OBJECTIVETo synthesize highly pure HBV post-transcriptional regulatory element (HPRE) via transcription in vitro by T7 RNA polymerase.

METHODSHPRE gene was amplified by PCR from a template containing HBV complete genomic DNA and cloned into plasmid pGEM-11zf. The cloned DNA sequence was transcribed by T7 RNA polymerase.

RESULTSThe construction of HPRE gene recombinant plasmid and production of HPRE via transcription in vitro was successful.

CONCLUSIONIn vitro transcription by T7 RNA polymerase can be used to synthesize highly pure HPRE.

DNA-Directed DNA Polymerase ; DNA-Directed RNA Polymerases ; Hepatitis B virus ; genetics ; RNA Processing, Post-Transcriptional ; RNA Splicing ; RNA-Binding Proteins ; physiology ; Transcription, Genetic ; Viral Proteins

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

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.
Amino Acid Sequence ; Cloning, Molecular ; Conserved Sequence ; DNA, Plant ; genetics ; Dendrobium ; enzymology ; genetics ; Molecular Sequence Data ; Phylogeny ; RNA-Directed DNA Polymerase ; chemistry ; genetics ; Retroelements ; genetics ; TATA Box ; genetics

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

OBJECTIVETo explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4.

METHODSDouble-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR.

RESULTSSerotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05).

CONCLUSIONObvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.

China ; DNA, Complementary ; genetics ; DNA, Viral ; Genes, Viral ; Humans ; Polymerase Chain Reaction ; RNA, Double-Stranded ; genetics ; RNA, Viral ; RNA-Directed DNA Polymerase ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Vaccines ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Serotyping

OBJECTIVETo investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads.

METHODSHBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing.

RESULTSIn HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups.

CONCLUSIONSHBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.

Antiviral Agents ; pharmacology ; Genotype ; Hepatitis B virus ; drug effects ; genetics ; Lamivudine ; pharmacology ; Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; genetics ; Sequence Analysis, DNA

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

OBJECTIVETo detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.

METHODSUsing the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).

RESULTSAmong the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.

CONCLUSIONNot only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; genetics ; Humans ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phosphoric Acids ; Polymerase Chain Reaction ; Rifampin ; pharmacology

OBJECTIVETo study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.

METHODSBased on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.

RESULTSThe major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.

CONCLUSIONHIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.

Anti-HIV Agents ; pharmacology ; Bayes Theorem ; Drug Resistance, Viral ; genetics ; Evolution, Molecular ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; RNA-Directed DNA Polymerase ; genetics

To produce the reverse transcriptase of moloney murine leukemia virus (MMLV-RT) through gene recombination, MMLV-rt gene was amplified by polymerase chain reaction (PCR) with specifically designed primers bearing restriction enzyme sites. Five mutation sites increasing the solution of the target protein were introduced through Site-directed mutation. After verification by sequencing, the gene was cloned into the expression vector pET15b to construct the recombinant plasmid pET15b-MMLV-rt. Purified MMLV-RT was obtained by affinity chromatography (Ni3+-NTA beads). Molecular weight and purity of MMLV-RT were analyzed with SDS-PAGE. Enzyme activity was characterized with RT-PCR. We successfully constructed the recombinant plasmid pET15b-MMLV-rt and obtained the MMLV-RT fusion protein with 6His on the N-terminus. Recombinant protein was purified through Ni3+-NTA beads based affinity chromatography, the purity of which was 96%. The Activity of the enzyme was high. MMLV-RT of 96% purity was obtained with the prokaryotic expression technique, which serves as the basis for mass production of this enzyme.
Animals ; Mice ; Moloney murine leukemia virus ; enzymology ; genetics ; RNA-Directed DNA Polymerase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism ; Recombination, Genetic