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 have isolated and sequenced a genomic clone from Saccharomyces cerevisiae that shows structural features of a novel retrotransposon, designated Ty4. The element is 6.2 kilobases in length, and its genetic organization of the deduced functional domains is similar to Ty1 and Ty2 and thus different from Ty3. In contrast to hitherto known Ty elements from yeast, Ty4 is flanked by long terminal tau-element repeats instead of delta or sigma sequences. Ty4 contains two overlapping open reading frames. The first open reading frame, TYA4, is 1230 base pairs long and encodes a protein with a motif found in the nucleic acid-binding gag-protein of retroviruses. The second 4395-base pair open reading frame, TYB4, encodes a polyprotein that has domains with significant homology to retroviral protease, integrase, reverse transcriptase, and RNase H, structurally arranged in that order. The deduced amino acid sequence shows the greatest similarity with Ty2 and Ty1. The overall identity of the deduced functional protein domains is 28% with Ty2, 25% with Ty1, 19% with copia from Drosophila, and 18% with Ty3. Examination of genomic DNA from several laboratory strains indicates that Ty4 is present in two to four copies. Ty4 mRNA is of low abundance as compared to other Ty retrotransposons. At the 3' end of Ty4, two "solo" delta-elements, a full length and an overlapping, truncated one, are associated.
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PMID:Ty4, a new retrotransposon from Saccharomyces cerevisiae, flanked by tau-elements. 132 82

The element; Ty4 is a retrotransposon present in low copy number in the genome of Saccharomyces cerevisiae [Stucka et al., Nucleic Acids Res. 17 (1989) 4993-5001]. We have determined the complete nucleotide sequence of one such element from a particular strain and compared it to the other two elements occurring in this strain. The genomic organization of Ty4 is homologous to that found in other retrotransposons of the Ty1-copia group. The internal part of the element contains two large open reading frames (TY4A and TY4B) overlapping by 226 bp in a + 1 mode. TY4A reveals characteristics of the gag portion of retrotransposons and retroviruses, while TY4B consists of a protease, an integrase, a reverse transcriptase, and an RNase H domain (in that order). Our analyses suggest that only one of these copies might be transpositionally active. Sequence comparisons at the amino acid level show that the domains in Ty4 diverge considerably from those of other retrotransposons. The greatest similarity is seen between the reverse transcriptases (50%), the proteases (40%), and the integrases (30%) of Ty4, Ty1/2 and copia, respectively, whereas the degree of similarity for all other entities of these elements is much lower. Considering evolutionary aspects of the retrotransposons, we have to conclude that Ty4 has diverged at an early stage from the progenitors of other known retroelements and represents a novel and independent subgroup of the Ty1-copia class of retrotransposons.
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PMID:Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons. 133 37

We have developed a highly efficient new method for the amplification of alpha- and beta-chain human T-cell receptor (TCR) cDNAs. This method is designated non-palindromic adaptor polymerase chain reaction (NPA-PCR). cDNA was synthesized from total RNA isolated from mononuclear leucocytes, using either an oligo (dT)15-NotI or a C alpha-NotI or a C beta-NotI primer and RNase H-negative reverse transcriptase. The double-stranded cDNA was ligated with the non-palindromic adaptors EcoRI-XmnI [d(ATTCGAACCCCTTCG)] and XmnI G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoRI-XmnI site in both 5' and 3' ends. These two non-palindromic adaptors, EcoRI-XmnI and XmnI G strand, are complementary to each other and both are required for ligation. The EcoRI-XmnI adaptor was removed from the 3' end by treatment with NotI restriction nuclease, whereas it was retained at the 5' end. The non-palindromic adaptor EcoRI-XmnI was used as the 5' amplification primer. C alpha or C beta constant region primers were used as 3' amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5 alpha competent cells. Over 1000 white colonies per 0.1-0.25 micrograms of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the alpha- or the beta-chain TCR cDNAs. Between 40 and 62% of the colonies (range from five donors) were positive after screening with either a C alpha or a C beta probe, located 5' to the C alpha and C beta amplification primers. A total of 50 amplified alpha- or beta-chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of alpha beta TCR. Two new J alpha gene segments were identified. Approximately 30% of the alpha-chain positive clones have 5' untranslated region, and most of the remaining alpha- or beta-chain TCR clones started from the initiation codon or near the 5' end. NPA-PCR has several advantages over existing PCR methods for the amplification of cDNAs with unknown or variable 5' end, such as the T-cell antigen receptors and the immunoglobulins. Among these advantages is that only one 5' end extension primer is required. Because of the large number of TCR V alpha and V beta families, a large number of different 5' end primers are required for amplification of alpha beta TCR cDNAs by conventional PCR.
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PMID:Development of the non-palindromic adaptor polymerase chain reaction (NPA-PCR) for the amplification of alpha- and beta-chain T-cell receptor cDNAs. 134 68

Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.
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PMID:Construction of a cDNA library from microdissected guinea pig organ of Corti. 135 71

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
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PMID:Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 137 87

We constructed a series of BspMI cassettes that simplify the introduction of specific point mutations in the polymerase domain of human immunodeficiency virus type 1 reverse transcriptase. A series of point mutants were constructed by using these cassette vectors. The RNA-dependent DNA polymerase and RNase H activities of 20 point mutations in the conserved portion of the polymerase domain were assayed. All the mutations analyzed are conservative substitutions of evolutionarily conserved amino acids. The mutations were divided into four classes. The first class has little effect on either polymerase or RNase H activity. The second class affects RNase H but not polymerase activity, while the third class has a normal RNase H activity with diminished polymerase activity. The fourth class affects both activities.
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PMID:Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. 137 May 46

Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism.
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PMID:RNase H activity associated with reverse transcriptase from feline immunodeficiency virus. 137 May 49

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.
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PMID:Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. 137 May 51

We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template approximately 100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (ii) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) alpha-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an alpha-oligomer bound to the sequence immediately adjacent to the primer-binding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.
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PMID:Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides. 137 May 86

In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells. A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays. Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time. Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio. Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity. Exogenous RNase H added to the transcription reaction ablated the signal. Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes. The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes.
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PMID:Detection of enteroviruses in cell cultures by using in situ transcription. 137 Aug 49


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