OBJECTIVE: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). METHODS: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. RESULT: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. CONCLUSION: This system will be one of the useful vector system for cervical cancer gene therapy.
Animals ; Antigens, Viral, Tumor ; Cell Cycle ; Cell Line ; Clone Cells ; DNA ; DNA, Complementary ; DNA, Single-Stranded ; Genetic Therapy* ; Humans ; Oncogene Proteins ; Oncogenes ; Parvovirus ; Phenotype ; Plasmids ; Retinoblastoma Protein ; Satellite Viruses ; Uterine Cervical Neoplasms*

DNA, Circular ; DNA, Viral ; Hepatitis B virus ; genetics

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.
Biotechnology ; Chimera ; DNA ; DNA, Single-Stranded* ; Genome, Bacterial* ; Retroelements ; Reverse Transcription ; RNA ; RNA-Directed DNA Polymerase

DNA, Circular ; isolation & purification ; DNA, Viral ; isolation & purification ; Hepatitis B ; diagnosis ; Hepatitis B virus ; genetics ; Humans

<b>OBJECTIVEb>Express and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

<b>METHODSb>The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

<b>RESULTSb>Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

<b>CONCLUSIONSb>We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

<b>OBJECTIVEb>To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.

<b>METHODSb>10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.

<b>RESULTb>Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.

<b>CONCLUSIONb>10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

DNA, Catalytic ; metabolism ; DNA, Single-Stranded ; metabolism ; Hepatitis B virus ; enzymology ; genetics ; Open Reading Frames ; RNA, Messenger ; metabolism ; RNA, Viral ; genetics ; metabolism ; Transcription, Genetic

Virus isolate G6 was obtained from Hibiscus rosa-sinensis showing yellow and leaf curl symptoms in Guangzhou, Guangdong Province. The complete nucleotide sequence of DNA-A was determined to be 2 737 nucleotides encoding six potential ORFs. Comparison showed that G6 DNA-A had more than 89% sequence identify with all isolates of Cotton leaf curl Multan virus (CLCuMV) and shared the highest sequence identify (96.1%) with CLCuMV isolate 62. G6 DNA-A had 87.1%-89.8% sequence identity with those of CLCuRV isolates, while less than 87% identities with other begomoviruses. Phylogenetic analysis of G6 DNA-A and selected begomoviruses showed that G6 was most closely related to CLCuMV isolates, and they clustered together as a separate branch. Satellite DNA molecule (G6 DNAbeta) was found to be associated with G6 using the primers beta01 and beta02. G6 DNAbeta contains 1346 nucleotides, with a potential functional ORF (C1) in complementary sense DNA. Pairwise comparison indicated that G6 DNAbeta had the highest sequence identities with CLCuMV DNAbeta (92.1%) and CLCuRV DNAbeta (88.7%), but less than 80% sequence identities with other reported satellite DNA molecules. Phylogenetic analysis indicated that G6 DNAbeta was most closely related to CLCuMV DNAbeta and the two DNAbetas clustered together as a separate branch, and formed the main branch with DNAbeta of CLCuRV and MYVV-Y47. It is concluded that G6 infecting Hibiscus rosa-sinensis is an isolate of CLCuMV.
Base Sequence ; DNA, Satellite ; chemistry ; DNA, Viral ; chemistry ; Geminiviridae ; classification ; genetics ; Gossypium ; virology ; Hibiscus ; virology ; Phylogeny

<b>BACKGROUNDb>Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).

<b>METHODSb>Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.

<b>RESULTSb>The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.

<b>CONCLUSIONb>The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Cells, Cultured ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Real-Time Polymerase Chain Reaction ; methods

<b>OBJECTIVEb>To investigate the levels of hepatitis B virus (HBV) total DNA and covalently closed circular (ccc) DNA in liver transplant recipients due to HBV-associated liver diseases and detemaine their clinical significance.

<b>METHODSb>Sixty patients undergoing liver transplantation (LT) due to HBV-associated liver diseases were enrolled for the study. Levels of HBV total and ccc DNA in plasma, liver and PBMCs were measured by RT-PCR.

<b>RESULTSb>The ratio of male:female participants was 48:12. The mean age was 52.98+/-9.40 years old, and the median duration post-LT was 72 (25-128) months. 59 of the patients had no detectable HBV DNA in plasma.Four patients had detectable levels of total HBV DNA in PBMCs, but no detectable ccc DNA. Five patients had detectable levels of total HBV DNA in liver, and two of those also had detectable levels of ccc DNA. One patients who had detectable HBV DNA in PBMCs suffered HBV recurrence.

<b>CONCLUSIONb>The liver transplant recipients with detectable levels of HBV total and ccc DNA in PBMCs and liver should be considered high risk for HBV recurrence following LT.

DNA, Circular ; DNA, Viral ; Female ; Hepatitis B ; Hepatitis B virus ; Humans ; Liver Transplantation ; Male ; Middle Aged ; Recurrence

<b>OBJECTIVEb>To investigate the sequence polymorphism of mtDNA HV1,HV2 overlapping fragments and coding region encompassing position 8430-8673 in Hebei Han population.

<b>METHODSb>Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with sequencing method was used to detect the haplotype distribution of mtDNA in 100 Hebei Han individuals.

<b>RESULTSb>Ninety-one haplotypes were noted in 100 unrelated individuals. The gene diversity is 0.9985 and the random match probability is 0.0115. Compared with the Anderson sequence, 65 sites of different nucleotide sequences were noted, of which 44 sites were previously registered in MITOMAP, 12 sites were not registered and the gene mutations were different from MITOMAP at 9 positions.

<b>CONCLUSIONb>The obtained data suggest that these loci are valuable genetic markers for personal identification and thus could be used as basic data for the forensic application of mtDNA in Hebei province.

China ; DNA, Mitochondrial ; genetics ; Humans ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational