Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.
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PMID:The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme. 7 Apr 28

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function. 169 96

Evidence is presented that DNA polymerase of avian myeloblastosis virus has an obligatory zinc requirement for activity. Previous studies indicate that the purified polymerase contains zinc in a stoichiometry of about 1 g-atom/mole. We now find that the enzyme-bound zinc is exchangeable with radioactive (65)Zn; after isoelectric focusing, the radioactive (65)Zn is coincident with polymerase activity. Dialysis of the (65)Zn-labeled polymerase against the chelator, 1,10-phenanthroline, results in a progressive loss of radioactive (65)Zn and polymerase activity. Thereupon, incubation of the inactivated enzyme with Zn(2+) fully restores activity. Thus, the DNA polymerase present in an oncogenic RNA virus, like animal DNA polymerases, can be rigorously classified as a zinc metalloenzyme. DNA polymerase of avian myeloblastosis virus is inactivated by 1,10-phenanthroline at a much faster rate than the bacterial and animal DNA polymerases that have been tested. It may, therefore, be possible to inactivate selectively DNA polymerases from animal tumor viruses by brief exposure to appropriate metal chelators.
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PMID:Reverse transcriptase: correlation of zinc content with activity. 414 May 13

Bisbrusatolyl malonate, which was shown previously to be active against P-388 lymphocytic leukemia cell growth, was investigated for inhibitory effects on nucleic acid and protein synthesis. DNA and RNA synthesis as well as protein synthesis were markedly inhibited at 10,25, and 50 mu mole final concentrations in vitro. The major sites of inhibition of nucleic acid synthesis appeared to be DNA polymerase, messenger and transfer RNA polymerases, orotidine-5'-monophosphate decarboxylase, phosphoribosyl pyrophosphate amino transferase, and dihydrofolate reductase. Moderate inhibition of nucleotide kinase activities and oxidative phosphorylation processes occurred after drug treatment. Cyclic adenosine monophosphate levels were reduced. Protein synthesis was inhibited during the elongation step of peptide synthesis. The data suggested that bisbrusatolyl malonate interfered with the peptide bond formation. However, the ongoing polypeptide synthesis must be completed before the drug can bind to the ribosome effectively.
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PMID:Antitumor agents XLVII: The effects of bisbrusatolyl malonate on P-388 lymphocytic leukemia cell metabolism. 627 24

Survival data from 379 patients with chronic hepatitis B were analyzed to determine life expectancy for the patient from the time of first contact. One hundred twenty-one patients had chronic persistent hepatitis, 128 had chronic active hepatitis, and 130 had chronic active hepatitis with cirrhosis. The frequency of symptoms (p less than 0.001), stigmata of chronic liver disease (p less than 0.001), and liver function test abnormalities (p less than 0.001) increased as the histologic features worsened, whereas the percentage of patients with circulating hepatitis B DNA polymerase declined (p less than 0.001). Women were uncommon in our series and had less severe disease than men (p less than 0.02). Fifty-one patients had died by the time of this analysis. The estimated 5-year survival rates were 97% for patients with chronic persistent hepatitis, 86% for those with chronic active hepatitis, and 55% for those with chronic active hepatitis with cirrhosis. The usual cause of death was liver failure and its sequelae. A multivariate analysis found age of 40 years or more, total bilirubin level of 1.5 mg/dL or more, ascites, and spider nevi to be factors that identified patients at a higher risk of death. The prognosis for patients with chronic hepatitis B is similar to that for patients with chronic hepatitis of other causes.
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PMID:Survival in chronic hepatitis B. An analysis of 379 patients. 648 92

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:CC-1065 bonding to intracellular and purified SV40 DNA: site specificity and functional effects. 804 19

Reconstitution experiments were performed by using an ordinary dye-primer protocol of a template spiked with known amounts of truncated fragments. We observed that as little as 0.2 mole-percentage of the truncated fragment caused sequence interpretation problems. Two protocols were developed for sequencing with dye-labeled terminators; this eliminates the problems with truncated fragments, which are adapted to a one-dye chemistry. One was designed for single extension sequencing using T7 DNA polymerase and one for cycle sequencing. To avoid precipitation and centrifugation and to facilitate automation, the dye-terminator protocols included the use of a biotinylated sequencing primer. Thus, the Sanger fragments were recovered and, by magnetic separation, washed and released by formamide, EDTA, and heat treatment before loading on the electrophoresis gel. Integrated procedures for sequencing PCR products using one-dye-labeled terminators suitable for automation are described. High quality data in terms of long reads and detection of polymorphisms is obtained. The protocols serve as attractive alternatives to internal labeling and dye-primer approaches.
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PMID:Truncated fragments in polymerase chain reaction-based DNA sequencing. 1021 63

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.
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PMID:Selective labeling and detection of specific RNAs in an RNA mixture. 1267 21

A method for the accurate determination of the fraction of surface-attached DNA duplexes exhibiting a single-fluorophore-labeled nucleobase is introduced. The fluorescence signals obtained from surface plasmon field-enhanced fluorescence spectroscopy along with the optical properties of the sensor architecture determined by surface plasmon resonance were employed for the calculation. A Cy5-labeled nucleotide was incorporated into DNA at a well-defined position via template-directed DNA synthesis performed by a DNA polymerase. The sample-to-sample variations associated with the optical properties of the employed metal films caused a small variation in the strength of the evanescent field. This variation was accounted for by evanescent field integration over the DNA layers. The exponential-type relationship between the fraction of DNA with Cy5-dCTP incorporation at the surface and the mole fraction of the Cy5-dCTP in solution indicates the preferential incorporation of nonlabeled nucleotides by the DNA polymerase.
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PMID:Concentration of dye-labeled nucleotides incorporated into DNA determined by surface plasmon resonance-surface plasmon fluorescence spectroscopy. 1530 86

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.
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PMID:RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi. 1820 35


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