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)

Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.
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PMID:Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90. 750 26

Stem cell factor (SCF), or c-kit ligand, is a multipotent growth factor that has been implicated in an important role in various aspects of animal development, including maintenance of the viability of primordial germ cells. A porcine SCF (pSCF) cDNA was generated from porcine uterine endometrial mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), and its nucleotide sequence was determined. The 952-bp pSCF cDNA contained an open reading frame encoding 274 amino acids. The deduced amino acid sequence of pSCF is approximately 86%, 83%, and 82%, identical to human, rat, and mouse SCFs, respectively; and it contains the four conserved cysteine residues and Asn-linked glycosylation sites. One additional amino acid was identified in pSCF, Glu130, which is not in the human (hSCF), rat (rSCF), or mouse (mSCF) sequences. Northern analysis of poly(A)+ RNA obtained from Day 16 pregnant endometrium revealed a transcript of approximately 6.5 kb. The size of this transcript is consistent with the size of full-length SCF mRNA, and the occurrence of alternatively spliced pSCF mRNAs were not detected by RT-PCR/Southern hybridization analysis of endometrial and ovarian total cellular RNA (tcRNA). Porcine SCF mRNA has been localized by in situ hybridization in porcine endometrial stromal tissue. Pregnancy does not appear to be a prerequisite for pSCF mRNA expression in endometrial tissue since it was detectable in tissue and tcRNA obtained from pregnant and nonpregnant gilts. The biological significance of uterine pSCF expression is currently unclear, but it probably participates in intracellular communication within the uterus.
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PMID:Porcine stem cell factor/c-kit ligand: its molecular cloning and localization within the uterus. 750 58

Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.
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PMID:Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. 750

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.
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PMID:Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors. 750

Tad is a LINE-like retrotransposon of Neurospora crassa. The element was originally detected and cloned using the am gene as a transposon trap in hybrid strains derived from a cross of Adiopodoume (a wild collected strain) and a laboratory strain devoid of Tad elements. We report the cloning and sequencing of an active Tad element, Tad1-1, which is capable of independent transposition. Transposition was demonstrated by screening for transfer of the element from a donor nucleus that contained the Tad1-1 element as the only active Tad, into a naive nucleus within a forced heterokaryon. We also report here the sequence analysis of Tad1-1, and its comparison with the sequence of another active element, Tad3-2. These elements are approximately 7 kb in length. They contain two long open reading frames (ORFs) encoded on the strand of the same polarity as the full-length transcript. ORF1 encodes a putative protein of 486 amino acids. Homology to the first ORF of other LINE elements is confined to three cysteine-rich motifs, located near the carboxy-terminus, that are thought to be involved in binding nucleic acids. The second ORF is 1156 amino acids in length and shows homology to the reverse transcriptase domains of various retroviruses and retrotransposons. Tad1-1 and Tad3-2 differ in only ten positions over their whole length.
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PMID:Tad1-1, an active LINE-like element of Neurospora crassa. 751 93

Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E-BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses.
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PMID:Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase. 753 30

hLH-2, a transcription factor that contains double cysteine rich regions (LIM motifs) and a homeobox (Hox) DNA-binding domain shows aberrant high expression in all cases of chronic myelogenous leukemia (CML). This gene has been mapped to the chromosome 9q33-34.1, the same region as the reciprocal translocation that creates the breakpoint cluster region (BCR)-ABL chimera of the Philadelphia chromosome (Ph'). To investigate the possible involvement between the BCR-ABL fusion gene and hLH-2 in the pathogenesis of CML, an hLH-2-negative CML cell line, JK-1 that carries double Ph' chromosomes, was examined. Like other CML cells, high BCR-ABL fusion mRNA levels are expressed, but no transcript of hLH-2 was detected in JK-1 cells as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with the CML cell line, K-562, an additional rearrangement of the BCR gene was observed in JK-1 as determined by Southern blot hybridization; however, the hLH-2 gene was normal. These findings raise interesting questions about the possible roles of either the abnormal BCR gene or other genetic events such as the complex chromosomal abnormalities that result in hLH-2 being turned off in JK-1 cells.
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PMID:A structurally abnormal breakpoint cluster region gene in a transcription factor, hLH-2-negative human leukemia cell line. 760 May 33

To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.
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PMID:The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects. 855 Jul 89

We investigated the effects of glutathione (GSH), the major naturally occurring thiol, and a pharmacologic thiol precursor of GSH, N-acetyl cysteine (NAC), on the expression of human immunodeficiency type 1 (HIV-1) in primary cord blood and adult donor monocyte-derived macrophages (MDM). HIV-1 infection of cord blood and adult MDM was accomplished after incubating 10-15-d-old cultures for 4 h with a monocyte-tropic strain of HIV-1 (Bal). After 1 wk in culture cell supernatants were tested for reverse transcriptase (RT) activity. MDM were exposed to 5, 10 and 20 mM concentrations of both GSH and NAC before infection, during infection, and after infection was established. GSH and NAC suppressed the replication of HIV-1 in both primary cord blood and adult donor MDM in a concentration dependent fashion. These suppressive effects were more pronounced in cord-derived cells than in adult-derived cells. In cells treated with GSH or NAC before infection, there was no significant rise in RT activity as compared with controls. Similarly, when cells were treated with GSH and NAC and simultaneously infected, there was also no significant rise in RT activity after 1 wk in culture. In cells treated after infection was established, RT values were suppressed 80-90% that of untreated controls. This effect persisted for 1-2 wk after exposure to GSH and NAC. Untreated controls demonstrated syncytium formation and lost characteristics of spreading and elongation 2 wk after HIV-1 infection, whereas most of the treated cells remained free of syncytium and retained cytoplasmic spreading, adherence, and elongation. These data are consistent with other studies of thiol suppression of HIV-1 replication and demonstrate a similar observation for primary cultured cord MDM. These results may offer new approaches toward cellular protection after infection with HIV-1.
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PMID:Thiol suppression of human immunodeficiency virus type 1 replication in primary cord blood monocyte-derived macrophages in vitro. 767 9

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


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