA that represent all human genes would help in both gene discovery and functional analysis.Constructing a high-quality cDNA library with good representation of full-length cDNAs is very important for cDNA microarrays. Part of the full-length and partial cDNAs representing known, novel and control genes cloned from the cDNA library was used to construct cDNA microarrays. Microarray analysis on a genomic scale was used to profile changes in gene expression accompanying human glioma. four patients were screened for expression of 4366 human genes, leading to the identification of 4 genes with elevated expression and 11 genes with reduced expression in the glioma tissues. Greater than 0.4% of the 15 genes with altered expression were identified in three or more of the patients examined. This highly concordant expression profile included human genes encoding proteins involved in the function of peroxisomes, serun control, polycyclic aromatic hydrocarbon (PAH) carcinogenesis, cell growth and differentiation, metastasis, the function of the immune system, apoptosis, and remodeling of the cytoskeleton. The newly identified genes afford a quantitative view of the changes that accompany human glioma at the genomic level, enable deeper insights into the molecular basis of disease, and provide an extensive list of potential early-onset molecular markers for improved diagnosis.Two novel gene cloned from the cDNA library were identified and preliminarily analysis. One was PKI (the form the Camp-dependent protein kinase inhibitor) gene. The deduced amino acid sequence of the human PKI P showed 30% identity to human PKI, 23% identity to human PKI and 74% identity to murine PKI. Residues important for the high affinity of PKIs aswell as for nuclear export of the catalytic (C) subunit of Camp-dependent protein kinase (PKA) were found to be conserved in human PKI . Using a bacteria expression vector constructed to synthesize the complete sequence of the human PKI has produced human PKI P protein with high purity, which inhibits phosphotransferase activity of C subunit in vitro. Northern blot analysis showed that three sizes of human PKI messages were detected: 0.7kb, 1.4kb and 1.8kb. Their expression levels varied in different tissues. By radiation hybrid panel mapping, the human PKI gene was localized to chromosome 6q21-q22 between markers D6S304 and D6S1712.The other novel gene identified is a new number of the human cyclophilin family. In agreement with the Human Genome Organization (HUGO) Nomenclature Committee, the gene has been named peptidylprolyl isomerase (cyclophilin)-like 3 (approved symbol, PPIL3). PPIL3 gene have two splicing variants (PPLI3a and PPIL3b) encoding two proteins which show 52% and 72% identified to the cyclophilin isoform 10 of C. Elgans, respectively. The PPIL3 genes was identified on a completely sequenced BAC (GenBank accession AC005037) from chromosome 2q33 between STS marker stSG2762 (proximal) and SHGC-3074(distal), oriented toward the telomere. PPIL3 gene consisted of 8 exons spanning more than 18 kb of genomic DNA. RT-PCR analysis indicated that PPIL3 was ubiquitously expressed in adult human tissues. Activity analysis of the recombinant PPIL3b protein shows that PPIL3b protein has nuclease activity of PPIL3b protein is stimulated by Ca2+ and/of Mg2+ and inhibited by K+YNa+, these data suggest that PPIL3b may be involved in degradation of the genome during apoptosis.