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)

We have used the program FOLD, which employs the Zuker folding algorithm, to identify regions of stable secondary structure in three chicken proto-oncogene mRNAs: c-src, c-myc, and c-fos. We have found that use of reverse transcriptase to synthesize a cDNA template for amplification by the polymerase chain reaction is successful only if the region chosen for amplification does not contain stem structures with calculated free energies less than -14 kcal/mol.
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PMID:Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. 169 49

Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src protein (pp60c-src) indicated that in pp62S1-src the 8 carboxy-terminal amino acid residues of the total of 533 in pp60c-src are replaced by 43 residues translated from the env sequence at the wrong frame. In pp62S2-src, on the other hand, the 14 carboxy-terminal amino acids of pp60c-src are replaced by the 38 carboxy-terminal residues of reverse transcriptase. The mechanism of c-src transduction and the structural changes necessary for pp60c-src activation are discussed.
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PMID:Activation of the cellular src gene by transducing retrovirus. 309 13

The precise src deletions in six transformation-defective (td) deletion mutants derived from the Schmidt-Ruppin strain of Rous sarcoma virus were determined by sequence analysis. Examination of the parental viral sequences neighboring the junctions of deletions in these td mutants revealed that these regions contained either directly repeated or inverted complementary sequences ranging from 9 to 28 nucleotides. Five td mutants were found to contain deletions flanked by directly repeated sequences, of which the 3' direct repeat was retained whereas the 5' direct repeat was deleted in the resulting td viral RNA. In the deletions of two td mutants where inverted complementary sequences were present at junctions of the deletions, both copies of the inverted complementary sequence were deleted in the td viral RNA. It is proposed from these observations that deletions of these mutants have been generated during the synthesis of minus-strand viral DNA by reverse transcriptase by jumping over a sequence flanked by direct repeats or by skipping a stem-and-loop structure formed via inverted complementary sequences on the viral RNA template. Data provide further information on the sequences in the td viral genome that are required for the generation of recovered sarcoma viruses (rASVs) by recombination with c-src. Sequence data of td viruses revealed that retaining as few as 82 nucleotides of the 3' src coding sequence is sufficient, whereas retaining as much as one-third of the 5' src but none of the 3' src coding sequences is not sufficient, for the generation of rASVs. Those that generate replication-competent rASVs retain, in addition to the 3' src region, a portion of the 5' src and/or its immediate upstream sequence that is homologous to exon 1 of the c-src DNA. These two sequence domains apparently provided 5' and 3' homologous regions for recombination between td viral genome and c-src DNA resulting in nondefective rASVs. Td109, which was shown previously to generate only replication-defective rASVs, retains 296 nucleotides of the 3' src sequence but lacks all the 5' src and 316 nucleotides of its immediate upstream region. It is concluded that the 5' src coding sequence and its immediate upstream region are not essential for the generation of rASVs. However, retaining a portion of those sequences is required for the generation of replication-competent rASVs.
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PMID:Mechanisms for the generation of src-deletion mutants and recovered sarcoma viruses: identification of viral sequences involved in src deletions and in recombination with c-src sequences. 609 66

The RNA-dependent DNA polymerase (the reverse transcriptase) was solubilized from three related strains of avian sarcoma virus (ASV B77, ASV tsLA334, and ASV QV2) as well as avian myeloblastosis virus (AMV) and a chicken endogenous virus (RAV-O), by a combination of non-ionic detergent treatment and CsCl step-gradient centrifugation, and was subsequently separated into individual enzyme forms by poly(C)-agarose column chromatography. The newly developed two-step method allowed us to purify the three molecular forms (alpha-, alpha beta- and beta-form) of highly active enzyme rapidly and quantitatively from all the five virus strains examined. The molar ratio of the three enzyme forms differed among the virus strains: For the three sarcoma viruses, the major species was the alpha beta-form enzyme, the putative holoenzyme, and the alpha- and beta-form enzymes were less than a few percent and 15-25%, respectively, while the alpha-form enzyme content was higher for the two leukosis viruses than for the three sarcoma viruses. Both the total DNA polymerase activity and the content of the two enzyme subunits in purified virions of the three sarcoma virus was in the following order: ASV tsLA334 greater than ASV B77 greater than ASV QV2, which paralleled the virus yield at a permissive temperature in roller bottle cultures of chick embryo fibroblasts. No alteration was found in the thermolability of DNA polymerases between tsLA334, which carries ts mutations affecting both virus growth and cell-transformation, and other viruses.
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PMID:Reverse transcriptase associated with avian sarcoma-leukosis viruses. I. Comparison of intra-virion content of multiple enzyme forms. 617 23

The three enzyme forms (alpha, alpha beta-, and beta-form) of the RNA-dependent DNA polymerase (the reverse transcriptase) from three strains of avian sarcoma virus (ASV B77, ASV tsLA334, and ASV QV2) and one exogenous (avian myeloblastosis virus (AMV)) and one endogenous avian leukosis virus (Rous-associated virus type-0 (RAV-0) were compared with each other in subunit structure and catalytic properties. Despite the gross similarity in the subunit molecular weight (Mr(alpha) = 65,000 and Mr(beta) = 92,000), minor differences were found in the molecular size of the subunit as determined by SDS-gel electrophoresis, the order being ASV tsLA334 less than or equal to ASV QV2 less than ASV B77 less than or equal to RAV-0 = AMV. The structural differences were supported by analysis of peptide fragments after treatment with S. aureus V8 protease. Although the general catalytic properties of the purified enzymes from the five virus strains were similar, the selectivety of template-primer differed in the RAV-0 enzymes. The template-primer selectivity of the reverse transcriptases from all five virus strains tested was also found to be greatly influenced by the reaction temperature for DNA synthesis, resulting in a temperature-dependent increase of poly(dG) synthesis over [(A)m] . [(dT)12-18]-dependent poly(dT) synthesis. The RAV-0 enzymes required a lower temperature for DNA synthesis, particularly for [(C)n] . [(dG)12-18]-dependent poly(dG) synthesis.
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PMID:Reverse transcriptase associated with avian sarcoma-leukosis viruses. II. Comparison of subunit structure and catalytic properties. 617 24

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.
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PMID:Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium. 792 41

The v-myb oncogene of the avian myeloblastosis virus (AMV) is unique among known oncogenes in that it causes only acute leukemia in animals and transforms only hematopoietic cells in culture. AMV was discovered in the 1930s as a virus that caused a disease in chickens that is similar to acute myelogenous leukemia in humans (Hall et al., 1941). This avian retrovirus played an important role in the history of cancer research for two reasons. First, AMV was used to demonstrate that all oncogenic viruses did not contain a single cancer-causing principle. In particular, although both Rous sarcoma virus (RSV) and AMV could replicate in cultures of either embryonic fibroblasts or hematopoietic cells, RSV could transform only fibroblasts whereas AMV could transform only hematopoietic cells (Baluda, 1963; Durban and Boettiger, 1981a). Second, chickens infected with AMV develop remarkably high white counts and therefore their peripheral blood contains remarkably large quantities of viral particles (Beard, 1963). For this reason AMV was often used as a prototypic retrovirus in order to study viral assembly and later to produce large amounts of reverse transcriptase for both research and commercial purposes. Following the discovery of the v-src oncogene of RSV and the demonstration that it arose from the normal c-src proto-oncogene, a number of acute leukemia viruses were analysed by similar techniques and found to also contain viral oncogenes of cellular origin (Roussel et al., 1979). In the case of AMV, it was shown that almost the entire retroviral env gene had been replaced by a sequence of cellular origin (initially called mab or amv, but later renamed v-myb) (Duesberg et al., 1980; Souza et al., 1980). Remarkably, sequences contained in this myb oncogene were shared between AMV and the avian E26 leukemia virus, but were not contained in any other acutely transforming retroviruses. In addition, the E26 virus contained a second sequence of cellular origin (ets) that was unique. The E26 leukemia virus was first described in the 1960s and causes an acute erythroblastosis in chickens, more reminiscent of the disease caused by avian erythroblastosis virus (AEV) than by AMV (Ivanov et al., 1962).
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PMID:Transformation by v-Myb. 1037

The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV-1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg(2+) ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140-149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV-1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV-1 integrase active site by the substrate, a strand of DNA.
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PMID:Similarities in the HIV-1 and ASV integrase active sites upon metal cofactor binding. 1068 51

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.
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PMID:Decreased c-Src expression enhances osteoblast differentiation and bone formation. 1103 78

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