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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The lily retrotransposon del 1-46 is 9345 base pairs (bp) long. It has long terminal repeats (LTRs) of 2406 bp (left) and 2415 bp (right), which differ in sequence by 1.4%. Sequences similar to those involved in priming DNA synthesis in retroviruses occur in the internal region. Near the left LTR is a sequence complementary to 18 residues at the 3' end of methionine initiator tRNA of three plant species, and a run of 12 purines occurs close to the right LTR. One internal reading frame of del 1-46 has relatively few stop codons. The 1462-codon product from this frame has motifs, in N to C terminus order, corresponding to those identified with RNA binding, protease, reverse transcriptase, RNase H, and integrase functions in retroviruses and certain other retrotransposons. Amino acid sequence comparisons of three conserved pol regions show del to be closely related to the Ty3 retrotransposon of yeast (37-40% identity). del is also related to the gypsy group of Drosophila (17.6, 297, gypsy/mdg4, and 412), showing closer identity with their reverse transcriptase (32-38%) and RNase H (36-45%) domains than with their integrase domain (21-26%). It is proposed that a gypsy group ancestor exchanged the integrase region with a more distantly related element since its divergence from a del/Ty3 common ancestor. The occurrence of related retrotransposons in three different kingdoms (plants, animals, and fungi) strongly implies their horizontal transmission in recent evolutionary time.
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PMID:Plant retrotransposon from Lilium henryi is related to Ty3 of yeast and the gypsy group of Drosophila. 254 87

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.
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PMID:Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes. 299 63

The mutD(dnaQ) gene in Escherichia coli codes for the epsilon subunit of the DNA polymerase pol III holoenzyme. Previous work has shown that this gene lies adjacent to the gene coding for RNase H (rnh). The two products are translated from diverging promoters. Here we report on the 1.6 kb (1 kb = 10(3) bases or base-pairs) sequence of the region coding for both genes, and the transcripts encoded by them. mutD codes for two transcripts, one of whose origins lies within the rnh structural gene. Both transcripts overlap and are complementary to a region of the rnh transcript. Thus, they can potentially form double-stranded helices with rnh. Of the two possible double-stranded structures, the shorter does not interfere with a likely rnh ribosome binding site, while the longer one does. We suggest that this unique organization may regulate rnh translation rates.
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PMID:DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene. 302 34

Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.
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PMID:Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains. 748 84

The gypsy group of long-terminal-repeat retrotransposons contains elements having the same order of enzyme domains in the pol gene as do retroviruses. Elements in the gypsy group are now known from yeast, filamentous fungi, plants, insects, and echinoids. Reverse transcriptase and RNase H amino acid sequences from elements in the gypsy group--including the recently described SURL elements, TED, Cft1, and Ulysses,--were aligned and analyzed by using parsimony and bootstrapping methods, with plant caulimoviruses and/or retroviruses as outgroups. Clades supported at the 95% level after bootstrapping include (1) 17.6 with 297 and (2) all of the SURL elements together. Other likely relationships supported at lower bootstrap confidence intervals include (1) SURL elements with mag, (2) 17.6 and 297 with TED, and this collective group with 412 and gypsy, (3) Tf1 with Cft1, (4) IFG7 with Del, and (5) all of the retrotransposons in the gypsy group together, to the exclusion of Ty3. In contrast with an earlier analysis, our results place mag within the gypsy group rather than outside of a cluster that contains gypsy group retrotransposons and plant caulimoviruses. Several features of retrotransposon genomes provide further support for some of the aforementioned relationships. The union of SURL elements with mag is supported by the presence of two RNA binding sites in the nucleocapsid protein. Location of the tRNA primer binding site and the presence of a long open reading frame 3' to the pol gene support the 17.6-297-TED-412-gypsy cluster.
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PMID:Phylogenetic relationships of reverse transcriptase and RNase H sequences and aspects of genome structure in the gypsy group of retrotransposons. 750 45

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.
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PMID:Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry. 753 31

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
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PMID:Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus. 754 60

The number of Alu transcripts that accumulate in HeLa and other human cells is normally very low; however, infection with adenovirus type 5 increases the expression of Alu elements dramatically, indicating that the potential for polymerase III (pol III)-dependent Alu transcription in vivo is far greater than generally observed (B. Panning and J.R. Smiley, Mol. Cell. Biol. 13:3231-3244, 1993). In this study, we employed nuclear run-on in combination with a novel RNase H-based assay to investigate transcription from uninfected and adenovirus type 2-infected nuclei, as well as genomic DNAs from uninfected and infected cells. When performed in the presence of excess uninfected nuclear extract, such assays revealed that (i) the vast majority of transcriptionally competent Alu elements in nuclei are masked from the pol III transcriptional machinery and (ii) the induction of Alu expression upon adenovirus infection can be largely accounted for by an increased availability of these elements to the pol III transcription machinery. We also investigated the role of H1 histone for silencing of Alu genes and, in comparison, mouse B2 repetitive elements. Depletion of H1 led to an approximately 17-fold activation of B2 repetitive elements but did not change Alu transcription relative to that of constitutively expressed 5S rRNA genes. These results are consistent with the view that Alu repeats are efficiently sequestered by chromatin proteins, that such masking cannot be accounted for by nonspecific H1-dependent repression, and that adenovirus infection at least partially overrides the repressive mechanism(s).
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PMID:Adenovirus type 2 preferentially stimulates polymerase III transcription of Alu elements by relieving repression: a potential role for chromatin. 762 22

We produced a series of monoclonal antibodies against the human immunodeficiency virus (HIV-1) reverse transcriptase by immunizing mice with either purified recombinant HIV-1 p66 protein or with recombinant vaccinia virus which expresses HIV-1 pol sequences. The antibodies generated were specific for the reverse transcriptase protein, and recognized only the p51 and p66 subunits of the enzyme in each of the HIV-1 viral lysates and lysates of HIV-1 infected cells. The antibodies did not cross-react with HIV-2 reverse transcriptase. Most important, several of the antibodies are unique, in that they are the first that can bind to sites close to the N-terminal. This latter region has been suggested to form part of the polymerase domain of the reverse transcriptase. None of the antibodies could neutralize either the RNA-dependent DNA polymerase or RNase H activities of either p66 or p51/66 proteins. The binding patterns of these various antibodies to p66 and p51/66 were dependent on each of three independent variables: the source of antigen amployed, the individual specificity of the antibody, and the method employed to detect reactivity. These monoclonal antibodies provide useful reagents for the study of reverse transcriptase native structure-function relationships.
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PMID:Generation and characterization of murine monoclonal antibodies reactive against N-terminal and other regions of HIV-1 reverse transcriptase. 768 57

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4


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