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)

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

Perlecan is a modular heparan sulfate proteoglycan that harbors five domains with homology to the low density lipoprotein receptor, epidermal growth factor, laminin and neural cell adhesion molecule. Using a monoclonal antibody directed against the laminin-like domain of perlecan, we have recently shown that perlecan is widely expressed in all lymphoreticular systems. To investigate further this observation we have studied the expression of perlecan in two human leukemic cell lines. Using reverse transcriptase-PCR, ribonuclease protection assay, and metabolic labeling we detected significant perlecan expression in the multipotential cell line K562, originally derived from a patient with chronic myelogenous leukemia. In contrast, the promyelocytic cell line HL-60 expressed perlecan at barely detectable levels. These results were intriguing because the K562 cells do not assemble or produce a classical basement membrane. Following induction with either sodium butyrate or the phorbol diester 12-0-tetradecanoylphorbol-13-acetate (TPA), K562 and HL-60 differentiate into early progenitor cells with erythroid or megakaryocytic properties, respectively. Following treatment of K562 and HL-60 cells with either of these agents, perlecan expression was markedly increased in K562 cells. In contrast, we could detect perlecan protein synthesis in HL-60 cells only at very low levels, even after induction with TPA or sodium butyrate. Collectively, these results indicate that perlecan is actively synthesized by bone marrow derived cells and suggest that this proteoglycan may play a role in hematopoietic cell differentiation.
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PMID:The proteoglycan perlecan is expressed in the erythroleukemia cell line K562 and is upregulated by sodium butyrate and phorbol ester. 754 67

We have recently identified a new exon of the CD44 gene and demonstrated abnormal retention of a noncoding section, intron 9, in mRNA from bladder carcinomas. To analyze this further, the present study examined CD44 gene expression in cell lines from 14 esophageal, 3 colonic, and 4 breast carcinomas and in fresh samples from 20 colorectal carcinomas and corresponding normal colonic mucosa, using reverse transcriptase followed by the polymerase chain reaction (RT-PCR). This confirmed that there was abnormal assembly of several exons of the gene in cell lines and in tumor tissues from these organs. However, the most striking new finding was that intron 9 was present in RNA from 11 esophageal, 3 colon, and 1 breast carcinoma cell line, respectively. This was confirmed by RNase and DNase digestion analysis. Moreover, it was detected both in nuclear and cytoplasmic mRNA fractions, indicating that abnormal splicing of pre-mRNA occurs in cancer cells. The abnormal retention of intron 9 in CD44 gene transcripts was also demonstrated in tumor tissues from 16 (80%) of 20 patients with colon carcinoma, but there was no correlation with Dukes' stage. The biological significance of these observations is not yet understood. However, it is clear that, as with the abnormal expression pattern of CD44 variant exons, intron 9 retention is a good-candidate molecular diagnostic tool for colorectal carcinomas.
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PMID:Abnormal retention of intron 9 in CD44 gene transcripts in human gastrointestinal tumors. 754 38

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.
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PMID:Formation of stable and functional HIV-1 nucleoprotein complexes in vitro. 756 74

Protein kinase C (PKC), a widely-distributed enzyme implicated in the regulation of many physiological processes, consists of a family of at least twelve isoenzymes which differ in tissue distribution, subcellular localization, regulatory properties, etc. In addition to this heterogeneity at the protein level, we identify here for the first time a PKC zeta pseudogene (psi PKC zeta) transcript, specifically expressed in the brain, which is identical with PKC zeta except for sequence divergence within the first variable domain (V1). The authenticity of this unique V1 sequence (V1') in mRNA was confirmed by RNase protection and reverse transcriptase PCR (RT-PCR) analysis. When translated in-frame with PKC zeta, a stop codon is located 28 amino acids towards the N-terminus of the divergence point and the intervening sequence lacks an expected initiating methionine. psi PKC zeta is non-functional in terms of protein synthesis since Western blotting with an antibody directed against the C-terminus of PKC zeta failed to reveal a protein smaller than PKC zeta, and synthetic psi PKC zeta RNA failed to support protein synthesis in a translation system in vitro. PCR amplification of rat genomic DNA demonstrated lack of an intron at the junction between V1' and the first constant domain (the V1'-C1 border), and genomic DNA Southern blot analysis using PKC zeta and psi PKC zeta-specific probes indicated that they have different loci. psi PKC zeta, therefore, is not derived from the PKC zeta gene by alternative splicing, but rather is the product of a distinct gene. In Northern blot analysis, brain PKC zeta mRNA was identified as a low-abundance 3.1 kb transcript, while the abundant 2.5 and 4.7 kb mRNAs previously reported to encode PKC zeta are, in fact, psi PKC zeta transcripts. Analysis of rat brain, heart, lung, liver, kidney and skeletal muscle revealed psi PKC zeta mRNA only in brain. PKC zeta transcripts were most abundant in lung and kidney (2.7 and 4.7 kb mRNAs), correlating with the tissue profile of PKC zeta immunoreactivity in Western blots. Probes complementary to the common V5 and C1 domains detected both PKC zeta and psi PKC zeta transcripts. Interestingly, the C1 probe also detected an abundant novel 1.75 kb mRNA in brain and heart, suggesting the existence of an additional PKC zeta-related species. This work, therefore, also emphasizes the importance of careful choice of oligonucleotide and cDNA probes to study PKC zeta mRNA.
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PMID:Identification of a brain-specific protein kinase C zeta pseudogene (psi PKC zeta) transcript. 757 16


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