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)

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.
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PMID:Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus. 57 97

Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.
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PMID:In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. 138 71

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.
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PMID:Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. 170 76

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.
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PMID:Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. 216 81

Drosophila retrotransposons have been shown to have genes for enzymes similar to the reverse transcriptase of retroviruse. They may possibly be involved in genome replication on translocation (15). Identification was made of a primer tRNA for the putative reverse transcription of a Drosophila retrotransposon, 297, and its genomic counterparts using a homology to the putative primer binding site of 297. Our nucleotide sequence analysis indicated a species of Drosophila serine tRNA to have two distinct properties similar to those characteristic of retroviral primer tRNA: its 3'terminal 18 nucleotides are exactly complementary to the putative primer binding site of 297 and its 19th base from the 3' end is modified. These results appear to support the notion stated above and suggest this serine tRNA to be the most likely candidate for a potential primer tRNA of 297.
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PMID:Identification and nucleotide sequence determination of a potential primer tRNA for reverse transcription of a Drosophila retrotransposon, 297. 242 Dec 56

TSP-1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP-1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP-1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.
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PMID:A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus-associated reverse transcriptase. 244 61

In central equatorial Africa the frequency of uninterpretable or atypical Western blots (WB)--ie. antibodies to gag proteins only--can represent up to 50% of enzyme-linked immunosorbent assay (ELISA)-positive samples. To date the significance of such serology remains unknown. Nevertheless, an unusual HIV-1 strain has been isolated from the blood of a healthy Gabonese individual who presented an atypical WB. This virus, identified as isolated HIV-1OYl, grew to low titres of reverse transcriptase activity (less than 50,000 cpm/ml) and was not obviously cytopathic. Radioimmunoprecipitation and peptide ELISA studies indicated that the lack of env-specific reactivity was probably due to the absence of antibodies to the viral glycoproteins, rather than the virus encoding a highly divergent envelope protein. Molecular cloning and sequencing of the provirus proved it to be a string of HIV-1 which was genetically closer to European and North American than to African strains. Furthermore the envelope protein sequence contained all the features of a typical HIV-1 env gene. However, the tat gene derived from the proviral clone was functionally defective. Site-directed mutagenesis of this gene showed that this was due to the substitution of an essential cysteine residue for a serine. Polymerase chain reaction amplification of the tat gene, as well as parts of the gag and env gene sequences of HIV-1OYl, showed that essentially all of the proviruses were defective. These data emphasize the need to view HIV isolates as populations of distinct genomes capable of complementing each other.
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PMID:A highly defective HIV-1 strain isolated from a healthy Gabonese individual presenting an atypical western blot. 255 49

To test the utility of the polymerase chain reaction in identifying single base mutations in a gene known to give rise to an altered enzyme and drug resistance phenotype, a human colon adenocarcinoma cell line resistant to methotrexate, with a known single base mutation (Srimatkandada et al., J. Biol. Chem. 264:3524, 1989) was examined. Poly A+ RNA was used for cDNA synthesis with reverse transcriptase, deoxynucleoside triphosphates, and 5 microM 3' primer that anneals outside the coding region of the human dihydrofolate reductase. The RNA:DNA hybrid was used as a template for the polymerase chain reaction with the addition of a 5' primer and Thermus aquaticus (Taq)I DNA polymerase. These primers flank the coding region of the human dihydrofolate reductase and define a region of 650 bases. The polymerase chain reaction was carried out for 40 cycles resulting in full length transcripts in microgram amounts clearly visible by ethidium bromide staining on agarose gels. DNA was isolated by standard methods, and double-stranded DNA was sequenced by the chain-termination method using TaqI DNA polymerase. A single point mutation was discovered at position 91 (T----C) resulting in a substitution of serine for phenylalanine at codon 31, as determined previously by classical cDNA cloning and sequencing. Sequence analysis indicated that this base transition resulted in the loss of Eco RI and Xmn I sites and the gain of a HinfI site in the cDNA, which were confirmed by restriction digests.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line using the polymerase chain reaction. 264 Jan 57

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.
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PMID:Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids". 299 55

The psbA gene coding for the herbicide binding QB protein of photosystem II has been sequenced previously (Karabin et al. 1984). A herbicide resistant mutant of Euglena, Euglena gracilis ZR, was studied by sequencing part of the psbA gene and its corresponding mRNA. Sequencing reactions were done by annealing a psbA specific, end-labeled DNA-oligomer to total chloroplast DNA or RNA and extending this primer with reverse transcriptase in the presence of the four dideoxynucleotides. An amino acid substitution from serine to alanine at position 265 was detected. All known herbicide resistant higher plants sequenced to date and the Chlamydomonas mutant DCMU-4 show a change at exactly this same position, but the substitution in higher plants is from serine to glycine.
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PMID:The psbA gene of DCMU-resistant Euglena gracilis has an amino acid substitution at serine codon 265. 312 2


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