Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of silkmoth chorion mRNAs with calf thymus RNase H (EC 3.1.4.34; RNA-DNA-hybrid ribonucleotidohydrolase) in the presence of oligo(dT) specifically and effectively removes the 3'-terminal poly(A) sequences. Excision of non-poly(A) fragments cannot be detected. Under these conditions, RNase H leads to increased electrophoretic homogeneity of rabbit globin mRNA, presumably as a result of removal of poly(A) sequences that are inherently variable in length. Treatment with RNase H converts the three diffuse zones of messages for the several chorion proteins into multiple sharp bands.
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PMID:Electrophoretic patterns of deadenylylated chorion and globin mRNAs. 105 86

Using BspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain. The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant RTs were assayed. The elucidation of the structure of HIV-1 RT makes it possible to determine the locations of specific mutations in the three-dimensional structure of HIV-1 RT [E. Arnold, A. Jacobo-Molina, R. G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature (London) 357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T. A. Steitz, Science 256:1783-1790, 1992]. The mutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase activity without significantly affecting RNase H activity.
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PMID:Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase. 127 5

The spatial and temporal relationship between the polymerase and RNase H activities of human immunodeficiency virus type 1 reverse transcriptase has been examined by using a 40-mer RNA template and a series of DNA primers of lengths ranging from 15 to 40 nucleotides, hybridized to the RNA, as substrates. The experiments were executed in the absence and presence of heparin, an efficient trap to sequester any free or dissociated reverse transcriptase, thus facilitating the study of events associated with a single turnover of the enzyme. The results indicate a spatial separation of 18 or 19 nucleotides between the two sites. To examine the effect of concomitant polymerization on the RNase H activity, the substrate was doubly 5' end labeled on the RNA and DNA. This enabled the study of RNase H activity as a function of polymerization in a single experiment, and the results in the absence and presence of heparin indicate a tight temporal coupling between the two activities.
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PMID:Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship between the polymerase and RNase H activities. 127 94

RNase D was recently reported as a new enzymatic activity associated with HIV-1 reverse transcriptase (RT), cleaving RNA at two positions within the double-stranded region of the tRNA primer-viral RNA template complex (Ben-Artzi et al., Proc. Natl. Acad. Sci. USA 89 (1992) 927-931). This would make RNase D a fourth distinct activity of HIV-1 RT, in addition to RNA- and DNA-dependent DNA polymerase and RNase H. Using a specific substrate containing tRNA(Lys,3) hybridized to the primer binding site, we were able to detect the reported RNase D activity in our preparations of recombinant HIV-1 RT. This activity was also present in several active-site mutants of RT, suggesting that it is independent of the RNase H and polymerase functionalities of RT. Furthermore, we found that the cleavage specificity of RNase D is the same as that of RNase III isolated from E.coli. A likely explantation of these results--that the observed RNase D activity is attributable to traces of RNase III contamination--was further strengthened by the finding that the recombinant preparations of HIV-1 RT can specifically cleave a phage T7-derived double-stranded RNA processing signal, which has been used as a model substrate for detection of E.coli RNase III. Moreover, RT purified from an RNase III- strain of E.coli displayed no cleavage of the tRNA primer-RNA template complex.
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PMID:RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III. 128 Aug 10

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) result from the assembly of a common subunit alpha and a unique subunit beta, expressed in the same cell by single, structurally-related genes. In order to compare the intrinsic stability of the alpha, LH beta and FSH beta mRNA transcripts, we used cultured rat pituitary cells incubated in presence of actinomycin D. Hybridization with 32P-labelled rat cDNA probes showed that the cell content of all three mRNAs decreased with time, but at different rates. Apparent half-lives, estimated as the time necessary to observe a 50% mRNA decay, were 1.0 +/- 0.13 h for FSH beta, 6.5 +/- 0.25 h for alpha and 44 +/- 0.5 h for LH beta, stability thus exhibiting an inverse relation to the sizes of the corresponding mRNAs (approximately 1700, 800 and 700 nucleotides, respectively). Northern analysis revealed that the decline in mRNA abundance was associated with a progressive decrease in the length of mRNAs, most clearly visible for alpha and LH beta. For the most stable LH beta mRNA, shortening was apparent as early as 2 h after exposure to actinomycin D thus preceding neatly the decrease in amount starting at about 10-12 h. In vitro RNase H digestion demonstrated that shortening resulted from a reduction of the length of the poly(A) tract. These data establish that the three mRNAs coding for gonadotropin subunits have different stabilities although they share substantial homology. Diversity in size and sequence essentially resides in untranslated regions in which, we suggest, specific motifs and protein factors may interact to determine mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential stability of mRNAs coding for alpha and gonadotropin beta subunits in cultured rat pituitary cells. 128 Nov 25

The molecular events involved in antisense-mediated inhibition of retroviral transcription were studied by analyzing the in vitro effect of antisense oligodeoxynucleotides on reverse transcription by Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptase (RT). Oligonucleotides have been designed to be complementary to three targets located in the 5' region of the HIV-1 RNA genome: the transactivating response element (TAR), the U5 region and a sequence contiguous to the primer binding site (PrePBS). Antisense oligodeoxynucleotides were used with their 3'-OH end either free or blocked by a dideoxynucleotide in order to avoid cDNA synthesis. Experiments with two recombinant forms of HIV RT, carrying or not RNase H activity, showed that antisense oligonucleotides can arrest reverse transcription by an RNase H-independent mechanism. The AntiTAR oligonucleotide did not affect reverse transcription. In contrast, the AntiU5 and AntiPrePBS oligonucleotides led to an efficient inhibition of both forms of HIV RT. In the case of the AntiU5, the inhibition obtained in the absence of the RNase H activity indicates that this effect can be related to features of the RNA secondary structure. The AntiPrePBS oligonucleotide did bind to its target only in the presence of PBS primer. Use of shifted oligonucleotides showed that the AntiPrePBS inhibitory effect depends on a cooperative annealing with the AntiPBS primer on the template.
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PMID:In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription. 128 17

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
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PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).
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PMID:Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843. 128 6

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.
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PMID:Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product. 128 95

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.
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PMID:Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging. 130 4


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