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

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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)

Rabbit globin mRNA was copied by AMV reverse transcriptase in the presence of various concentrations of deoxyribonucleotides (dNTPs). The cDNAs were analyzed by electrophoresis under denaturing conditions in formamide-polyacrylamide gels. Discrete size products were detected, ranging from 65 to 650 nucleotides-that is, up to the full length of the mRNA template. Increasing the concentrations of all four dNTPs stimulated formation of full-length transcripts and made the incomplete copies less abundant. Hybridization and nuclease digestion experiments indicated that the full-size product is indeed a complete transcript of globin mRNA. Similar results were obtained with chorion mRNAs. The possible usefulness of the discrete partial transcripts is discussed.
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PMID:Full length and discrete partial reverse transcripts of globin and chorion mRNAs. 4 72

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.
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PMID:The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA. 5 72

Controlled disruption of 60S AMV RNA with formamide was used to prepare 50-55S and 30-40S RNAS. When the activities of these RNAs as templates for AMV reverse transcriptase were compared it was found that 50-55S RNA was 1-5 times and 30-40S RNA 2 to 3 times more active than 60S RNA. The 30-40S RNA produced by heating, instead of formamide disruption, was inactive as a template but activity was restored by addition of oligo(dT). 40% of the 4S RNA initially associated with the 60S RNA remained associated with all the RNA species obtained by formamide treatment but was lost on heating. It is concluded that this RNA acts as resident primer whereas the other 60% of the 4S RNA is less firmly bound and appears to have little or no primer activity. Removal of the less firmly bound 4S RNA increases the template activity of the viral RNA.
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PMID:Stepwise dissociation of high molecular weight avian myeloblastosis virus RNA: 30-40S RNA subunits--the best natural template-primer for viral reverse transcriptase. 5 91

The polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E. coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported. We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase. The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA. Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels. These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end. Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8.
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PMID:Polyadenylation and reverse transcription of influenza viral RNA. 8 38

Total poly(A)-mRNA from polyribosomes of MOPC 21 mouse myeloma were investigated. Poly(A)-mRNA was released by two successive chromatography on oligo (dT)-cellulose. A 14S fraction of total poly(A)-mRNA was obtained and partially purified by sucrose gradient centrifigation followed by acrylamide gel electrophoresis. As estimated from the electrophoretic analysis, the 14S mRNA has three components, one of which appears to be 18S rRNA and two others--mRNAs with molecular weight of 5.2.10(5) and 3.8.10(5), respectively. Total poly(A)-mRNA and partially purified 14S mRNA were active when employed as a template in a reverse transcription and cell-free system from wheat germ. DNA complementary to the 14S mRNA was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA was heterogeneous in size with the average size of about 800 nucleotides when analyzed by gel electrophoresis in 98% formamide. The maximal length was about 1100 nucleotides that consistent with full template length. About half of the translation product directed by the 14S mRNA migrated as mature L-chain Ig (upon polyacrylamide gel electrophoresis in sodium dodecylsulfate). The presented data suggested that 14S mRNA species contain mRNA L-chain Ig.
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PMID:[mRNA of mouse plasmacytoma. Reverse transcription and translation in cell-free systems]. 8 67

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.
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PMID:Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C. 40 23

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.
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PMID:Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C. 40 24

Starting from poly(A)-containing RNA prepared from the fat body of larvae of the blowfly Calliphora vicina, we have purified an mRNA coding for the protein calliphorin, which is a major blowfly protein accounting for approximately 9% of the total poly(A)-containing mRNA activity in the fat body of 5-days-old larvae, as demonstrated by translation in vitro. The mRNA for this protein was purified by sucrose gradient centrifugation under denaturing conditions and identified by cell-free translation. The peak fraction of the gradient shows three bands of about 20 S after formamide/acrylamide gel electrophoresis. Using reverse transcriptase and Calliphora mRNA isolated directly from the acrylamide gel electrophoresis or from sucrose gradients, we synthesized a cDNA probe. This cDNA hybridizes with the template, showing a major kinetic component with a molecular weight of 2.8 x 10(6), fitting well with the three bands observed in the electrophoresis. Hybridization of the cDNA with total sonicated Calliphora DNA shows annealing only at very high c0t values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.
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PMID:Purification and reverse transcription of the messenger RNA coding for the insect protein, calliphorin, isolated from larvae of the blowfly, Calliphora vicina R.-D. 42 1

Polyadenylated RNA isolated from a clone of Trypanosoma brucei was shown to direct the synthesis of a variety of polypeptides in a cell-free system. A predominant 58,000 dalton polypeptide was immunoprecipitated with antisera to the T. brucei variant specific surface antigen (VSSA). The mRNA that directed the synthesis of the VSSA was 2.0 kilobases (kb) long as measured by polyacrylamide gel electrophoresis in 98% formamide. Complementary DNA was prepared with avian myeloblastosis virus reverse transcriptase and the nucleotide sequence complexities of the total polysomal poly(A)+RNA and a gel purified VSSA mRNA were measured. 20% of the total cellular poly(A)+RNA contained abundant sequences with an apparent complexity of 9.6 kb; 42% of the purified VSSA mRNA contained abundant sequences with a complexity of 7.2 kb. Complementary DNA synthesized from gel purified VSSA mRNA was hybridized to total cellular poly(A)+RNA isolated from an unrelated T. brucei clone expressing a different variant antigen. A portion of the low complexity RNA sequence component was absent in the heterologous mRNA population but the same plateau of hybridization was achieved (93%). The abundance of some of the low complexity mRNAs appears to be T. brucei clone specific.
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PMID:A characterization of mRNA activites and their sequence complexities in Trypanosoma brucei: partial purification and properties of the VSSA mRNA. 70 49

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47 degrees C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.
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PMID:Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA. 247 18


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