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)

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.
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PMID:Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification. 240 38

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.
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PMID:An alternative nonradioactive method for labeling DNA using biotin. 252 11

The use of biotinylated DNA hybridization probes for clinical detection of bovine herpesvirus-1 was investigated. Biotinylated DNA hybridization probes were prepared from bovine herpesvirus-1 DNA purified from infected cell cultures. The viral DNA was nick translated in the presence of biotin-dUTP with DNA polymerase to incorporate biotin into the newly synthesized strand. The probe was tested for specificity in in situ hybridization assays with bovine herpesvirus-1 DNA. Hybridization was detected using avidin-fluorescein single sandwich systems and an avidin-globulin with anti-globulin-fluorescein double sandwich system. Hybridization was detected by specific fluorescence of infected cells. Fluorescence was present only in bovine herpesvirus-1-infected cell culture and not in noninfected cell culture or cell cultures infected with several other viruses. The assay was performed in 6 hr.
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PMID:Rapid fluorescence detection of in situ hybridization with biotinylated bovine herpesvirus-1 DNA probes. 256 8

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.
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PMID:Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. 257 94

Aspects of the regulation of DNA replication and mitosis have been studied using a cell-free extract of Xenopus eggs. The extract is characterized by repeated cycles of DNA replication and mitosis, which are accompanied by periodic synthesis and degradation of cyclins as well as fluctuations in the level of Histone H1 kinase activity. DNA replication in this system is dependent upon the formation of a nucleus. However, while nuclear structures are clearly required for initiation, a complete nuclear membrane does not appear to be necessary. Indirect immunofluorescence and DIC microscopy indicate that nuclear reformation from chromosomes occurs asynchronously around individual chromatids. Lamin polymerization, biotin-11-dUTP incorporation and association of polymerases with chromatin occur before membrane formation is complete. S phase nuclei are typified by the co-distribution of both anti-DNA polymerase alpha and anti-PCNA antibodies as discrete spots of fluorescence which align the chromatin. However, as DNA replication is terminated, PCNA fluorescence fades and DNA polymerase alpha dissociates from the chromatin and is redistributed throughout the nucleoplasm. By inhibiting DNA replication with aphidicolin, both DNA polymerase alpha and PCNA remain associated with the chromatin throughout prolonged incubation. Under these conditions mitosis is delayed by up to 70 min, although both the general rate of protein synthesis and more importantly the rate of cyclin synthesis and histone kinase activation are unaffected. Upon nuclear envelope breakdown and lamin dispersal, cyclins degrade; however, no chromosomes are formed, and both PCNA and DNA polymerase alpha remain associated with the chromatin. Also, histone kinase activity is maintained at elevated levels.
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PMID:DNA replication and cell cycle control in Xenopus egg extracts. 257 47

Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.
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PMID:Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I. 266 60

Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.
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PMID:Non-radioactive hybridization probes prepared using M13 phage vector and the universal sequencing primer. 267 2

The synthesis of a biotinylated derivative of dCTP, viz. N4-[(N-biotinyl)-4-amino-butoxyl]-2'-deoxycytidine 5'-triphosphate (I), is described. DNA polymerase I (Klenow fragment) incorporates (I) in DNA chains instead of thymidine, although with a lower efficiency than previously described biotinylated dUTP derivative (II), whereas highly purified DNA polymerase alpha from human placenta uses as substrate derivative (II) but not (I). A DNA fragment bearing biotin residues in one of strands was synthesized with the use of DNA polymerase alpha and dUTP derivative (II); its cloning in the plasmid vector pBR322 revealed that the DNA nucleotide sequence remained intact.
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PMID:[Inclusion of biotinylated analogs of dUTP and dCTP in DNA by DNA-polymerases. Cloning DNA fragments, containing biotinylated deoxyribouridine in E. coli]. 268 53

In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA-protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions.
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PMID:Mechanisms of in situ nick translation of chromosomes using restriction endonucleases. 301 9

The herpes simplex virus type 2 (HSV-2)-induced deoxyuridine triphosphate nucleotidohydrolase (dUTPase) was purified approximately 600 +/- 43-fold using a combination of affinity, hydrophobic, absorption, and ion-exchange chromatography techniques. The only substrate for the dUTPase was dUTP with a Km of 3.6 +/- 1.1 microM. There was no apparent divalent cation requirement, but the HSV-2-induced dUTPase was inhibited by EDTA (0.1 mM) and this inhibition was reversed by either Co2+ (0.5 mM) or Mg2+ (0.5 mM). The HSV-2-induced dUTPase was distinguished from the HSV-1-induced and cellular dUTPases based upon differences in sensitivity to substrate inhibition, thermostability, and electrophoretic migration in nondenaturing polyacrylamide gels. Analysis of HSV-1 temperature-sensitive (ts) mutants demonstrated that ts A15 and ts K13 did not induce significant amounts of dUTPase activity at the permissive or nonpermissive temperatures. Mutants with defects in HSV-induced DNA polymerase or in the major DNA binding protein induced dUTPase at both temperatures. In contrast ts mutants defective in the alpha polypeptide VP175 (ICP4) did not induce normal levels of dUTPase at the nonpermissive temperature. The location of a gene encoding for the type specificity of the HSV induced dUTPase was mapped to the left 20% of the genome in Us in the region 0.060 to 0.100 or from 0.148 to 0.204.
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PMID:Characterization of a herpes simplex virus type 2 deoxyuridine triphosphate nucleotidohydrolase and mapping of a gene conferring type specificity for the enzyme. 302 79


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