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)

A 1.67-kb segment of the equine infectious anemia virus pol gene, encoding a 66-kDa reverse transcriptase (RT), was cloned and expressed in Escherichia coli. Recombinant RT, purified by a combination of metal chelate affinity chromatography and ion-exchange chromatography, displays both RNA-dependent DNA polymerase and RNase H activity. The affinity of purified RT for its replication primer, tRNA(3Lys) was equivalent to that observed for human immunodeficiency virus RT. Our data suggest that an additional domain between RT-RNase H and integrase on the equine infectious anemia virus pol open reading frame is not an integral component of the RT polypeptide.
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PMID:Purification and characterization of recombinant equine infectious anemia virus reverse transcriptase. 171 38

We investigated TEL/AML1 fusion mRNA in 108 children with acute lymphoblastic leukemia (ALL) (86 B-lineage ALL, 15 T-ALL, two mixed lineage ALL, and five other phenotypes) using reverse transcriptase-polymerase chain reaction (RT-PCR). TEL/AML1 transcripts were found in 14 patients (13%) including three relapsed patients, and were unexceptionally limited to B-lineage ALL patients. The incidence of TEL/AML1 transcripts among B-lineage ALL was 16% (14/86). The reciprocal AML1/TEL transcripts were detected in 12 (86%) of the 14 cases expressing a TEL/AML1 transcript. In three cases, the TEL gene was fused to exon 3 of the AML1 gene, and to exon 2 in the remaining cases. To evaluate the amount of TEL/AML1 molecules for the quantification of a minimal residual disease (MRD), a plasmid vector which contained either a long TEL/AML1 PCR product (464 bp) or a short one (425 bp) was used as a competitor. We amplified RNAs obtained from bone marrow (BM) at complete remission or from peripheral blood stem cell (PBSC) harvests in two representative cases. For one PBSC harvest showing a positive result, a competitive PCR was carried out to quantify the amount of MRD. A 1:4 dilution series of competitor vectors was constructed, and each vector was added to a PCR reaction which contain a constant amount of cDNA obtained from the PBSC harvest. An equivalent point was compared to that of corresponding samples at diagnosis. Using this method, MRD in the PBSC harvest was 3.9:10(3). Our results elucidated the incidence, lineage-specificity, and variant forms of TEL/AML1 fusion transcripts in childhood ALL. Since the percentage of other chromosomal translocations in childhood ALL is not more than 5%, TEL/AML1 transcript would be the most feasible clone-specific marker for these patients. In addition, our method could be a powerful tool for quantification of the TEL/AML1 transcript and for the detection of MRD.
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PMID:Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia. 875 64

Two mutants of HIV-1 reverse transcriptase (RT) associated with high-level resistance of the virus to AZT (RT-AZT: D67N, K70R, T215Y, K219Q, and M41L) or 3-TC (RT-3TC: M184V) were expressed in Escherichia coli and purified. None of these mutants showed significant changes in the affinity and kinetics of binding to a DNA/DNA primer/template. RT-AZT was investigated in detail with respect to its kinetics of incorporation of nucleotides. No change in the relative rates of TMP and AZTMP incorporation could be detected for RT-AZT with respect to wild type RT. These results imply that there is no increased discrimination against AZTTP in the mutant. This was found for DNA/DNA and DNA/RNA primer/template. Additionally, rapid kinetics of incorporation of 3'-amino-3'-deoxythymidine 5'-monophosphate (a possible metabolite of AZT) were investigated and compared with TMP incorporation, but no difference in its relative rates of incorporation between wild type RT and RT-AZT was detected. In contrast, the already very slow rate of incorporation of 3-TCMP seen with wild type enzyme was drastically reduced (by a factor of 23 and 36 with DNA/DNA primer/template and DNA/RNA primer/template, respectively) for RT-3TC, showing a clear correlation between in vitro and in vivo effects. The affinity of 3-TCTP to the RT-3TC-primer/template complex was not affected by the mutation M184V. A 1.6-fold cross-resistance to ddATP, the converted form of the prodrug ddI, could also be shown for RT-3TC, but no cross-resistance to ddCTP was detected. Additionally, rapid kinetics of AZTMP incorporation by RT-3TC were investigated. There was an indication of a slightly higher rate of incorporation of AZTMP by RT-3TC than wild type RT.
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PMID:Single-step kinetics of HIV-1 reverse transcriptase mutants responsible for virus resistance to nucleoside inhibitors zidovudine and 3-TC. 925 28

In this study, expression of the prolactin receptor (PRL-R) gene in the ovaries of cycling and pregnant red deer (Cervus elaphus) hinds was investigated. A 1.9-kilobase (kb) cDNA encoding the cervine long-form PRL-R was amplified by reverse transcriptase polymerase chain reaction from corpus luteum (CL) and liver poly(A)+ RNA. Northern hybridization revealed a major mRNA transcript of 3.5 kb in both tissues. PRL-R mRNA transcripts were localized by in situ hybridization in 15-micron frozen sections of red deer ovaries, collected during the estrous cycle and early pregnancy, with homologous 45-mer [35S]dATP-labeled sense and antisense oligonucleotide probes. Specific hybridization was assessed by measurement of autoradiograph optical density (OD) in CL, follicles and stroma. PRL-R mRNA expression was higher (p < 0.001) in the CL (OD = 22.2 +/- 3.77, n = 11 CL) than in follicles (OD = 2.8 +/- 0.10, n = 224 follicles) and was undetectable in the stroma (OD < 1, limit of detection). No differences in abundance of PRL-R mRNA were observed between follicles divided on the basis of size, health vs. atresia, or stage of estrous cycle or pregnancy, or between CL from pregnant and nonpregnant hinds. In the follicles, PRL-R mRNA was localized to the theca layer. These results suggest a direct role for PRL in red deer ovarian physiology during the estrous cycle and pregnancy.
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PMID:Expression and localization of prolactin receptor messenger ribonucleic acid in red deer ovary during the estrous cycle and pregnancy. 931 91

An endonuclease named DNase gamma has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675-681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase gamma. A 1.6 kb mRNA coding for the DNase gamma precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase gamma mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase gamma reveals that full-length DNase gamma, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase gamma, from which the N-terminal precursor has been removed, has the same properties as purified DNase gamma: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase gamma cDNA, exogenously introduced DNase gamma is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase gamma in DNA fragmentation and nuclear structural changes during apoptosis.
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PMID:Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase gamma. 962 Aug 74

To extend our understanding of amplicon structure in methotrexate-resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3'-end of the Type 1 dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an approximately 0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.
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PMID:Similarities to a LINE element shared by Anopheline and Culicine mosquitos map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells. 975 71

In this study, reverse transcriptase polymerase chain reaction was used to amplify human endothelin receptor A (ETA) and ETB receptor mRNA. A truncated ETA receptor transcript with exons 3 and 4 skipped was found. The skipping of these two exons results in 109 amino acids being deleted from the receptor. The truncated receptor was expressed in all tissues and cells examined, but the level of expression varied. In melanoma cell lines and melanoma tissues, the truncated receptor gene was the major species, whereas the wild-type ETA was predominant in other tissues. A 1.9-kb ETA transcript was identified in melanoma cell lines by Northern blot, which was much smaller than the transcript in heart and in other tissues reported previously (4.3 kb). The cDNA coding regions of the truncated and wild-type ETA receptors were stably transfected into Chinese hamster ovary (CHO) cells. The truncated ETA receptor-transfected CHO cells did not show binding affinity to endothelin 1 (ET-1) or endothelin 3 (ET-3). The function and biological significance of this truncated ETA receptor is not clear, but it may have regulatory roles for cell responses to ETs.
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PMID:Truncated human endothelin receptor A produced by alternative splicing and its expression in melanoma. 982 Jan 69

To investigate the potential of autologous lymphocytes to eliminate chronic myelogenous leukemia (CML) cells following activation and targeting by CD3-monoclonal antibody, we cultured Ficoll-isolated peripheral blood cells from 11 patients with CML in the chronic phase in permutated combinations of interleukin (IL)-2, CD3-monoclonal antibody (OKT3), and interferon (IFN)gamma. The efficiency of CML cell elimination was studied by means of flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Cultures containing only OKT3 and IL-2, with or without IFNgamma, resulted in tumor cell reduction to the level of RT-PCR negativity. The length of the culture period required to reach a RT-PCR-negative state ranged from 3 to 33 days. A 1- to 2-log reduction in leukemic cells could be achieved by culture medium alone. In contrast, 3- to 4-log reductions in CML cells were observed following in vitro culture and ex vivo T cell activation with a given sensitivity for RT-PCR detection of 1 bcr/abl+ cell in 10(4). The feasibility of purging chronic myelogenous leukemia cells in a short time was associated with a low number of platelet counts (r=0.6457; p < 0.05). CML cell reduction was associated with expansion of CD25+/CD4+//CD8+/-/CD56+/- lymphocytes. These findings may be of relevance for immunotherapy procedures.
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PMID:Activation of autologous lymphocytes of patients with chronic myelogenous leukemia in the chronic phase with cytokines and CD3 monoclonal antibody results in bcr/abl- blood leukocyte cultures as determined by reverse transcriptase-polymerase chain reaction. 984 83

A 1 kb EcoRI restriction fragment cloned from a band visible in an agarose gel of Pinus lambertiana (sugar pine) genomic DNA is present in both subgenera of Pinus with at least 10(4) copies/genome. A full-length copy of this repeated element recovered from a P. radiata (Monterey pine) genomic DNA library was found to possess all of the sequence features associated with gypsy-like retrotransposons. This report describes the biology and history of the IFG (Institute of Forest Genetics) family of retrotransposons. The characterized IFG7 is 5937 bp long. Immediately interior to its 5' and 3' long terminal repeats are sequences consistent with primer binding sites for reverse transcription of the RNA genome. Presumptive gene products associated with retrotransposition appear to be coded in a single reading frame and are in the same order as the gypsy-like retrotransposons and retroviruses. The 1.0 kb EcoRI fragment of IFG elements codes for the 3' half of IFG's reverse transcriptase and the entire RNase H domain. Southern blot analysis suggests IFG was present in Pinaceae before its division into its modern genera. Sequence analysis of IFG 1.0 kb RI fragments and southern analysis also suggest that IFG continued to evolve in Pinus with restriction fragment length polymorphism (RFLP) subfamilies appearing early in the history of each subgenus often correlating with subdivisions of Pinus. Features shared with other plant retrotransposons are also discussed.
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PMID:IFG, a gypsy-like retrotransposon in Pinus (Pinaceae), has an extensive history in pines. 1009 71

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded and imported into the mitochondrion. A heterogeneous population of RNAs having characteristics of precursor tRNAs have previously been identified within the mitochondrion of T. brucei, suggesting that import occurs via a precursor molecule. In order to identify nuclear genes encoding tRNAs targeted to the mitochondrion, individual mitochondrial tRNAs were separated using two-dimensional gel electrophoresis and enzymatically sequenced. A 1.1-kilobase pair genomic DNA fragment was cloned containing three tRNA genes, tRNA(1)(Ser), tRNA(Leu), and tRNA(2)(Ser). Dicistronic precursors containing the tRNA(1)(Ser) and tRNA(Leu) transcripts with a 59-nucleotide intergenic sequence were identified by reverse transcriptase and polymerase chain reactions and the 5' end of the precursors determined. The dicistronic precursor tRNA is present both in the cytosol and the mitochondrion supporting a model for tRNA import involving precursor tRNA transcripts.
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PMID:A nuclear encoded and mitochondrial imported dicistronic tRNA precursor in Trypanosoma brucei. 1040 59


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