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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.
...
PMID:[DNA synthesis on the heterogeneous nuclear RNA template catalysed by DNA polymerase of avian myeloblastosis virus]. 5 56

[3H]Uridine-labeled Rauscher leukemia virus was used to infect mouse embryo fibroblasts. After the infected cells were separated into nuclear and cytoplasmic fractions nucleic acid was extracted by sodium dodecyl sulfate-phenol-chloroform treatment and analyzed by Cs2SO4 and sucrose density gradient centrifugation. Between 45 and 70 min after infection a transient and synchronized shift of the acid-insoluble radioactive peak toward the RNA-DNA hybrid region occurred in both the nuclear and cytoplasmic fractions. The density of the cytoplasmic hybrid shifted to 1.56 g/ml (RNA equals about 50%), while the sedimentation rate decreased from 36 S to 14 S; however, the density of the nuclear hybrid shifted to 1.58-1.48 g/ml (RNA equals 57-17%, respectively), while its sedimentation rate remained about 65 S. The hybrids in both the nuclear and the cytoplasmic fractions still showed hybrid density after heat denaturation. The processes of the early stages of RNA tumor virus infection are discussed with regard to the functions of viral RNA-dependent DNA polymerase (reverse transcriptase) and a possible integration of viral genetic information into the host chromosome.
...
PMID:Fate of viral RNA of murine leukemia virus after infection. 16 22

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.
...
PMID:Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction. 128 41

Intact RNA from various rat organs was isolated by an efficient and rapid method. This method of RNA isolation is a modification of an earlier method that uses guanidinium isothiocynate followed by extraction in the presence of sarcosyl, acetate and phenol. The RNA obtained by the method reported here was comparable with the RNA prepared by the CsCl2 ultracentrifugation method and the commercially available kit based on published methods. The quality of RNA was found suitable for Northern blotting analysis, RNase protection assays and reverse transcriptase-polymerase chain reaction (RT-PCR). Since reverse transcriptase is active in the buffer used for Taq DNA polymerase, only one reaction needs to be set up. We also found that the use of aurintricarboxylic acid in the RNA preparation prevents the degradation of RNA during storage. Expression of low density lipoprotein (LDL) receptor, apolipoprotein (apo) AI, AII and AIV mRNAs were quantified in various rat organs. Our results indicated that rat LDL receptor mRNA is expressed in several organs whereas apoAI and AIV mRNAs were expressed mainly in the liver and intestine. However, apo AII mRNA is expressed mainly in the liver. Unlike mice and some species of monkeys, in the rat apoAI mRNA is expressed at 5-6 times higher levels in the intestine compared to liver. Apo AIV mRNA abundance was also found to be several fold higher in intestine compared to hepatic tissues. We present here, for the first time, data on the absolute amounts of LDL receptor, apoAI, AII and AIV mRNA in various rat organs which were quantified by a novel RNase protection/solution hybridization assay.
...
PMID:Expression of low density lipoprotein receptor, apolipoprotein AI, AII and AIV in various rat organs utilizing an efficient and rapid method for RNA isolation. 137 76

A rapid and simple protocol for the isolation of RNA from transfected tissue culture cells is described. The protocol employs a guanidinium thiocyanate/phenol mixture to lyse cells directly from tissue culture plates and extract the total RNA. A total of six simple steps, which can be accomplished within 2.5 hours, are required. The protocol reproducibly yields 20-40 micrograms RNA from 0.5 x 10(6) - 1 x 10(6) cells per sample. The quality of the RNA obtained is sufficient for reverse transcriptase assays such as oligonucleotide-directed primer extension and random-primed cDNA synthesis.
...
PMID:Rapid, small-scale RNA isolation from tissue culture cells. 171 28

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.
...
PMID:Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver. 616 9

The expression of the cytokine genes was studied under physiological conditions in normal adult mice using RT-PCR method capable of detecting low levels of mRNA. Total RNA was prepared from brain, spinal cord, lung, spleen, liver and kidney of 6 to 8 week-old specific pathogen-free BALB/c mice by acid guanidinium thiocyanate-phenol-chloroform (AGPC) method. cDNA was synthesized by M-MLV reverse transcriptase, and amplified using the specific oligonucleotide primers for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, IFN-beta and IFN-gamma by PCR method. Although IL-1 mRNA was detected in all the organs, IL-3 mRNA was not detected in any organs tested. IL-2 or IL-4 mRNA and IL-5 mRNA were produced only in spleen and lung, respectively. However, IL-6, TNF-alpha and IFN mRNA were detected in some different organs. These results suggest that many kinds of cytokine mRNA were produced in vivo under physiological conditions in normal mice.
...
PMID:[Expression of cytokine messenger RNA in mice in physiological conditions]. 751 36

The induction of the cytokine mRNA after infection with Herpes simplex virus (HSV) was studied using RT-PCR method capable of detecting low levels of mRNA. Total RNA was prepared from spleen lymphocytes 3 h after infection with HSV-1 (+GC virulent variant and -GCr attenuated variant of Miyama strain) by acid guanidinium thiocyanate-phenol-chloroform (AGPC) method. cDNA was synthesized by M-MLV reverse transcriptase, and amplified using the specific oligonucleotide primers for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, IFN-beta and IFN-gamma by PCR method. After HSV-1 infection, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-4, IL-6 and TNF-alpha mRNA were significantly induced, but IL-2, IL-3 and IL-5 mRNA were not induced. Although IFN-alpha, IFN-gamma and IL-6 mRNA were more strongly induced by infection with +GC virulent variant than -GCr attenuated variant, there was no significant difference in the expression of other cytokine mRNA between two variants. These results demonstrate that cytokine mRNA in addition to IFN was induced by HSV infection, and suggest that cytokines as well as IFNs may play a role in the defense mechanism against HSV infection.
...
PMID:[Induction of messenger RNA of cytokines by Herpes simplex virus infection in mice]. 751 38

The expression of the cytokine and IFN-related genes was studied in mouse embryo using RT-PCR method capable of detecting low levels of mRNA. Total RNA was prepared from the days 7 embryos by acid guanidinium thiocyanate-phenol-chloroform (AGPC) method. cDNA was synthesized by M-MLV reverse transcriptase, and amplified using the specific oligonucleotide primers for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, IFN-alpha/beta receptor (IFN-alpha/beta R), IFN-gamma receptor (IFN-gamma R), interferon reguratory factor (IRF)-1, IRF-2 and 2'-5' oligoadenylate synthetase (2-5AS) by PCR method. Although the expressions of IL-1 beta, IL-4, IL-5, IL-6, TNF-alpha and IFN-gamma mRNA were detected in all the embryos tested, the expressions of IL-2, IL-3, IFN-alpha and IFN-beta mRNA were not detected at all. On the other hand, the expressions of IFN-related genes such as IFN-alpha/beta R, IFN-gamma R, IRF-1, IRF-2 and 2-5AS mRNA, were also detected. These results suggest that these cytokine may play an important role in early embryonic development.
...
PMID:[Expression of cytokines and interferon-related genes in the mouse embryo]. 754 Jan 2

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.
...
PMID:Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans. 768 67


1 2 3 4 5 6 7 Next >>

---