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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report deals with the test of a series of 5-halogenated derivatives of ara-UTP for the inhibition of DNA polymerase gamma and viral reverse transcriptase. The compounds newly synthesized and tested were; ara5-FUTP, ara5-C1UTP, ara5-BrUTP and ara5-IUTP. The results were: 1) All these compounds were inhibitory to DNA polymerase gamma and reverse transcriptase. The mode of inhibitions was, in all cases, competitive against dTTP. 2) Ki values for these inhibitors with DNA polymerase gamma were lower than those with reverse transcriptase. 3) Ara5-ClUTP was less inhibitory to reverse transcriptase than other derivatives.
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PMID:Recognition of structure of 5-halogenated derivatives of ara-UTP by DNA polymerase gamma and reverse transcriptase. 9 43

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
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PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

We present an improved approach to the screening of eucaryotic libraries for differential gene expression. Previous techniques have generated probe via the harvesting of cellular poly(A)+ RNA and synthesizing labeled cDNA probe using reverse transcriptase. In our approach we prepare labeled RNA probe via in vitro transcription. Unlike cDNA preparation, in vitro transcription (i) directly reflects the ongoing rate of gene expression, and (ii) allows one to assess expression of genes whose transcripts are not polyadenylated. To make this approach practical for the screening of a large library, we modified and optimized existing in vitro transcription techniques, enhancing manyfold the [alpha-32P]UTP incorporation into mRNA, while almost completely suppressing rRNA incorporation. In addition, we developed a simple procedure for making precise replicate dot blots of very large quantities of lambda-phage library DNA. By combining our techniques of in vitro transcription and replicate blotting, we are able to approach detection of a twofold difference in gene expression over a greater than 1000-fold range in overall expression. Our single-clone amplification and dot-blotting technique resulted in nearly the same number of lambda-phage DNA copies per dot for all members of the library. This feature allows us to assign genes to different expression classes, as well as to detect any alterations in expression. We demonstrate our approach by screening the drosophila genomic DNA library with in vitro transcripts from drosophila tissue culture cells. Screening of the entire drosophila genomic library at the 99% probability level is readily achieved.
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PMID:Screening of lambda library for differentially expressed genes using in vitro transcripts. 241 9

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
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PMID:Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts. 628 36

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.
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PMID:Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. 753 90

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.
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PMID:An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs. 765 59

Two new cell lines developed from chinook salmon with plasmacytoid leukaemia have been found to be producing a virus. The virus has been identified as a retrovirus based on: type of c.p.e. induced in culture; morphology and density of the particle; presence of Mn(2+)-dependent, poly(rA)-directed reverse transcriptase activity which was associated with a density of 1.16 to 1.18 g/ml in sucrose; electrophoretic pattern of the polypeptides from purified virions; elevated [3H]UTP labelling of RNA in the cell cultures occurring at a density of 1.16 to 1.18 g/ml in sucrose. This report describes the first isolation of a retrovirus from a salmonid cell line.
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PMID:Isolation of a retrovirus from two fish cell lines developed from chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukaemia. 840 55

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5' non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.
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PMID:Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells. 858 37

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) agonists. High-molecular mass glycoconjugates (HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml. The purified HMMG material exhibited a single major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 microM) 4-5-fold over a baseline of 4 ng/min. The three dose-effect relations were nearly identical (K0.5 approximately 4 microM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.
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PMID:P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways. 867 Jan 74

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.
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PMID:A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase. 881 Oct 91


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