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)

The nucleotide sequence of the entire 5' untranslated region of human gamma-globin mRNA has been determined. This was accomplished by analyzing complementary DNA (cRNA) synthesized from the mRNA with reverse transcriptase. The CDNA was labeled at its 3' end with 32"p using terminal deoxynucleotidyl transferase, digested with the restriction endonuclease Hae III and the end-labeled fragment isolated ans sequenced by the method of Maxam and Gilbert. Including the initiation codon AUG, the 5' untranslated region of human gamma-globin mRNA contains 57 nucleotides, compared to 41 in alpha- and 54 in beta-globin mRNA. There is very little homology between alpha and gamma sequences in the 5' region. There is considerable homology between beta- and gamma-globin mRNAs in the regions proximal and distal to the initiation codon, but the entire sequence shows less homology than the human and rabbit beta-globin mRNAs. The hexanucleotide sequence CUUCUG is found near the 5' ends of all three human globin mRNAs, suggesting a possible role of this sequence or ribosomal binding. Both guanosine and cytidine were found at the 19th nucleotide position from the 5' end of the gamma mRNA. We believe this heterogeneity arises from the difference in nucleotide sequence between the A gamma and G gamma loci.
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PMID:The nucleotide sequence of the 5' untranslated region of human gamma-globin mRNA. 31 62

Double stranded human globin cDNA was synthesized by use of viral reverse transcriptase from globin mRNA of cord blood of premature infants requiring exchange transfusions. The cDNA was introduced into plasmids and the recombinant DNA plasmids used to transform E. coli X1776. A number of transformants were obtained. Plasmid DNA from selected colonies was isolated and characterized for the type of globin cDNA it contained by three types of procedures: 1) hybridization to previously characterized 3H-labeled alpha,beta and gamma cDNA; 2) analysis of the size and nature of fragments produced by digestion of the plasma DNA by different restriction endonucleases; and 3) by rapid DNA sequence analysis of selected DNA fragments produced by restriction endonuclease digestion. Analysis by these techniques of plasmid DNA from different colonies has definitively identified the presence of human alpha, beta or gamma cDNA sequences in different plasmids.
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PMID:Insertion of synthetic copies of human globin genes into bacterial plasmids. 34 45

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.
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PMID:Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA. 34 4

We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A).
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PMID:Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA. 90 79

It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA (msDNA) whose 5' end is covalently linked to RNA (msdRNA) by a 2'-5' phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotide-long single-stranded DNA with monophosphate on its 5' terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5' side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-long DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5' monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autocatalytically processed.
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PMID:Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate. 128 91

Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.
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PMID:Recent studies on retrovirus-like particles in Chinese hamster ovary cells. 128 76

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).
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PMID:Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase. 137 51

The gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.
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PMID:Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. 168 46

We have analysed the mechanism of ribonuclease H (RNaseH) induced cleavage of a defined RNA-DNA hybrid by human immuno-deficiency virus (HIV-1) reverse transcriptase (RT). An in vitro transcribed RNA labelled at the 3' end was hybridized to a pentadecameric DNA oligonucleotide complementary to an internal region of the RNA. Upon incubation of this RNA-DNA hybrid with recombinant p66 or p66/p51 HIV-1 reverse transcriptase, RT-RNaseH mediated cleavage is observed at most nucleotides within the short hybridized stretch, resulting in a spectrum of RNA fragments extending from the 3' label to this region and differing in length by one nucleotide. The same RNA, this time labelled at the 5' end, yields only one or two major cleavage products corresponding to RNA species extending from the 5' label to the middle of the hybridized region. Such a result can be explained by the action of both endonuclease and 3'----5' exonuclease activities inherent to the C-terminal domain of p66 RT. To investigate how RNaseH cleavage is coupled to reverse transcription, a combination of deoxynucleoside triphosphates was used which allowed controlled extension of the primer DNA. Concomitantly with the elongation of the oligonucleotide primer, RNaseH cleavage proceeds towards the 5' end of the RNA with identical increments, suggesting a simultaneous action of both activities.
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PMID:HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity. 169 Oct 93

The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction. In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes. The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added. We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase. The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching. In the absence of RNase H, switching still occurred but the efficiency was lowered. Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H. Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching. We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product. This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.
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PMID:Template switching by reverse transcriptase during DNA synthesis. 169 39


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