The The Human Scdr10b, Lhfpl1 And Cypj Gene Cloning And Protein-coding Features And Functions

Based on the conservation of genome in evolution, genes in different species with the same function often share homology with different degrees. The homology is instructive to clone human novel genes and study their function. Besides, using homology among genes to clone new members of gene families becomes an efficient strategy. The thesis include three parts, In the first and second part of this study, we report the identification and characterization of two novel human genes SCDR10B and LHFPL1. In the third part, we study the biochemical character and structure of cyclophilin J.The short-chain Dehydrogenases/Reductases(SDR)family is a very large family of enzymes, most of which are known to be NAD+- or NADP+-dependent oxidoreductases. In the first part of this study, using homologous strategy, we cloned a novel human gene SCDR10B, The deduced protein of this gene has a notable SDR pattern, It is highly homologous to 11β-hydroxysteroid dehydrogenase, which suggest it is a novel member of hydroxysteroid dehydrogenase subfamily. SCDR10B was expressed in prokaryocyte and purified by chromatography. The purified proteins immunized rabbits and polyclonal antibody was obtained. It was shown that SCDR10B can catalyse the conversion of active cortisol to inactive cortisone in the presence of NADP+. Northern blot analysis of human SCDR10B was carried out on Multiple Tissue membranes with mRNA samples from 8 adult human tissues to confirm its natural existence and its tissue distribution. It was found to be 1.7kb in length and highly expressed in the brain. The expressive changes of SCDR10B on their transcriptional levels in tumor cell lines were detected, it is found highly expressed in lung cancer cell lines, the result of immunohistochemistry also showed it is up-regulated in lung cancer tissues, which suggest SCDR10B may be related to lung cancer. Using the immunohistochemistry method, SCDR10B also found to be transcripted in Glimoa, meningioma, normal cerebral cortex and hippocampal neurons.Lipomas, benign neoplasms of adipose tissue, are among the most common mesenchymal tumors in human. A major cytogenetic subgroup of these tumors is characterized by chromosomal aberrations, aberrations involving chromosomal region 12q13-15 have been shown to affect the high mobility group protein gene HMGIC. Rescently, LHFP(lipoma HMGIC fusion partner) was identified as the translocation partner of HMGIC in a lipoma with t(12,13). In the second part of this study, we cloned a novel gene LHFPL1 mapped to Xq23 which is highly homologous to LHFP. RT-PCR amplification in seventen human tissues revealed that LHFPL1 is expressed widely in all tissues, especially highly in lung, thymus, skeleton muscle, colon and ovary. In addition, it was demonstrated that LHFPL1 is transcribed at low level in liver tumor cell lines. LHFPL1 encode a protein with a signal peptide sequence and three transmembrane regions, and it is localized at endoplasmic reticulum. It is suggested that LHFPL1 maybe act as a secretory transmembrane protein. In addition, It would be intriguing to investigate whether LHFPL1 plays roles in related cancer via the similar mechanism of LHFP.Cyclophilins comprise a highly abundant family of proteins that are expressed in every organism studied to date. They have been shown to act as peptidylprolyl cis-trans-isomerase whose activity can be blocked by CSA. Recently, it was reported that cycolphilins can degrade DNA, and nuclease activity inherent to cyclophilins is similar to that described for apoptotic nuclease. In the third part of this study, A novel member cyclophilin J in this family was cloned, expressed and purified, and the high purity CYPJ protein was obtained and analyzed. It was shown that CYPJ has the peptidylprolyl cis-trans-isomerase activity. we also evaluated the nucleolytic activity of recombinant CYPJ, and the result indicate that CYPJ can degrade both plasmid and genome DNA. Nucleolytic activity of CYPJ is stimulated by Ca2+ and Mg2+, but inhibited by EDTA. We observed a direct interaction between...