The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.
DNA-Directed DNA Polymerase/antagonists & inhibitors ; DNA-Directed DNA Polymerase/metabolism* ; Hepatitis B Virus/enzymology* ; Human

A common DNA polymerase can replicate DNA which functions normally. However, if DNA suffers damage, the genome can not be replicated by a common DNA polymerase because DNA lesions will block the replication apparatus. Another kind of DNA polymerases in organism, Y-family DNA polymerases which is also called translesion synthesis (TLS) polymerases, can deal with this problem. Their main functions are bypassing the lesions in DNA, replicating the genome and saving the dying cells. This thesis presents a historical review of the literature pertinent to the structure, functions and roles of Y-family DNA polymerases.
DNA Damage ; DNA Repair ; DNA Replication ; DNA-Directed DNA Polymerase ; classification ; metabolism ; physiology ; Humans ; Mutagenesis ; Mutagens ; Proliferating Cell Nuclear Antigen ; genetics

Base Sequence ; DNA ; chemistry ; DNA-Directed DNA Polymerase ; metabolism ; Fluorescent Dyes ; chemistry ; Humans ; Molecular Sequence Data ; Nanostructures ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods

OBJECTIVETo investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance.

METHODSAfter L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L).

RESULTSMTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control.

CONCLUSIONThis study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.

Cell Survival ; drug effects ; Cells, Cultured ; DNA Damage ; drug effects ; DNA Repair ; DNA-Directed DNA Polymerase ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Hydroquinones ; adverse effects ; Mutagens

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

Avian Myeloblastosis Virus ; enzymology ; Escherichia coli ; genetics ; metabolism ; Genome, Bacterial ; MicroRNAs ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
Bacteriophages ; genetics ; DNA, Complementary ; biosynthesis ; DNA, Neoplasm ; biosynthesis ; DNA, Recombinant ; biosynthesis ; Gene Library ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; RNA-Directed DNA Polymerase ; metabolism ; Transcription, Genetic ; genetics

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
Bacteriophages/genetics ; DNA, Complementary/*biosynthesis ; *DNA, Neoplasm/biosynthesis ; DNA, Recombinant/biosynthesis ; *Gene Library ; Genetic Vectors ; Leukemia, Promyelocytic, Acute/*genetics ; Leukemia, Promyelocytic, Acute/metabolism ; Leukemia, Promyelocytic, Acute/pathology ; RNA-Directed DNA Polymerase/metabolism ; Transcription, Genetic/genetics

HBV infections leads to severe public health problems around the world, especially in China. Improved understanding of the molecular mechanisms of HBV reverse transcription is fundamental for optimization of treatment and solution to drug-resistance. Recently, the main structural basis involved in the process of HBV reverse transcription and the cis-elements were revealed by means of biochemistry and genetics. The entire process of reverse transcription is completed mainly through the first template switch mediated by the P- epsilon structure; the second template switch mediated by 5E/3E and M structure; and the third template switch mediated by 5' r / 3' r structure. The important structure and the cis-elements involved in this process are the focus of this review, at the same time, an overview of the progress in relevent studies is demonstrated to show the whole picture of the HBV reverse process.
Animals ; Hepatitis B ; virology ; Hepatitis B virus ; enzymology ; genetics ; metabolism ; Humans ; RNA, Viral ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Viral Proteins ; genetics ; metabolism

Currently, animal somatic cell reprogramming into the induced pluripotent stem cell (iPS) is one of the hottest research target in the field of cell biology. We focused on the analysis of telomerase reverse transcriptase (TERT) gene expression during goat somatic fibroblasts reprogramming, and investigated the relationship between the expression of TERT and the pluripotency of reprogrammed cells. RNA samples of fetal tissues isolated from Guanzhong milk goat fetus, and the induced goat reprogramming cell clones were used to determine the relative expression levels of TERT by the real-time RT-PCR method. Goat embryonic fibroblasts (GEF) collected from the Guanzhong milk goat with normal karyotype were induced by 4 transcription factors to become reprogramming cells. The expression of TERT in reprogramming cells was detected by Real-time RT-PCR. The results showed that the expression of TERT in testis tissue was higher than that in epithelial tissues (P < 0.01). The expression level of TERT was higher in AP staining positive cells than that in AP staining negative cells (P < 0.01). This result indicated that TERT activity played an important role in cell reprogramming.
Animals ; Cellular Reprogramming ; Fibroblasts ; cytology ; Gene Expression Regulation ; Goats ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; Telomerase ; metabolism