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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple and reproducible method of polymerase chain reaction (PCR) assay was established to detect trinucleotide repeat expansion for Huntington's disease (HD) using a new DNA polymerase and buffer system. The system consists of an extremely heat stable DNA polymerase (Pfu), and a buffer supplemented with ammonium sulfate and dimethyl sulfoxide. Previous methods to amplify expanded alleles for HD have been very complex in PCR conditions, but the reproducibility was sometimes very low because of repetitive sequences around the primer sequences. With the present method, strong bands for the disease alleles were reproducibly visible in a conventional agarose gel stained with ethidium bromide without using isotopes. Three cases with sporadic HD and a case with senile chorea showed expanded alleles for HD with smaller sizes of the expansion than cases with typical HD. These results showed that the present method provides a simple and reproducible way to detect HD allele, and some cases with sporadic HD and senile chorea had expanded HD alleles.
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PMID:A reproducible assay of polymerase chain reaction to detect trinucleotide repeat expansion of Huntington's disease and senile chorea. 871 30

The 36K protein attached at the 5' end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0 center dot 8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.
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PMID:The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase. 890 36

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.
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PMID:Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry. 891 80

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198-204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.
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PMID:Use of Vibrio spp. for expression of Escherichia coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes simplex virus type 1. 893 1

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase alpha-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase.
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PMID:Purification and properties of a DNA primase from Nicotiana tabacum. 944 86

A gene, top encoding Thermus thermophilus HB27 (Top) DNA polymerase, was cloned in E. coli and its nucleotide sequence was determined. Based on its deduced amino acid sequence, Top DNA polymerase is a 93.8 kDa protein comprising 834 amino acid residues. Top DNA polymerase showed high amino acid homology with those of other DNA polymerases from the Thermus sp., for example, 87.3% identity with Taq DNA polymerase. Codon usage in the top gene was similar to those of the proteins from other Thermus strains. The G + C content in the third position of the codons was as high as 93%. The top gene under the control of the tac promoter was expressed in E. coli [plasmid pTOP9]. DNA amplification using the recombinant Top DNA polymerase performed the same as other thermostable DNA polymerases from Thermus strains. The optimum temperature for its reaction was 76 degrees C. An interesting observation was that the recombinant Top DNA polymerase was slowly cleaved into two fragments of about 60 kDa and 35 kDa at 4 degrees C and -20 degrees C. The larger fragment possessed polymerase activity like the Klenow fragment of E. coli DNA polymerase I. To prevent the cleavage of the Top DNA polymerase, a variety of protecting agents were examined. Among those examined, (NH4)2SO4 (100 mM) solution demonstrated an outstanding ability to block its cleavage for a prolonged period.
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PMID:Top DNA polymerase from Thermus thermophilus HB27: gene cloning, sequence determination, and physicochemical properties. 963 46

The activity of DNA polymerase alpha-primase complex from calf thymus and Klenow fragment of E. coli DNA polymerase 1 has been studied in reverse microemulsions formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and Triton X-114 in decane. DNA polymerases were not active in AOT, CTAB, and SDS reverse microemulsions, but these enzymes catalyzed DNA synthesis in Brij 58 and its mixture with other surfactants. We have also found the system composed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration of 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v), in which DNA polymerases revealed maximum activity. The above system was optically transparent, fluid, and stable during a few hours with a water-surfactants molar ratio up to 160. The pH dependence of DNA polymerase activity was not significantly different in comparison with water; however, DNA polymerase was sensitive to ionic strength in microemulsions. The dependence of DNA polymerase activity on w0 was the curve with a few optima. DNA polymerases synthesized more products in water than in reverse microemulsions, and the processivity of Klenow fragment decreased. An increase of the water content resulted in an increase of DNA polymerase processivity.
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PMID:Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions. 965 93

DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.
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PMID:Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase. 975 14

The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source. Native data have been collected at 100 K. A heavy-atom derivative search is in progress.
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PMID:Crystallization and preliminary X-ray diffraction studies of DNA polymerase from the thermophilic archaeon Sulfolobus solfataricus. 975 20

Herpes simplex virus DNA polymerase (HSV pol) holoenzyme consists of a large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3'-5' exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme-DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditions in vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme-DNA complex.
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PMID:DNA and protein interactions of the small subunit of herpes simplex virus type 1 DNA polymerase. 988 18


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