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)

Delineation of major T helper cell recognition sites of human immunodeficiency virus (HIV-1) proteins represents one important step in the design of an efficient acquired immune deficiency syndrome (AIDS) vaccine. Towards this end, we have explored the immunogenicity of HIV-1BRU proteins in the mouse model. Preliminary experiments revealed that inbred mice primed with whole inactivated HIV-1 developed strong CD4+ T cell proliferative responses to a variety of recombinant viral proteins including reverse transcriptase (RT). To characterize further the mouse T cell responses to this protein, several Ad- or Ed-restricted T hybridoma cells (THC) were established from BALB/c or DBA/2 mice. These THC were tested for their capacity to recognize a series of 15-mer synthetic overlapping peptides spanning three segments of HIV-1 RT that had been preselected on the basis of either alpha-helicity, amphipaticity, and/or for containing rare amino acid sequence patterns. Peptides corresponding to a C-terminal region (residues 528-560) of RT were recognized by several of the THC established from RT-primed mice. Furthermore, a non-alpha-helical peptide from this region (A3, 528-543) was capable of priming mice with different H-2 haplotypes for both peptide A3 and native RT CD4+ T cell recognition. In addition to the recently identified RT determinant 203-219 capable of triggering both mouse and human CD8+ CTL, the present results identify a good candidate for an immunodominant RT epitope capable of eliciting RT-specific T helper cell responses.
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PMID:Identification of a major human immunodeficiency virus-1 reverse transcriptase epitope recognized by mouse CD4+ T lymphocytes. 171 May 63

In addition to their properties as sequence-specific inhibitors of gene expression, sequence nonspecific phosphorothioate oligodeoxynucleotides have been shown to protect against the cytopathic effects of HIV-1. Although these compounds are effective inhibitors of HIV-1 reverse transcriptase in vitro, it is not certain that they exert their cytoprotective effect only in this manner. Initial binding of the HIV-1 virion to cells involves the interaction of the viral envelope protein gp120 with CD4. In this report, we describe flow cytometric data and a solid-phase ELISA assay that document the ability of a phosphorothioate deoxycytidine 28-mer to interfere with this interaction by competing with gp120 binding to CD4. The biological importance of this interaction is demonstrated by the fact that phosphorothioate oligodeoxycytidine inhibits syncytium formation resulting from HIV-1-induced cell fusion. These data suggest that phosphorothioate oligodeoxynucleotides may exert their cytoprotective effects, perhaps at least in part, by interfering with the binding of HIV-1 to the target cells.
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PMID:Phosphorothioate oligodeoxycytidine interferes with binding of HIV-1 gp120 to CD4. 171 Nov 18

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid-phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells.
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PMID:Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro. 171 43

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.
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PMID:A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi. 171 80

We used oligodeoxynucleotides to prevent reverse transcription of beta-globin mRNA by reverse transcriptase of avian myeloblastosis virus. Unmodified oligomers hybridized to the template arrested synthesis of cDNA in a dose dependent manner. The longer the oligomer the more efficient the inhibition, 50% inhibition being achieved at 0.3 and 30 microM of a 17- or a 12-mer, respectively. The use of complementary oligonucleotides with a 3' end blocked either by a dideoxy residue or by a dodecanol group also induced inhibition of cDNA synthesis.
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PMID:Inhibition of reverse transcription by unmodified and modified antisense oligodeoxynucleotides. 172 38

The combination of S-dC28 (a phosphorothioate oligodeoxcytidine 28 mer) with AZT, recombinant interferon alpha-A (IFN-alpha A) or dextran sulfate (DS) against replication of human immunodeficiency virus type 1 (HIV-1) were studied in MT4 cells, using both p24 core antigen and reverse transcriptase (RT) assays. Under the standardized conditions, the anti-HIV-1 dose-effect relationships of all test drugs showed sigmoidal curves with the following EC50 values: for the p24 core antigen assay, S-dC28, 0.03 microM; AZT, 0.004 microM; IFN-alpha A, 9.2 U/ml; DS, 0.26 micrograms/ml; for the RT assay, S-dC28, 0.04 microM; AZT, 0.01 microM; IFN-alpha A, 11.6 U/ml; and DS, 0.31 micrograms/ml. A computer software based on the median-effect principle and isobologram techniques were used to quantitatively analyze drug interactions by calculating the combination index (CI) where CI less than 1, = 1, and greater than 1 indicates synergism, additive effect and antagonism, respectively. For p24-ELISA, the interaction of S-dC28 and AZT in combination produced a slight antagonism on HIV-1 replicative inhibition with CI values of 1.29-1.10; for RT assays, at EC50-EC95 levels, the CI values are 1.96-1.11. For p24 core antigen assay, the combination of S-dC28 with IFN-alpha A exhibited a dose-dependent anti-HIV synergism with CI values of 1.15-0.87 at EC75-EC95 levels. The RT assays for the same combination showed a broad synergistic effect with CI values of 0.62-0.60, at EC50-EC95 levels. S-dC28 plus DS showed a nearly additive effect based on both assay methods.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential alteration of the anti-HIV-1 effect of phosphorothioate oligonucleotide S-dC28 by AZT, interferon-alpha, and dextran sulfate. 176 Feb 31

alpha-Anomeric oligonucleotides are resistant to nucleases and display parallel hybridization with complementary DNA and RNA sequences. Although alpha-DNA/beta-RNA duplexes are not substrates for RNases H, alpha-oligos are able to inhibit translation through a RNase H independent mechanism. alpha-Oligos and their alpha-phosphorothioate analogs (12 mer) targeted against the splice acceptor site of the HIV-TAT gene were potent inhibitors of de novo HIV infection. Furthermore alpha-phosphorothioate and dithioate homo-oligomers exhibit an in vitro, nonsequence-specific, inhibitory effect on HIV reverse transcriptase.
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PMID:Alpha-oligonucleotides: a unique class of modified chimeric nucleic acids. 177 67

Strong evidence points to mutation induction as one mechanism by which changes are introduced into normal cells to convert them into cancer cells. To understand the mechanisms by which mutations are induced in human cells by carcinogens, we are determining the kinds and spectra of mutations induced in the coding region of the hypoxanthine(guanine)phosphoribosyltransferase (hprt) gene. This region, composed of 654 bp, represents nine exons from a 44 kbp gene. To be able to analyze a large number of independent mutants rapidly and economically, we have optimized the conditions for copying mRNA directly from lysates of a small number of cells (e.g., 100) from a 6-thioguanine-resistant clone using reverse transcriptase and oligo(dT)12-18 primers. Then two 20-mer primers, specific for the cDNA of the hprt gene, are used to amplify the first and second strand cDNA 5 x 10(7)-fold during 30 cycles of polymerase chain reaction (PCR). The product (2 to 10 ng) is then purified by ultrafiltration, diluted 1:10, and subjected to an additional 30 cycles of PCR, using two 20-mer primers located just interior to the first set. The amplification product, 5 to 10 ug, is sequenced directly using three other end-labeled primers and Sequenase. To date, we have analyzed 26 mutants induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha,epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and found that 24/26 involved base substitutions. 97% of these involved G.C, predominantly G.C----T.A, distributed over seven exons, with many of the substitutions located in exon 3.
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PMID:Amplification of mRNA of the hprt gene from lysates of mutant human cells and direct DNA sequencing to determine the spectrum of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha, epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. 211 60

The possibility has been mentioned that a change in the structure is responsible for the deviant behavioral activity of gamma-endorphin in extracts of postmortem brain and pituitary gland samples of schizophrenic patients. This paper describes the investigation of this possibility by means of: amino acid composition analysis of alpha- and gamma-endorphin isolated from a pituitary gland of a schizophrenic patient; and nucleotide sequence analysis of the gamma-endorphin coding region of pro-opiomelanocortin (POMC) mRNA from two other pituitary glands, using the primer extension method. Both methods require no more than a single pituitary to obtain reliable results. alpha- and gamma-endorphin were isolated from an acid extract by gel filtration and two subsequent HPLC steps. In addition, the gamma-endorphin region of beta-endorphin was analyzed by enzymatic cleavage of beta-endorphin and isolation of the resulting fragment. Single-stranded gamma-endorphin cDNA was synthesized by reverse transcriptase using total cellular pituitary RNA and a 5' 32P-labeled oligodeoxyribonucleotide primer (20-mer) hybridizing close to the gamma-endorphin coding region of POMC mRNA. Single-stranded cDNA was digested with restriction enzyme HaeIII which generated a 148 nucleotides long radioactive cDNA fragment containing the gamma-endorphin cDNA sequence. The sequence of the 148 nucleotides fragment was determined. Neither the amino acid composition analysis nor the amino acid sequence derived from the cDNA nucleotide sequence revealed differences between schizophrenics and controls. Thus, no evidence was found for changes in the amino acid sequence of pituitary gamma-endorphin in these analyses, which include 3 cases of schizophrenia.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:gamma-Endorphin and schizophrenia: amino acid composition of gamma-endorphin and nucleotide sequence of gamma-endorphin cDNA from pituitary glands of schizophrenic patients. 242 70

The miscoding properties of a 5-bromodeoxyuridine (dB) containing DNA template during in vitro replication have been investigated. 5-bromodeoxyuridine was introduced site-specifically into the amber 16 codon of a 25-mer oligodeoxynucleotide representing part of the sequence of phi x174am16(+)DNA. The dB containing oligodeoxynucleotide served as a template for in vitro replication by DNA polymerase alpha, DNA polymerase I (Escherichia coli) and AMV reverse transcriptase. The amber 16 revertant assay was used to detect the presence of misincorporated bases in the replication products. For all three DNA polymerases, the presence of dB does not constitute a significant barrier to replication. Errors at the position of dB substitution were found to originate exclusively from dGTP:dB mispairing during in vitro replication thus inducing A-T----G-C transitions. The dGTP:dB mismatches are formed at a 2-4-fold higher frequency as compared to dGTP:T mismatches. Our results indicate that the miscoding potential of dB-substituted DNA templates during replication is only weak at the specific site observed.
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PMID:The fidelity of DNA polymerases during in vitro replication of a template containing 5-bromouracil at a specific site. 246 57


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