Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (EIAV) was investigated using a modification of the dideoxy fingerprinting (ddF) technique. PCR-amplified regions of the gag gene from EIAV isolates were ligated into plasmid vectors and used to transform bacteria. The single dideoxynucleotide sequencing step was performed using plasmid DNA prepared from individual bacterial colonies using an 35S end-labeled primer and Taq DNA polymerase. Analysis of the products of this reaction was conducted using non-denaturing polyacrylamide gel electrophoresis. Polymorphism within this gene was suggested by the presence of several distinct electrophoretic profiles. Significantly, each profile could be correlated with variations in nucleotide sequence, which demonstrates that cycle ddF (CddF) offers a rapid and sensitive approach to identify polymorphism in PCR-amplified products.
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PMID:Application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus. 781 1

The purpose of this study was to characterize quantitative changes in circulating infected cells over the natural history of human immunodeficiency virus (HIV) disease in relation to clinical/immunological outcome. HIV-1 gag DNA polymerase chain reaction (PCR) and peripheral blood mononuclear cell (PBMC) co-cultures were performed on limiting dilutions of cryopreserved PBMC from specimens collected at enrollment and after 5 years of follow-up from nine seropositive subjects classified as rapid progressors, nine intermediate progressors, and 10 nonprogressors. Limiting dilution PCR was also performed on serial pre/postseroconversion specimens from 18 seroconvertors. By quantitative DNA PCR analysis, the infected cell burden was significantly higher at enrollment in the RP [mean of 330 PCR units (PCRU)/10(6) PBMCs] than in the IP (160 PCRU/10(6) PBMCs) and NP (73 PCRU/10(6) PBMCs) groups (p = 0.05). When results were analyzed on an individual level with proportional hazard regression, baseline PCRU (p = 0.05) and CD4 slope (p = 0.0007) were significantly associated with developing acquired immune deficiency syndrome (AIDS) in 5 years, but baseline tissue culture infectious units (TCIU) was not. The increase in PCR-positive cells after 5 years was modest in all three groups (two- to fivefold), whereas the proportion of PCR-positive cells that yielded virus in culture increased significantly (21- to 31-fold) over time in all three groups. Infected cell burden in postseroconversion specimens was relatively stable within each subject, but varied greatly (from 1.6 to 1,024 PCRU/10(6) PBMCs) among subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Circulating HIV-1-infected cell burden from seroconversion to AIDS: importance of postseroconversion viral load on disease course. 790 63

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.
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PMID:Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames. 797 96

ACH-2 cells, an immortalized human T-cell line, contain a single integrated copy of the HIV-1 provirus. Here, the structure of HIV-1 chromatin was probed using a DNA cleavage reagent. Nuclei were isolated from ACH-2 cells and treated with methidiumpropyl-EDTA (MPE)-iron(II) to produce limited DNA cleavage. Primers were selected at approximately 300 bp intervals along the HIV-1 DNA, and sites of preferential cleavage were mapped by carrying out 50 cycles of primer extension using a thermo-stable DNA polymerase in the presence of [32P]dATP. By comparing the resulting cleavage pattern with patterns derived from human cell lines not containing HIV-1 sequences, it was possible to map the arrangement of nucleosomes across the integrated HIV-1 genome. Particularly regular spacing was seen in the 3' end of the pol and env coding regions, and several extended blocks spared of nucleosomes were found in gag and pol, the largest being an approximately 450 bp region in gag. For comparison, and to examine nucleosome placement on HIV-1 DNA when it is not integrated, overlapping segments of HIV-1 DNA were cloned into an EBV-oriP plasmid, grown as stable episomes in a human B lymphoblastoid cell line, and the same analysis using MPE-iron(II) cleavage and primer extension carried out. The major features of nucleosome placement on these EBV/HIV minichromosomes was very similar to that observed in the integrated HIV-1 genome arguing for a strong sequence dependence for nucleosome placement along HIV-1 DNA.
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PMID:Nucleosomal arrangement of HIV-1 DNA: maps generated from an integrated genome and an EBV-based episomal model. 860 34

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.
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PMID:Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique. 961 1

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and pol regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3' coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-maf proto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3' noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.
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PMID:Sequence and insertion sites of murine melanoma-associated retrovirus. 1051 25

An 11-mo-old captive-bred male neutered bobcat (Felis rufus) presented with lethargy, anorexia, leukopenia, neutropenia, lymphopenia, and nonregenerative anemia. The animal was diagnosed as feline leukemia virus (FeLV) positive by immunofluorescent antibody and enzyme-linked immunosorbant assay (ELISA) testing. It died despite supportive care. Pathologic examination revealed multifocal non-suppurative encephalitis, diffuse interstitial pneumonia, multifocal hepatocellular necrosis, non-suppurative peritonitis, and lymphoid depletion. FeLV was isolated from peripheral blood mononuclear cells, bone marrow, spleen, and lymph node. FeLV-specific gag sequences were amplified by DNA polymerase chain reaction (PCR) and aligned with known domestic cat FeLV's. The source of the virus was speculated to be a domestic cat that was a surrogate nurse. Case reports of FeLV in nondomestic felids are few, and FeLV does not appear to be enzootic in wild felids, except European wildcats (Felis silvestris) in France and Scotland. Introduction of FeLV into free-living and captive nondomestic felid populations could have serious consequences for their health and survival. Measures to prevent the introduction of this virus to nondomestic felids are warranted.
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PMID:Feline leukemia virus in a captive bobcat. 1127 97

For the amplification of HIV-1 gag gene, and other challenging targets, a simple new hot start PCR protocol is presented which consists simply and entirely of the buffer system. This novel buffer composition and reaction assembly protocol for PCR includes magnesium and phosphate combined at high concentration in addition to standard buffer reagents. The resulting magnesium-containing precipitate provides a hot start for PCR, since the magnesium in the precipitate is unavailable to DNA polymerase until thermal cycling. No extra manipulations at the thermal cycler or changes to standard thermal cycling profiles are necessary. Upon normal cycling, the magnesium becomes fully available within the first 3 cycles. The method effectively prevents premature primer extension by several DNA polymerases or mixtures of DNA polymerases tested, including Klentaql, KlentaqLA, Pfu, and full-length wild-type DNA polymerase. Once the precipitate is formed, the hot start buffer is stable and functional for at least a week at -20 degrees, 4 degrees , or 25 degrees. We demonstrate that the method is as effective as a manual hot start (addition of magnesium at or above primers'annealing temperatures) for several target gene amplifications which require a hot start.
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PMID:Magnesium precipitate hot start method for PCR. 1221 33

An internally controlled high-input PCR method, termed HIV-1 Mega-PCR was developed to lower the detection limit of HIV-1 DNA polymerase chain reaction (PCR) and to improve its value as a complementary diagnostic test. It is based on PCR amplification of two target sequences in the gag gene of HIV-1 following the selective capture of the targeted sequence and removal of unselected DNA from up to 500 microg of DNA. Efficient selection and amplification was monitored by inclusion of two mimic plasmids. The method was evaluated with buffy coat cells from healthy blood donors which were spiked with blood from 106 different HIV-1-infected individuals, and with 107 HIV-1 seronegative control buffy coats. All specimens from HIV-infected individuals were positive by a PCR protocol using 1 microg of patient DNA. Amplification of 1 microg DNA of the 106 spiked, diluted samples resulted in 68 double positive, 14 single positive, and 24 double negative reactions. In the Mega-PCR, the average input was 260 +/- 84 microg DNA containing an estimated 1.1 +/- 0.6% of spiked patient DNA. Of the 106 samples tested by Mega-PCR, 102 were positive and three negative. One failed to select the mimic plasmid. Among the 107 negative buffy coat controls, none was false-positive and four exhibited a failure of the internal reaction control. Application of HIV-1 Mega-PCR to clinical specimens from seroreverting newborns of HIV-infected mothers and seroindeterminate, PCR-negative specimens revealed no indication for HIV infection, whereas three samples from confirmed, HIV-1-infected but PCR negative individuals showed evidence of the presence of HIV-1 DNA. Mega-PCR lowers the detection limit of an individual analysis to approximately 0.01 HIV-1 DNA copies/microg of applied DNA and may help to confirm or exclude HIV-1-infection in difficult situations diagnostic.
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PMID:Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR). 1463 4

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.
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PMID:The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses. 1645 31


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