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)

Plasmid pLC1437a contains DNA from Escherichia coli K12 including fol, the structural gene for dihydrofolate reductase. The fol gene was mapped on this plasmid relative to several restriction endonuclease cleavage sites. fol was also cloned from strain RSO and the nucleotide sequence for the entire fol gene and its flanking regions from this strain was determined. The amino acid sequence predicted from the nucleotide sequence differs in only a few respects from the reported amino acid sequence of dihydrofolate reductase from E. coli B. The major RNA transcripts initiated at the fol promotor in vivo are approximately 550 and 590 nucleotides long. In addition to these, several longer transcripts (up to 1400 nucleotides) are present in lesser amounts. A new procedure is described for 3' end labeling of DNA fragments having blunt ends using E. coli exonuclease III and avian myeloblastoma virus reverse transcriptase.
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PMID:Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. 615 75

The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.
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PMID:The 5' terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae. 615 79

Reverse transcriptase from avian retrovirus has a physically associated DNA endonuclease with novel substrate and cofactor requirements. A similar endonuclease activity copurifies with pp32, a protein from viral cores that has been identified with the non-alpha region of the beta subunit of reverse transcriptase. Several temperature-sensitive mutants of avian retrovirus with thermolabile DNA polymerase were tested for thermal sensitivity of their DNA endonuclease activity. Two pol mutants of Rous sarcoma virus, ts335 and ts337, had thermolabile DNA endonuclease; a temperature-resistant revertant of ts335 had a heat-stable DNA endonuclease. DNA endonuclease is therefore a product of the pol gene and an integral part of the reverse transcriptase. A second class of pol mutants, typified by ts568 and ts553, had thermolabile DNA polymerase, but heat-stable DNA endonuclease.
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PMID:Virus-coded DNA endonuclease from avian retrovirus. 616 35

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.
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PMID:Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase. 616 71

To study the structure and function of the gene for parathyroid hormone, we obtained recombinant plasmids containing bovine parathyroid hormone cDNA. The nucleotide sequence at the 5' terminus (relative to the sense strand) of the cDNA insert in a recombinant plasmid, pPTHi4, was different from that previously reported for the bovine parathyroid hormone cDNA insert of another recombinant plasmid, pPTHm1 [Kronenberg, H. M., McDevitt, B. F., Majzoub, J. A., Nathans, J., Sharp, P. A., Potts, J. T., Jr. & Rich, A. (1979) Proc. Natl. Acad. Sci. USA 76, 4981-4985]. The first 50 nucleotides of the pPTHm1 insert were an inverted complement of nucleotides 2-51 of the pPTHi4 insert. the cDNA insert of another plasmid, pPTHi8, contained a sequence identical to nucleotides 2-51 of the pPTHi4 insert but also contained an additional 42 bases at the 5' terminus. The first 41 bases of the pPTHi8 insert were an inverted repeat of an internal sequence of the pPTHi4 insert corresponding to nucleotides 184-224. Restriction endonuclease analysis of pPTHi8 indicated that the internal sequence corresponding to this region was retained. The nucleotide sequence of a restriction fragment hybridized to parathyroid hormone mRNA and extended toward the 5' terminus of the mRNA with reverse transcriptase confirmed that the sequence at the 5' terminus of the pPTHi4 insert was an accurate copy of the parathyroid hormone mRNA sequence. These data suggest that two types of sequence rearrangements may occur at the 5' terminus, as occurred in pPTHm1, and (ii) an inverted repeat of an internal sequence, as occurred in pPTHi8.
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PMID:Introduction by molecular cloning of artifactual inverted sequences at the 5' terminus of the sense strand of bovine parathyroid hormone cDNA. 617 60

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

The early DNa products of reverse transcription have been analyzed from reconstructed reactions containing avian myeloblastosis virus 35S RNA . tRNAtrp complex and highly purified reverse transcriptase. We describe conditions for the synthesis of genome-length complementary DNA and two discrete species of plus strand DNA (the same chemical polarity as the viral RNA genome) about 300 and 400 nucleotides in length. Plus DNA400 and plus DNA300 were detected by molecular hybridization with DNA probes complementary to sequences from both the 3'- and 5'-ends of the viral RNA. Both species appear to be copied from the 5'-end of minus strand DNA by their hybridization properties and their early synthesis when only the 5'-end of minus strand DNA is available as template. Restriction endonuclease mapping of plus DNA400 and plus DNA300 rules out a precursor-product relationship between the two. Rather the results suggest a unique initiation site for both species, with plus DNA400 containing internal sequences not present in plus DNA300. Plus DNA400 and plus DNA300 appear to be analogous to early plus DNA species detected in cells early after retrovirus infection. Thus, purified reverse transcriptase appears to be enzymatically sufficient for synthesis of genome-length complementary DNA and initiation and synthesis of early plus strand DNA as observed in infected cells.
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PMID:Reverse transcription of avian myeloblastosis virus 35S RNA. Early synthesis of plus strand DNA of discrete size in reconstructed reactions. 617 40

Employing enzymatic reactions containing reverse transcriptase and appropriately defined substrates, we have demonstrated that the tRNATrp primer molecule required for the initiation of DNA synthesis is cleaved from viral DNA by an enzymatic activity associated with the reverse transcriptase molecule. Since the alpha subunit of reverse transcriptase facilitates release of the tRNATrp primer from viral DNA and this activity is inhibited by a known inhibitor of reverse-transcriptase-associated RNAase H, it appears that the RNAase H activity, rather than the DNA endonuclease activity, is involved in this reaction. The cleavage site for RNAase H-mediated removal of the tRNATrp primer from viral DNA is located at or near the tRNATrp-viral DNA junction, and transcription of most, if not all, of the tRNATrp-binding site into (+) polarity DNA occurs before RNAase-H-mediated cleavage takes place. These studies indicate that an additional function can be ascribed to the reverse-transcriptase-associated RNAase H activity, which in this instance acts like an endonuclease, not requiring the unblocked termini of an RNA-DNA hybrid molecule for its activity.
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PMID:Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. 618 6

Preparations of the alphabeta and the betabeta forms of reverse transcriptase from the Prague C strain of Rous sarcoma virus grown in chicken embryo fibroblasts, the alphabeta and the betabeta forms of the enzyme from the B77 strain of Rous sarcoma virus grown in duck embryo fibroblasts, and the alphabeta form of reverse transcriptase from avian myeloblastosis virus have been analyzed. All these enzyme preparations contain a Mn(2+) -activated endonuclease activity. The betabeta form of enzyme, in addition, contains a Mg(2+) -dependent endonuclease. Such an activity is barely detectable in the alphabeta form of enzymes. The endonuclease associated with reverse transcriptase introduces single- and double-strand breaks containing 3' OH and 5' P termini into RF I DNA. The conversion of RF I DNA to RF III DNA is more readily catalyzed by the betabeta form of reverse transcriptase. In contrast to a recently published report by Hizi et al. (J. Virol 41:974-981, 1982), we have failed to detect the conversion of RF I DNA to covalently closed relaxed circles (RF IV DNA) by any of the alphabeta form of enzymes tested. RF IV DNA was not produced by the betabeta form of reverse transcriptase either. We conclude that topoisomerization is not an intrinsic activity of reverse transcriptase. Although the conversion of RF I DNA to RF II DNA was found to be rapid, the endonuclease associated with reverse transcriptase acted slowly on RF II, RF III, and RF IV DNAs. Circular and linear single-stranded DNAs were also susceptible to cleavage by the endonuclease at a rate comparable to nicking of RF I DNA. This pattern of activity suggests that the endonuclease cleaves the RF I DNA in the single-stranded regions of the DNA induced by its supercoiling. The preference of the alphabeta and the betabeta forms of the endonuclease for viral DNA was tested with Rous-associated virus type 2 and Rous sarcoma virus transformation-defective Schmidt-Ruppin B strain DNA molecularly cloned in plasmid pBR322 and M13 DNA vectors, respectively. The rate of nicking of RF I DNA containing viral DNA or partial sequences of viral DNA with one or two tandem long terminal repeats was the same as when these sequences were not present in the host vectors. A similar lack of preference was observed with single-stranded M13 DNAs.
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PMID:Mechanism of action of the endonuclease associated with the alpha beta and beta beta forms of avian RNA tumor virus reverse transcriptase. 618 36

M13 recombinant DNA clones containing a 350-base sequence derived from the EcoRI fragment of two tandemly linked Rous-associated virus 2 (RAV-2) long terminal repeat (LTR) sequences have been used to map reverse transcriptase-associated endonuclease (RT-endonuclease) cleavage sites by primer extension studies. Under appropriate conditions, the alpha beta form of RT-endonuclease (composed of both the alpha and beta subunits) purified from Avian sarcoma virus (Pr-C and B-77 strains) introduces a specific break in the inverted complementary repeat sequence found at the junction of the LTRs. The cleavage sites occur in the same nucleotide sequence in (-) and (+) DNA strands; together they have the potential of generating a 6-base-pair staggered overlap that spans the junction. This supports the notion that the enzyme is involved in viral DNA integration. Other RT-endonuclease sites were analyzed. A second site, which occurs in the lac region of the M13 vector DNA upstream from the unique EcoRI cloning site, bears no apparent sequence homology to the site at the junction of the LTRs. However, it also lies within an inverted complementary repeat and, as is the case for the site in the LTR, the break occurs to the 5' side of the axis of symmetry. Cleavage at this second site is suppressed when the vector contains the RAV-2 LTR insert. Thus, the viral LTR appears to exert a cis effect that can influence a region over 300 base pairs away.
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PMID:Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase. 619 75


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