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)

A single RNase H enzyme was detected in extracts of Streptococcus pneumoniae. The gene encoding this enzyme was cloned and expressed in Escherichia coli, as demonstrated by its ability to complement a double-mutant rnhA recC strain. Sequence analysis of the cloned DNA revealed an open reading frame of 290 codons that encodes a polypeptide of 31.9 kDa. The predicted protein exhibits a low level of homology (19% identity of amino acid residues) to RNase HII encoded by rnhB of E. coli. Identification of the S. pneumoniae RNase HII translation start site by amino-terminal sequencing of the protein and of mRNA start sites by primer extension with reverse transcriptase showed that the major transcript encoding rnhB begins at the protein start site. Comparison of the S. pneumoniae and E. coli RNase HII sequences and sequences of other, putative bacterial rnhB gene products surmised from sequencing data revealed three conserved motifs. Use of these motifs to search for homologous genes in eucaryotes demonstrated the presence of rnhB genes in a yeast and a roundworm. Partial rnhB gene sequences were detected among expressed sequences of mouse and human cells. From these data, it appears that RNase HII is universally present in living cells.
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PMID:The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes. 919 Jul 96

The circular DNA plasmid, designated pAAT56, has been isolated from strain T88-56 of the Japanese pear pathotype of Alternaria alternata. We determined the complete nucleotide sequence (5354 bp) of pAAT56 and mapped its possible open reading frames (ORFs). Three long ORFs, ORF1 (1290 bp), ORF2 (1653 bp) and ORF3 (690 bp), and four smaller ORFs, ORF4 to ORF7 (> or = 300 bp), were predicted from the sequence. The potential peptides derived from the ORFs other than ORF2 show no homology to other known proteins from a database search. However, ORF2 has significant homology to the pol gene of retrotransposons. The polypeptide derived from ORF2 includes sequences homologous to the reverse transcriptase (RT) and ribonuclease H (RNase H) domains of the retrotransposon Pol peptide. Phylogenetic comparison of RT domains from the retroelements placed pAAT56 in the Ty3/gypsy group of long terminal repeat (LTR) retrotransposons, most closely linked with those of filamentous fungi. The PCR primers were designed on the basis of nucleotide sequences encoding the highly-conserved amino-acid sequences in RT domains among pAAT56 and fungal retrotransposons. The PCR amplified the DNA fragments that possibly encode RT from strains of filamentous fungi that have been reported to carry retrotransposons. These results suggest that pAAT56 has acquired the pol gene from a Ty3/gypsy-group retrotransposon.
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PMID:Structural analysis of the plasmid pAAT56 of the filamentous fungus Alternaria alternata. 942 6

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.
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PMID:Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity. 965 93

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.
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PMID:Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII. 978 7

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.
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PMID:Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene. 1035 84

We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.
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PMID:Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria. 1036 2

The genes encoding the alpha (63-kDa) and beta (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV alphabeta and alphaPol RTs within which one subunit was selectively mutated. When alphabeta heterodimers contained the Asp 181-->Asn mutation in their beta subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their alpha subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in alphabeta heterodimers mutated in the beta subunit but was lost when the mutation was present in the alpha subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the alpha subunit of the heterodimeric RSV RT alphabeta.
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PMID:Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase alphabeta: localization of the polymerase and RNase H active sites in the alpha subunit. 1070 41

Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.
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PMID:Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions. 1158 54

The senescence phenotype of Podospora anserina wild-type strains depends on mitochondrial (mt) genome stability. Characterization of activities implicated in the maintenance of the mt DNA is therefore essential for a better understanding of these degenerative processes. To address this question we looked for a nuclease activity in this fungal mitochondria. Here we describe the purification of an endo-exonuclease active on single-stranded, double-stranded and flap DNA. The Podospora nuclease also possesses an RNase H activity. Gel filtration chromatography showed a native molecular mass of 90 kDa for the P. anserina enzyme. The highly purified fraction shows a single polypeptide chain of 49 kDa on SDS-PAGE, indicating that the Podospora enzyme is probably active as a dimer. Purification and sequencing of the endolysine digestion peptides of the Podospora mt nuclease suggested that this enzyme could belong to the 5' structure-specific endo-exonuclease family. The possible involvement of this nuclease in mt DNA recombination during the senescence process is evoked.
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PMID:Purification and characterization of an endo-exonuclease from Podospora anserina mitochondria. 1195 15

The C-terminus of the HIV-1 reverse transcriptase heterodimer was reconstructed into a single polypeptide. The construct encodes the p51 thumb (T) and connection (C) subdomains joined through a linker region to the p66 connection (C) and RNase H (R) domain. The TCCR protein was purified from insoluble fractions of Escherichia coli lysates. The TCCR construct maintains Mn(2+)-dependent RNase H activity and specifically cleaves the substrate mimicking the tRNA removal required for second-strand transfer reactions.
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PMID:Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity. 1533 74


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