Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.
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PMID:RNA abundance measured by a lysate RNase protection assay. 138 Nov 96

Biologically active RNA has been isolated from several tissues implicated in the pathogenesis of the genetic disease cystic fibrosis. To ensure that mRNA from stomach and pancreas has not been degraded, it must be isolated within 2 h postmortem, while intact RNA can be isolated from lung material up to 20 h postmortem. It is imperative that tissue removed during autopsy be dispersed in a strong denaturant buffer (guanidine isothiocyanate) to inactivate nucleases. While stomach and pancreas yields of RNA are low, relative to the amount of gland material, yields of RNA from lung samples are sufficient for the further isolation of mRNA by oligo(dT)-cellulose chromatography. The biological integrity of subsequently isolated mRNA has been assessed by AMV-reverse transcriptase cDNA synthesis as well as by in vitro translation. Protein products generated in this manner show distinct differences from mRNA translation patterns of age- and sex-matched controls when compared with mRNA from CF samples. R0t analysis of cDNA libraries thus generated indicates that the complexity of such libraries is representative of the starting RNA species.
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PMID:Isolation of biologically active RNA from human autopsy for the study of cystic fibrosis. 376 47

Two polypeptides were isolated from the Mason-Pfizer monkey virus (MP-MV). The polypeptides, designated MP-MV p26 and MP-MV p15, were 26,000 and 15,000 molecular weight, respectively, based on gel filtration chromatography in the presence of 6 M guanidine hydrochloride. Radioimmunoassays developed for MP-MV p26 and p15 were, respectively, 10 and 100 times more sensitive than immunodiffusion and 1 and 10 times more sensitive than micro-complement fixation for the detection of MP-MV. Antigens of other reverse transcriptase-containing RNA viruses did not cross-react in either MP-MV radioimmunoassay. Further, antisera against these other viruses did not react with the radioiodine-labeled MP-MV polypeptides. The MP-MV radioimmunoassays should be useful in studying the natural occurrence of this virus and its relationship to primate tumors.
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PMID:Immunological properties of two polypeptides of Mason-Pfizer monkey virus. 413 67

Proteins of leukemia and sarcoma viruses from the chicken, mouse, hamster, and cat were analyzed by gel filtration in guanidine hydrochloride. The mammalian viruses were found to contain six major proteins, whereas the avian viruses contained seven proteins. Proteins of viruses from different mammalian species had the same molecular weights which also closely resembled the molecular weights of the six equivalent avian viral proteins. These results defined a basic similarity in protein composition for the C-type viruses of avian and mammalian origin. The two glyco-proteins of murine leukemia virus were identified immunologically as constituents of the viral membrane. Antisera prepared against other proteins distinguished individual internal viral antigens in immunodiffusion tests and also reacted in immunofluorescence with cytoplasmic components of infected cells. Antisera which reacted with the major internal viral proteins did not contain antibodies inhibitory to the viral reverse transcriptase.
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PMID:Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. II. Mammalian leukemia-sarcoma viruses. 433 24

Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a major species of 5.3 kb and a minor species of 7 kb. These RNAs are present in total RNA from sterna and differentiated limb bud cultures but are not detected in RNA from stage 24 limb bud which has not yet differentiated to cartilage or in RNA from calvaria. The time of appearance of these RNAs during the differentiation of limb mesenchyme in culture parallels the appearance of translatable type II procollagen mRNA.
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PMID:Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage. 628 Jan 34

mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.
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PMID:Characterization of a sheep elastin cDNA clone containing translated sequences. 632 Aug 24

Plasmodium vivax-infected blood samples were collected from patients in the field during malaria transmission season. Total RNA of the parasites was extracted by guanidine HCl/cesium chloride centrifugation. mRNA was purified through oligo-dT cellulose. Double stranded cDNA were synthesized with AMV reverse transcriptase by Huynh's method. lambda gt11 phage was used as the vector. A cDNA library of the erythrocytic stage P. vivax was constructed after recombination of DNA and package in vitro.
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PMID:[Construction of a cDNA library of the erythrocytic stage of Plasmodium vivax]. 795 56

Vesicular stomatitis virus of the New Jersey serotype (VSV-NJ) causes vesicular disease in cattle, pigs, and horses throughout the Americas. Vesicular disease is clinically indistinguishable from foot-and-mouth disease (FMD). Therefore, outbreaks of vesicular disease in FMD-free areas must be rapidly diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm the absence of FMD. Diagnosis is currently performed in high-containment (biosafety level 3) laboratories by using complement fixation and virus isolation in tissue culture. We describe here an alternative method for the detection of VSV-NJ RNA in clinical samples. This method includes a rapid acid guanidine-phenol RNA extraction procedure coupled with a one-tube polymerase chain reaction (PCR) using reverse transcriptase. By using this test, we were able to detect the largest number of positive samples (53 of 58), followed by complement (48 of 58) and isolation in tissue culture (43 of 58). The primers chosen for this assay amplify a 642-nucleotide region of the phosphoprotein gene of VSV-NJ but not of VSV-IN. Sequencing of the PCR product enables genetic typing of virus isolates and epidemiological studies. Since no infectious materials are necessary to perform this test and any infectious virus in clinical samples is destroyed by acid guanidine-phenol treatment, diagnosis can be safely performed in regular diagnostic laboratories.
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PMID:Rapid detection of vesicular stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction. 839 84

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.
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PMID:Regulation of phagosomal acidification. Differential targeting of Na+/H+ exchangers, Na+/K+-ATPases, and vacuolar-type H+-atpases. 936 53

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.
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PMID:Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart. 968 29


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