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 model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.
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PMID:Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H. 8 56

The expression of the mRNA for mouse testicular lactate dehydrogenase (LDH-X) was examined by RNA:cDNA hybridization in situ in the testis and by Northern analyses of meiotic and postmeiotic spermatogenic cell populations. Silver grains accumulated in cells inside the second layer from the periphery of the seminiferous tubule, confirming previous findings that LDH-X mRNA first appears in the spermatocyte and continues to accumulate until the late spermatid stage. Northern analyses showed that meiotic and postmeiotic cells contained 1.2 and 1.3 kb classes of hybridizing mRNA, respectively. RNase H digestion of oligo (dT)-hybridized RNA and poly(U)-Sepharose column chromatography with differential elution by formamide revealed that the difference in size of the two classes of mRNAs was due to the poly(A) tail length of the LDH-X mRNA. When the distribution of the LDH-X mRNA was examined across polysome gradients, both mRNAs were partially associated with polysomes. These results suggest that the changes in the polyadenylation of LDH-X mRNA were associated with the meiotic division during spermatogenesis in the mouse. They raise the possibility that the stable accumulation of the LDH-X mRNAs in the postmeiotic cells is enhanced by poly(A) tails of increased length.
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PMID:Changes in polyadenylation of lactate dehydrogenase-X mRNA during spermatogenesis in mice. 290 41

The poly(A) sequence of 30 to 40S Rous sarcoma virus RNA, prepared by digestion of the RNA with RNase T(1), showed a rather homogenous electrophoretic distribution in formamide-polyacrylamide gels. Its size was estimated to be about 200 AMP residues. The poly(A) appears to be located at or near the 3' end of the 30 to 40S RNA because: (i) it contained one adenosine per 180 AMP residues, and because (ii) incubation of 30 to 40S RNA with bacterial RNase H in the presence of poly(dT) removed its poly(A) without significantly affecting its hydrodynamic or electrophoretic properties in denaturing solvents. The viral 60 to 70S RNA complex was found to consist of 30 to 40S subunits both with (65%) and without (approximately 30%) poly(A). The heteropolymeric sequences of these two species of 30 to 40S subunits have the same RNase T(1)-resistant oligonucleotide composition. Some, perhaps all, RNase T(1)-resistant oligonucleotides of 30 to 40S Rous sarcoma virus RNA appear to have a unique location relative to the poly(A) sequence, because the complexity of poly(A)-tagged fragments of 30 to 40S RNA decreased with decreasing size of the fragment. Two RNase T(1)-resistant oligonucleotides which distinguish sarcoma virus Prague B RNA from that of a transformation-defective deletion mutant of the same virus appear to be associated with an 11S poly(A)-tagged fragment of Prague B RNA. Thus RNA sequences concerned with cell transformation seem to be located within 5 to 10% of the 3' terminus of Prague B RNA.
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PMID:Properties and location of poly(A) in Rous sarcoma virus RNA. 437 9

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.
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PMID:Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis. 633 Jun 82

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.
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PMID:Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms. 1518 70

Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV RT) enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RT IC(50) values) was directly correlated to the outcome of "mock" reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates.
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PMID:Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors. 2010 Apr 52