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)

Human epidermal growth factor (EGF) receptor mRNA was detected in cryopreserved tissue sections adherent to whole glass slides using in situ reverse transcriptase polymerase chain reaction. EGF receptor cDNA was synthesized in situ by reverse transcription using an EGF receptor-specific oligonucleotide primer. In situ polymerase chain reaction amplification in the presence of digoxigenin-11-dUTP and subsequent binding with an antidigoxigenin antibody conjugated to alkaline phosphatase allowed direct visualization. Because DNase, RNase, or proteinase K are not required, tissue integrity is maintained. EGF receptor mRNA is expressed in the basal layer of normal human skin epithelium and is significantly overexpressed in squamous cell tumor specimens, which is consistent with conventional analysis of EGF receptor expression. The assay is semiquantitative, quicker, more sensitive, and void of the nonspecific binding associated with in situ hybridization. In situ reverse transcriptase polymerase chain reaction using whole glass slides is ideally suited for detecting moderate to infrequently expressed transcripts in biopsy specimens.
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PMID:Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. 808 54

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.
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PMID:Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H. 750 4

Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.
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PMID:m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC. 751 60

Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent. Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction. In addition, the cleavage at a single site requires Mg2+. Cleavage in the presence of Mn2+ is less specific. Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction. Our work indicates that calf RNase HI is designed to recognize Okazaki fragments. It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.
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PMID:Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI. 752 96

Bacterial reverse transcriptase is responsible for the synthesis of multicopy single-stranded DNA (msDNA). Reverse transcriptases from retron-Ec73 and retron-Ec107 do not contain an RNase H domain. Cellular RNase H is therefore considered to be required to make the mature form of msDNA. We found that RNase HI, but not RNase HII, is required for the production of the mature form of both msDNAs.
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PMID:The role of ribonuclease H in multicopy single-stranded DNA synthesis in retron-Ec73 and retron-Ec107 of Escherichia coli. 752 2

Replication complexes that contained either murine leukemia virus reverse transcriptase (MLV RT) or a variant reverse transcriptase without a ribonuclease (RNase) H domain (delta RH MLV RT) were visualized by enzymatic footprinting. Wild-type MLV RT protected template nucleotides +6 to -27, and primer nucleotides -1 to -26 of primers that had first been extended by one or four nucleotides. Although it catalyzed DNA synthesis, delta RH MLV RT stably bound template-primer only under conditions of reduced ionic strength and protected the duplex portion only as far as position -15. Despite altered hydrolysis profiles, both enzymes covered primarily the template-primer duplex, contradicting recent predictions based on the structure of rat DNA polymerase beta.
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PMID:Footprint analysis of replicating murine leukemia virus reverse transcriptase. 752 42

The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+. Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E. coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E. coli RNase HI is an excellent substrate binding region because of its sequence and/or location. The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase.
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PMID:Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase. 753 Mar 60

Novel 3,4-dihydro-6-benzyl-4-oxopyrimidines (DABOs), variously substituted at both the C-2 and C-5 positions of the pyrimidine ring, proved to be specific inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro. Some compounds showed potency at micromolar doses, no cytotoxicity at the maximum testable doses and selectivity indexes comparable to that of 2'-3'-dideoxyinosine (ddI). Mode of action studies suggested that DABOs interfered with a step of the virus multiplication cycle following adsorption and preceding integration. Enzyme assays indicated that DABOs targeted HIV-1 reverse transcriptase: they inhibited the RNA-dependent DNA polymerase activity in a template-dependent manner and, to a lesser extent, the DNA-dependent DNA polymerase activity. No inhibition of the RNase-H associated activity was observed. When DABOs were assayed in combination with 3'-azido-3'-dideoxythymidine (AZT) or ddI against HIV-1 in cell cultures, a slightly synergistic inhibitory effect was observed. The combination of DABO 546 and AZTTP in enzyme assays showed that the two compounds were kinetically mutually exclusive.
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PMID:Characterization of the anti-HIV-1 activity of 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs), new non-nucleoside reverse transcriptase inhibitors. 753 70

Micrometastases of solid tumors are most commonly detected by immunocytochemistry using monoclonal antibodies directed against tissue-specific gene products like cytokeratin-18 (CK-18) and the carcinoembryonic antigen (CEA). While CK-18 is a marker for epithelia in general, CEA is mainly employed in the detection of gastrointestinal and breast carcinomas. To improve the sensitivity and specificity of micrometastasis detection, we planned to establish polymerase chain reaction (PCR) assays for both markers. Here we provide strong evidence for the existence of a CK-18 pseudogene, since specific amplification (i) was readily obtained from healthy bone marrow donors, (ii) did not require reverse transcription of CK-18 mRNA and (iii) was not abolished by RNase treatment. Using a CK-18-specific probe, Southern blot analyses revealed identical-size fragments for both genomic DNA and a CK-18 cDNA after digestion with appropriate restriction enzymes. On the other hand, the amplification of CEA mRNA (i) was never observed in bone marrow samples of healthy donors or patients without solid tumors, (ii) required intact mRNA and the reverse transcriptase reaction, and (iii) could not be obtained after RNase treatment. In reconstitution experiments, single CEA-expressing tumor cells were reliably detected among 2 x 10(7) normal bone marrow cells. We conclude that, due to the presence of pseudogene(s), PCR-based detection systems are not readily suitable for CK-18, while the CEA mRNA amplification should provide a sensitive and specific test for the presence of ectopic, and hence presumed malignant, CEA-expressing cells in body fluids.
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PMID:Diagnosis of micrometastases by the amplification of tissue-specific genes. 754 67

The reverse transcriptase of retroviruses contains an RNase H activity essential for the proper synthesis of the viral DNA copy of the RNA genome. We have previously characterized a number of point mutations altering the RNase domain of the Moloney murine leukemia virus reverse transcriptase (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). One such mutation, Y586F (a Y-to-F change at position 586), reduced RNase H activity, as assayed by in situ gel analysis, to about 5% of the wild-type level and prevented viral replication. We have now recovered a revertant virus with near-normal infectivity and in vitro enzymatic activity. The revertant contains a single substitution, N613H, distant in the primary sequence of the protein, but modeling with the Escherichia coli RNase H structure suggests that the reverted residue is close in space to the original substituted residue. Examination of the structure permits some suggestions as to how this second-site revertant restores enzyme activity.
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PMID:Reversion of a Moloney murine leukemia virus RNase H mutant at a second site restores enzyme function and infectivity. 754 47


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