Cloning And Characterization Of ARHT2, RHEBL1, STK40 And LDHAL Genes

With the completion of human genome project and the acculation and release of express sequence tags, the in silico clone became a regular method to rapidly get interesting genes. By differential display between liver cancer tissues and their adjacent no-tumor liver tissues, we obtained a lot ESTs during past several years' work. For find novel genes and study their function, using these ESTs as information probes, combining the EST strategy and experiment, we proceed to our work about human novel genes. The dissertation includes two parts. In the first part, by EST strategy, we reported the identification and characterization of two small GTPase genes ARHT2 and RHEBL1, and one serine/threonine kinase-like gene STK40. In the second part, by homologous cloning strategy, we identified and characterized a testis specific lactate dehydrogenase gene LDHAL.In the chapter one of the first part, we cloned a novel GTPase gene named ARHT2 by EST strategy. The Rho family is an important branch of the superfamily of Ras GTPase. ARHT is a new member of Rho family identified recently. It consists of two GTP-binding domains separated by a linker region containing putative calcium-bing EF hand motifs. Overexpression of a constitutivly active mutant of ARHT1 resulted in an increased apoptotic rate of cells. We get the ARHT2 genes of other species by homologous clone strategy. Northern blot analysis showed a 2.6kb transcript ubiquitously expressed in 16 adult human tissues, and it was significant highly in testis and prostate. Mouse Arht2 mRNA was ubiquitously expressed in 15 adult mouse tissues and significant highly in testis, too. RT-PCR analysis indicated that the Arht2 gene is expressed in all stage of mouse testis and reached the adult level of transcription at postnatal day 30. In situ hybridization revealed strong signals of Arht2 in residual bodies. In mouse testis, Arht2 may be involved in the differentiation of testis and spermiogenesis.In the chapter two of the first part, we cloned another novel GTPase gene named RHEBL1. RHEBL1 is a paraiogue of Rheb, the key regulatory component of mTOR signaling. RT-PCR amplification in seventeen humantissues revealed that RHEBL1 is ubiquitously expressed in all tissue dectected. Subcellular localization of RHEBL1 is in the cytoplasm. Interaction of RHEBL1 and B-Raf was demonstrated by coimmunoprecipitation and colocalization. RHEBL1 can enhance the kinase activity of B-Raf and augment the phosphorylation of ERK1/2, activate the c-myc mediated gene transcription. In addition, RHEBL1 and its active mutant Q64L have similar effect on activation of the transcriptional activities of NF-Ob, while a negative mutant D60K significantly attenuated this activation. It would be intriguing to investigate whether RHEBL1 play roles in mTOR via the similar mechanism of RHEB.In the chapter three of the first part, using EST strategy, we cloned novel human and mouse genes STK40, the deduced protein of these gene contain a pseudokinase domain. Sequence analysis revealed that STK40 lacked several residues that are indispensable for intrinsic kinase activity. Specifically, the DFG motif in VII subdomain was absent. Furthermore, the essential aspartic acid residue was replaced by asparagine. This replacement was conserved in the orthologues of STK40. The eukaryotic protein kinase family is one of the largest protein families represented in the human genome. The recent works showed that some proteins contained protein kinase-Me domain, but lacked several residues that are indispensable for intrinsic kinase activity. They could not phosphorylate the universal kinase substrate and maybe have not phosporylational function. These pseudokinases have their important physiological function. Northern blot analysis revealed that human STK40 have two transcripts: a 4.0kb transcript was ubiquitously expressed in all 16 adult human tissues, and highly expressed in skeletal muscle and testis; a 1.7kb transcript was exclusively expressed in testis. In vitro kinase assay showed the protein expressed in prokaryocyte could not phosphorylate the universal substrate MBP, but we transfect HA-STK40 in HEK293T cell and found that the precipitated protein could phosphorylate the MBP. We mutated . D197Y, A conserved aminoacids critical for kinase activity of serine/threonine kinase in the VIB subdomain. But the mutation did not affect its phosphorylation on MBP in invitro kinase. Base on these data, we believed that STK40 is not an authentic kinase. Subcellular localization of STK40 maily distributes in the nucleus and D197Y mutant mainly distribute in cytoplasm.