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)

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

Synthetic oligonucleotides complementary to putative retroviral primer-binding sites were used as hybridization probes to detect novel retroviruslike sequences. An 8.1-kilobase element with structural features of a retroviral provirus was isolated from a human genomic library by this approach. Nucleotide sequence analysis of its 600-base-pair long terminal repeats revealed characteristic motifs known as regulatory signals for RNA polymerase II transcription: CCAAT, TATA, and ATTAAA. In addition, a putative pol gene displays apparent homologies to conserved regions of retroviral reverse transcriptase. The 5' long terminal repeat is flanked at its 3' end by a putative primer-binding site for reverse transcription with homology to tRNA(Pro). This element is therefore termed HuRRS-P (human retrovirus-related sequence-proline). There are 20 to 40 copies of HuRRS-P homologous sequences in DNAs of human and simian origin.
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PMID:Isolation of novel human retrovirus-related sequences by hybridization to synthetic oligonucleotides complementary to the tRNA(Pro) primer-binding site. 243 28

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.
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PMID:Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica. 244 71

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

Based on the crystallographic structure of the active site in the reverse transcriptase (RT) of human immunodeficiency virus (HIV), a group of hydrophobic polyadenylic acid (5') derivatives were designed and synthesized as inhibitors of the enzyme. These compounds were found to inhibit all six of the RTs tested, with IC50 = 10(-11)-10(-8) M, but did not inhibit either RNA polymerase II (even at 10(-5) M) or DNA polymerase I up to 10(-6) M inhibitor concentration. The underivatized poly(A) did not inhibit any of the RTs tested under the same conditions. In aqueous solutions of purified HIV-1 RT, poly-2'-O-(2,4-dinitrophenyl)-oligo(A) was found to inhibit the enzyme reversibly and compete with the primer-template poly(A)-(dT)12, whereas poly-2'-O-(3-fluoro-4,6-dinitrophenyl)-poly(A) was found to inactivate HIV-1 RT irreversibly by covalent labeling. A comparison of physicochemical properties of the hybrids poly(A)-poly(dT) and dinitrophenyl-poly(A)-poly(dT) shows that the hydrophobic dinitrophenyl groups stabilize double helical structures. These inhibitors were also found to be effective in keeping susceptible lymphocytes viable in the presence of HIV-1 (wild type). The effective inhibitor concentrations (EC50) were found to be 0.2-2.6 microgram/ml. No toxic effect on the host cells was found even at 100-1000-fold higher inhibitor concentrations.
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PMID:Design of structure-based reverse transcriptase inhibitors. 751 57

We recently discovered an opposing initiator promoter (Inr) downstream of the sense promoter region of the eIF-2 alpha gene (Silverman, T., Noguchi, M., and Safer, B. (1992) J. Biol. Chem. 267, 9738-9742). By reverse transcriptase/polymerase chain reaction analysis of G0 and activated (G1) T-lymphocyte RNAs, overlapping sense and antisense transcripts are now identified. Sense transcription of the eIF-2 alpha gene proceeds from left to right to generate alpha-mRNA; antisense transcription proceeds from right to left to generate RNA, having a sequence complementary to eIF-2 alpha mRNA. Upstream indicates a position 5' relative to the transcription start site. Using DNase I footprint analysis and EMSA, we have found a potential cis-regulatory sequence immediately upstream of the Inr which binds a 43-kDa protein. In addition to conferring protection against DNase I (+457 to +474), the factor also generates hypersensitive sites directly over the Inr (+447 to +457). Insertion of the Inr footprint region into a luciferase reporter gene construct increases expression 150-fold. While mutation of the Inr conserved sequence decreases luciferase activity by 50%, mutation of the 43-kDa factor binding site inhibits luciferase activity by 20%. Sense orientation of the Inr footprint region decreases activity by 80%. The 43-kDa Inr-associated binding protein may be involved in allowing access of RNA polymerase II transcription complexes ot the initiation site of this TATA-less gene. A model for the regulation of eIF-2 alpha expression involving the rapid degradation of dsRNA generated by the relative activities of the two overlapping and opposing promoters is proposed.
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PMID:Characterization of an antisense Inr element in the eIF-2 alpha gene. 752 81

The genomes of Lilium species are very large, containing 30-40 million kilobase pairs of DNA. An abundant fragment of 3.5 kb was released by BamHI digestion of genomic DNA of Lilium speciosum. Analysis of 20 genomic clones containing sequences homologous to the fragment showed it to be part of a 4.45 kb dispersed repeat, which was named del2. Sequence analysis of one full element and regions of four others revealed del2 to be a non-LTR (long terminal repeat) retrotransposon. It is flanked by short direct repeats of from 4 to 13 bp and a run of adenines occurs at one end (the proposed 3' end), 63 bp downstream from a polyadenylation signal. A possible RNA polymerase II promoter similar to that found in Drosophila I and F group elements is present internally 30 bp downstream from the 5' end. Two degenerate open reading frames (ORFs) are present, the 5' ORF containing a gag-related cysteine motif, and the 3' ORF containing a different cysteine motif also found in most non-LTR retrotransposons. The 3' ORF also has regions with homology to reverse transcriptase sequences, which are most similar to those in Cin4 of maize, the L1 LINE elements of humans and mice and the R2 ribosomal DNA inserts of insects. The majority of del2 elements occur as the full 4.45 kb element. They account for an estimated 4% of the L. speciosum genome and are present in approximately 250,000 copies. del2-related sequences were also detected in 12 other monocot species.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An abundant LINE-like element amplified in the genome of Lilium speciosum. 768 Nov 39

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively.
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PMID:Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication. 889 79

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is a polypeptide that forms a ternary complex with IGFs and an acid-labile subunit. The hormonal regulation of the components of this complex is highly controversial, and both IGF-I and GH have been shown to mediate the expression/synthesis of IGFBP-3. This study investigates the regulation of IGFBP-3 protein, measured by RIA and Western ligand blot, and messenger RNA (mRNA) expression, measured by Northern analysis and reverse transcriptase-PCR, in SKHEP-1 human hepatocarcinoma cells. SKHEP-1 cells significantly increased the IGFBP-3 concentrations in conditioned medium (CM) when treated with GH (0.1-10 ng/ml), IGF-I (1-100 ng/ml), or Des(1-3)-IGF-I (1-100 ng/ml) in a dose-dependent manner (>3-fold). The increase in IGFBP-3 protein concentrations in CM was accompanied by a corresponding increase in IGFBP-3 mRNA levels. Interestingly, time-course studies showed that the GH-induced increase in IGFBP-3 mRNA preceded the IGF-I-induced increase (6 h for GH-induced IGFBP-3 mRNA; 12 h for IGF-I-induced IGFBP-3 mRNA). The half-life of IGFBP-3 mRNA was evaluated after transcriptional arrest by treatment with a RNA polymerase II inhibitor (5,6-dichloro-1beta-D-ribofuranosylbenzimidazole), and was found to be 14-18 h and unaltered by GH or IGF-I treatment. The induction of IGFBP-3 by GH was not due to the indirect action of locally synthesized IGF-I, because 1) no immunoreactive IGF-I was detected in the CM of control or GH-treated cells; 2) Northern blots revealed no IGF-I mRNA expression in SKHEP-1 cells; 3) reverse transcriptase-PCR did not detect any expression of the IGF-I gene; and 4) time-course studies showed an earlier increase in IGFBP-3 mRNA after GH treatment than after IGF-I treatment. Thus, the results obtained in this study are consistent with an IGF-I-independent regulation of IGFBP-3 gene expression by GH.
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PMID:Evidence for insulin-like growth factor (IGF)-independent transcriptional regulation of IGF binding protein-3 by growth hormone in SKHEP-1 human hepatocarcinoma cells. 907 3

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2, 4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1, 3-indandione, and others. While active against both 3'-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the cinnamoyl residue C=C-C=O in a syn disposition, differently from flavone derivatives characterized by an anti arrangement about the same fragment. Certain inhibitors, lacking one of the two pharmacophoric catechol hydroxyls, retain moderate potency thanks to nonpharmacophoric fragments (i.e., a m-methoxy group in curcumin) which favorably interact with an "accessory" region of IN. This region is supposed to be located adjacent to the binding site accommodating the pharmacophoric dihydroxycinnamoyl moiety. Disruption of coplanarity in the inhibitor structure abolishes activity owing to poor shape complementarity with the target or an exceedingly high strain energy of the coplanar conformation.
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PMID:Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling. 976 32


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