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

---

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

The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.
...
PMID:Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity. 170 18

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
...
PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.
...
PMID:[Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties]. 171 98

The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants.
...
PMID:Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. 171 22

We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase.
...
PMID:A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. 171 68

Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100-200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.
...
PMID:Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus. 197 13

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.
...
PMID:Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene. 217 91

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.
...
PMID:Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H. 255 16

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence. 256 Aug 8

The POMC gene is predominantly expressed in the pituitary gland; it is also expressed in various extrapituitary tissues. While POMC mRNAs of similar size (approximately equal to 1000 nucleotides) are present in the anterior and neurointermediate lobes of the pituitary, other POMC-expressing tissues contain POMC mRNAs of different sizes. Longer POMC mRNAs are observed in the hypothalamus. Using S1 nuclease mapping and mRNA deadenylation by RNase H, we have shown that these large hypothalamic POMC mRNAs have longer poly(A) tails than pituitary POMC transcripts but contain the same transcripted sequences. In contrast, the testes contain POMC transcripts which are smaller than pituitary POMC mRNA. RNase and S1 nuclease mapping analyses suggest that these short transcripts do not contain sequences transcribed from pituitary exons 1 and 2. Indeed, as revealed by primer-extension experiments, these transcripts appear to initiate within exon 3 sequences of the POMC gene. The heterogeneous 5'-ends of these short testicular transcripts map into the NH2-terminal portion of the precursor in the region encoding gamma MSH; if ever translated, these transcripts would produce a form of POMC that would be truncated at the NH2-terminus and therefore would be devoid of any signal peptide sequence. Interestingly, the sequence of the short testicular transcripts corresponds to that of the mouse POMC pseudogene, suggesting that this POMC pseudogene may have derived from genomic integration of testicular transcripts via a cDNA intermediate.
...
PMID:Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly(A) tails in hypothalamus. 285 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---