Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
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PMID:Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation. 5 Sep 2

Bleomycin (BLM) exclusively affects thymidine-containing compounds such as DNA and polydeoxyribonucleotides by releasing free thymine and leaving aldehyde functions. Molecular morphology and base sequence of the DNA strongly influence BLM activity. High BLM concentrations, besides modifying DNA into oligothyminic or athyminic nucleic acids, cause strand scissions. Enzymatic DNA and RNA synthesis is strongly influenced by BLM. The inhibition in DNA-dependent DNA polymerase and DNA-dependent RNA polymerase assays is of the non-competitive type. Protein biosynthesis in in vitro systems is not affected by BLM even at high concentrations. BLM turns out to be a strong inhibitor of DNase I and of DNase II; the inhibition is of the competitive type. The enzymatic activities of nucleases using RNA as substrate (RNase A, RNase B, Rnase T1, venom phosphodiesterase I and spleen phosphodiesterase II) are not influenced by this antibiotic. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro in low concentrations by cytostasis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells, DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo, using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This is attributed to the observed inhibition of DNA synthesis. RNA and protein synthesis as well as gene expression are not influenced by BLM under the conditions used. The selective inhibition of DNA synthesis in vivo may be caused by the following mechanisms: (1) competition of BLM with RNA; (2) blocking of the accessibility of DNA in chromatin to BLM, and (3) dependence from the repair processes. BLM inhibits growth of sarcomas, induced by oncogenic RNA viruses in vivo; well-developed tumours show regression after BLM treatment. Transformation of chick embryo fibroblasts by oncogenic RNA viruses in vitro and growth of these viruses is blocked by BLM; the most sensitive period for BLM inhibition is the time during the first period (integration of viral genome into cellular genome?) after infection.
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PMID:Effect of bleomycin on DNA, RNA, protein, chromatin and on cell transformation by oncogenic RNA viruses. 6 69

To date the only point mutations demonstrated to cause hemophilia are C to T transitions in TaqI sites. These were detected by screening Southern blots with cloned factor VIII probes. During the development of improved methods for detecting and analyzing mutations in genomic DNA, a novel G to C transversion mutation has been identified. This rare transversion results in a missense mutation, with proline being substituted for arginine in one of the active domains of the factor VIII molecule. The results suggest that the improved methods will be useful for detecting mutations in hemophilia as well as in other genetic disorders. In this method, specific DNA sequences in genomic DNA are amplified using oligonucleotide primers and a heat-resistant DNA polymerase. Mutations are detected and localized in the amplified samples by RNase A cleavage, and the altered region is then sequenced.
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PMID:A novel missense mutation in the factor VIII gene identified by analysis of amplified hemophilia DNA sequences. 312 81

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.
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PMID:Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method. 615 42

Cross-contamination with previously amplified products poses a serious limitation in the use of PCR for clinical testing and in certain research applications as well. In the present study we report the use of novel primers containing a 3'-terminal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating in any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and post-amplification sterilization can be carried out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, is also destroyed.
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PMID:Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences. 841 89

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.
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PMID:p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors. 949 22

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.
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PMID:Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system. 1006 45

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed bla(SHV) genes was used for the identification and genotyping of SHV beta-lactamases. For evaluation, bla(SHV) stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.
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PMID:Detection and genotyping of SHV beta-lactamase variants by mass spectrometry after base-specific cleavage of in vitro-generated RNA transcripts. 1651 75