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)

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

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

The minus strand of hepatitis A virus can be detected specifically by reverse transcription and polymerase chain reaction amplification in infected cell culture extracts. Several controls gave evidence that the amplified fragment actually used the minus strand as initial template. Non-thermostable reverse transcriptase was not efficient for this purpose because of self-priming of the positive-stranded viral RNA during the reverse transcription step. This problem was overcome by the use of the thermostable rTth DNA polymerase that also has reverse transcriptase activity in the presence of Mn2+.
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PMID:Separate detection of the two complementary RNA strands of hepatitis A virus. 753 50

A putative retrovirus was isolated from a dog with a severe, acquired immunodeficiency-like syndrome. The haematological abnormalities and immunological deficiencies included anaemia, leucopenia (lymphopenia and neutropenia), thrombocytopenia, decreased humoral immunity, and ineffective T-cell responses in-vitro. The necropsy findings included generalized lymphoid depletion, severe bone marrow hypoplasia, plasmacytic infiltrates in lymphoid and non-lymphoid organs, and severe secondary infections. Supernates of peripheral blood mononuclear cell cultures from the affected dog contained an agent with manganese-dependent reverse transcriptase (RT) activity that sedimented at a density of 1.122 g/ml. RT activity was also found post-mortem in extracts prepared from the bone marrow, lymph nodes, and small intestine. The lymph nodes and small intestine expressed a 3.8 kb mRNA that was recognized by a bovine leukaemia virus (BLV) pol DNA probe by Northern blotting. DNA isolated from the lymph nodes and small intestine from the affected dog showed distinct band patterns by Southern analysis, suggesting an exogenous retrovirus. The retrovirus could be propagated in normal canine peripheral blood mononuclear cells or short-term canine lymphocyte cell lines in-vitro, and was cytopathogenic for cells of canine, but not human, origin. These results suggest the existence of a pathogenic canine retrovirus capable of producing disease of the type associated with retroviruses in other species.
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PMID:Retrovirus-like activity in an immunosuppressed dog: pathological and immunological findings. 753 63

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

A purified preparation of endogenous RNA-dependent DNA-polymerase (reverse transcriptase) earlier identified in rat brain (Ivanov, V.A., Pakhotin, P.I., Bobkova, N.V., and Ilyin, Yu.V. parallel Dokl. RAN (1992). V. 323, P. 173-177) has been obtained. A comparative analysis of the enzyme and recombinant reverse transcriptase coded by the mobile genome element jockey earlier expressed in a heterological cell system (Ivanov V.V., Melnikov A.A., Siunov A.V., Fodor I.I., Ilyin, Yu.V. parallel EMBO J. (1991), V. 10, P. 2489-2495) has been carried out. Like retroviral RNA-dependent DNA-polymerases, these enzymes show preference for polyribonucleotides and can use poly(rCm) as template. Besides they are inhibited by SH-reagents and require bivalent cations (Mg2+ or Mn2+) and detergent and/or KCl as ionic strength carrier. The enzymes differ drastically from retrovirus reverse transcriptases by a number of catalytic properties (low optima of concentration of requisite cations and ionic strength, strong preference for Mn2+, highly efficiency in using poly(rCm), lack of associated RNase H activity) but exhibit a high degree of similarity among themselves with regard to the above properties. It is suggested that endogenous reverse transcriptase from rat brain is a product of expression of the mobile genome element of the LINE family.
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PMID:[Endogenous cellular reverse transcriptase in the rat brain. A comparative analysis of the authentic enzyme and recombinant reverse transcriptase coded by a mobile genetic element of the LINE class]. 757 75

Integrins alpha 1 beta 1 and alpha 2 beta 1 are major cellular receptors for collagens. The alpha 1 and alpha 2 subunits contain a approximately 200 amino acid inserted domain (I-domain) in their N-terminal region and, because of the homology between the I-domains and the collagen-binding A-domains of von Willebrand factor, it has been suggested that the I-domains might mediate the collagen-binding functions of alpha 1 beta 1 and alpha 2 beta 1. In order to fully investigate this hypothesis, we have generated recombinant human alpha 2 I-domain (r alpha 2I) by reverse transcriptase-polymerase chain reaction/bacterial expression and tested its ability to mediate the collagen-binding functions of alpha 2 beta 1. R alpha 2 I binds specifically to type I collagen in a concentration-dependent manner: binding is cation dependent and, like the complete receptor, is supported by magnesium and manganese ions but not by calcium ions. R alpha 2I is recognised by anti-functional anti-alpha 2 monoclonal antibodies 6F1, 5E8 and P1E6 in capture ELISAs, and anti-functional antibodies inhibited r alpha 2I-collagen binding. In addition, r alpha 2I inhibits cell spreading on collagen. R alpha 2I is therefore a collagen-binding domain and can account for many of the collagen-binding functions of integrin alpha 2 beta 1. We have also determined the collagen specificity of r alpha 2I and found that it binds types I, II and XI collagen.
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PMID:Integrin alpha 2 I-domain is a binding site for collagens. 761 81

The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography. HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein. Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA. Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end. The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates. The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide. In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved. In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein. After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.
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PMID:Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus. 768 24

A 25mer oligonucleotide containing a single N-(deoxyguanosin-8-yl-)-1-aminopyrene (dGAP), the major DNA adduct formed by reductively activated 1-nitropyrene, was synthesized. The adduct was located at nucleotide 21 from the 3' end. DNA synthesis on this template by human DNA polymerases alpha and beta, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site. Only when a 3'-->5' exonuclease-deficient Klenow fragment was used was incorporation of a nucleotide opposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent. Only a relatively small proportion of full-length product (< 5%) was detected. In the presence of Mn2+, the efficiency of bypass with this polymerase increased. When a 20mer primer was elongated in the presence of only one nucleotide triphosphate, deoxycytidylic acid was preferentially incorporated opposite the adduct. Deoxycytidine opposite the adduct was also preferred when a set of 21mer primers (containing each of the four nucleotides opposite dGAP) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these results, extension of a 15mer primer was carried out with all four deoxynucleotide triphosphates and the products were isolated. Maxam--Gilbert sequencing of each elongation product showed that primer extension occurred in an error-free manner. We conclude that dGAP is a strong block of DNA replication. However, when translesion synthesis occurs, it is largely accurate.
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PMID:DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene. 772 60

Integrins comprise a superfamily of alpha beta heterodimers that serve as cell signaling as well as adhesion molecules. We demonstrate that the alpha 3 beta 1 and alpha 6 beta 1 integrins are laminin/merosin receptors expressed in human thymocytes. By reverse transcriptase-PCR analysis, we determined that the alpha 3A beta 1, but not the alpha 3B beta 1, cytoplasmic structural variant of alpha 3 beta 1 is expressed in thymocytes. In contrast, both alpha 6A beta 1 and alpha 6B beta 1 cytoplasmic structural variants of alpha 6 beta 1 are expressed. A small percentage (10 to 15%) of human thymocytes bind to immobilized laminin, and even fewer (3 to 5%) bind to merosin, the laminin isoform normally present in the thymus. This binding, however, can be increased to 39 to 41% after activation of thymocytes with Mn2+ (or PMA). Binding to either laminin or merosin is completely inhibited by anti-beta 1 mAb or by a mixture of anti-alpha 3 and anti-alpha 6 mAbs, indicating that both alpha 3 beta 1 and alpha 6 beta 1 participate in thymocyte adhesion to the laminin family of extracellular matrix proteins. The protein kinase C inhibitors, calphostin C and staurosporine, inhibit Mn(2+)-enhanced thymocyte binding, suggesting that protein kinase C activity is crucial for the binding. Furthermore, the data indicate that at least two divalent cation binding sites serve to regulate integrin binding activity. Finally, we show that both immobilized laminin and merosin have costimulatory function for anti-CD3-induced thymocyte proliferation, and both anti-alpha 3 and anti-alpha 6 mAbs can block this proliferative response. The cooperative function of alpha 3 beta 1 and alpha 6 beta 1 evidenced in the laminin/merosin binding and proliferation assays suggests that thymocyte-merosin interactions may play an important role in thymic T cell development.
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PMID:Alpha 3 beta 1 and alpha 6 beta 1 integrins mediate laminin/merosin binding and function as costimulatory molecules for human thymocyte proliferation. 781 63


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