Isolation and characterization of the human D-glyceric acidemia related glycerate kinase gene GLYCTK1 and its alternatively splicing variant GLYCTK2.

Deficiency of human glycerate kinase leads to D-glycerate acidemia/D-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes--Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant--Glycerate Kinase 2 (GLYCTK2), which might be associated with D-glycerate acidemia/D-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in D-glycerate acidemia/D-glyceric aciduria.

Human THROMBOSPONDIN-1 gene contains a natural antisense transcript, and characterization of its expression in human multiple tissues and cells.

Human THROMBOSPONDIN-1 play versatile roles in platelet aggregation, angiogenesis, and tumorigenesis, which forms a disulfide-linked homotrimeric complex. Here we reported that the genomic lotus of TSP1 also transcribes a natural antisense transcript (NAT) with an overlapping and reverse complement region against TSP1. The NAT of TSP1 was expressed in human testis, lung, thymus, colon, placenta, kidney and skeleton muscle, revealed by PCR amplification. It was also expressed in some tumor cell lines. The identification of NAT of TSP1 would be of significant importance to understand the functions of TSP1, and it would also suggest the potential attempt of using RNA interference for related tumor therapy, for instance, breast cancer.

Human liver specific transcriptional factor TCP10L binds to MAD4.

A human gene T-complex protein 10 like (TCP10L) was cloned in our lab. A previous study showed that it expressed specifically in the liver and testis. A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity. In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen. This finding was confirmed by immunoprecipitation and subcellular localization experiments. As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells. Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.

Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system.

Many human genes determined by genomic sequencing have only few information about their functions. To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively. By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila. Fifty-four transgenic fly lines corresponding to 10 human genes have been established. When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes. This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila.

Isolation, expression pattern of a novel human RAB gene RAB41 and characterization of its intronless homolog RAB41P.

Small GTPases form a big family including Ras, Rho, Rac, Rab, Sar1/Arf subfamilies and Ran homologs, playing important roles in diverse cellular processes. Through data mining, a novel human RAB41 gene was predicted and subsequently isolated from human testis. The open reading frame of RAB41 is 636bp in length. RAB41 was composed of three exons, and it was mapped to chromosome 3q21.3 by comparing with the human genomic data. RAB41 protein contains a RAB domain. Similarity analysis indicated that RAB41 is closely similar to RAB19. The results of PCR amplification indicated that human RAB41 is widely expressed in brain, testis, lung, heart, ovary, colon, kidney, uterus and spleen but not in liver. By genomic searching, an intronless pseudogene homologous to RAB41 at chromosome 16--RAB41P was identified at human chromosome 16q11.2. Flanking the pseudogene RAB41P in human genome, there exist some transposable elements LINEs--L1, whose contribution in the generation of this intronless homolog is discussed.

[Effect of tumor necrosis factor alpha and beta on ionizing radiation-induced apotosis].

Present paper described the effect of human tumor necrosis factor alpha (hTNFalpha) and beta (hTNFbeta) on apoptosis induced by ionizing radiation in human embryonic lung cell line 2BS diploid cells and human lung cancer cell line A549 and SPC cells. The results showed that both hTNFalpha and hTNFbeta significantly inhibited 7Gy gamma-ray-induced apoptosis in 2BS cells. In contrast, hTNFalpha or hTNFbeta can increase the sensitivity of tumor cell lines (A549 and SPC cells) to radiation. Therefore, the results suggested that TNFalpha and TNFbeta had potential as a therapeutic agent to protect normal cell from the radiation-induced apoptosis and to sensitize the tumour cells for the damage effect of ionizing radiation.

Aggregation of the Intracellular Domain of the Human Thrombopoietin Receptor c-MPL.

Thrombopoietin (TPO) is the major cytokine which is involved in platelet production and exerts its effects via the receptor c-MPL. The yeast two-hybrid system has been used to study the aggregation of the intracellular domain of c-MPL in TPO signal transduction. First, the cDNA fragment of MPLP intracellular domain was amplified and cloned by using RT-PCR method from the total RNA of HEL cells. Then the cDNA fragment was sequenced and subcloned into two-hybrid vectors pAS2 and pGAD424, respectively, and the recombinants are named as pASMM and pGADMM. Co-transformation of these plasmids into yeast activated his3 and lacZ reporter genes, demonstrating in vivo interaction of the MPLP receptor intracellular domain itself. The aggregation may be important in TPO signal transduction.

Cloning and Expression of a Fused Gene Encoding TNFRI Death Domain and Chloramphenicol Acetyltransferase.

By designing four primers for an overlapping PCR, we created a fusion gene Ddlcat encoding human TNF receptor I death domain and chloramphenicol acetyltransferase (CAT). By DNA sequencing, the whole sequence of the fusion gene is confirmed to be correct. Two hours after induction with IPTG, we could see a 39 kD extra protein band on SDS-PAGE pattern. We proved that this 39 kD protein band is DdLcat protein by Western blotting. Then we purified this protein to the purity of 95% through Q-Sepharose chromatography.

Isolation and Cloning of 17 Novel C(2)H(2) Zinc Finger Gene Fragments.

A pair of degenerate primers were designed according to the DNA sequences of the C(2)H(2) zinc finger genes conservative domain and then homologious PCR was performed with the human genomic total DNA as template. The zinc finger fragments thus obtained were used as probes to screen cDNA libraries of human fetal kidney, muscle and marrow and 22 C(2)H(2) zinc finger gene fragments were selected. Among these fragments, 17 were confirmed to be novel zinc finger gene fragments by literature searches in the National Center Biotechnology Information (NCBI) database. Expression patterns of the clones K3-4 and K5-12 selected from kidney cDNA library were analyzed. The results showed that the expression level of K3-4 in kidney is obviously higher than in other tissues and K5-12 is expressed at different levels in 8 tissues.

High Expression of a cDNA Coding for Human Lymphotoxin alpha Derivative in E. coli and Purification of Its Protein Product.

Using PCR, a cDNA coding for human lymphotoxin alpha derivative (hLTalphaDa) lacking 27 amino acids at N-terminal of natural hLT was constructed. The expression construct was expressed in E. coli BL21 (DE3). The product of expression was in the form of inclusion bodies, and accounted for 60%-80% of total bacterial proteins. The product protein was purified to over 95% by treatments of inclusion bodies. The specific activity of hLTalphaDa was above 2x10(8) IU(per mg protein). Its cytotoxic activity can be neutralized by monoclone antibody against hLT. The anti-tumor effects of hLTalphaDa were also tested in vitro and in vivo.