JaLCDOI 10.18926/AMO/32639
FullText URL fulltext.pdf
Author Zhang, Bo| Seki, Shuji| Akiyama, Kosuke| Tsutsui, Ken| Li, Ting| Nagao, Kazutaka|
Abstract <p>DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG &#62; AG &#62; GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture.</p>
Keywords DNA damage cisplatin gel electrophoresis sequence gel analysis
Amo Type Article
Published Date 1992-12
Publication Title Acta Medica Okayama
Volume volume46
Issue issue6
Publisher Okayama University Medical School
Start Page 427
End Page 434
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 1336637
Web of Sience KeyUT A1992KE49600004
JaLCDOI 10.18926/AMO/32188
FullText URL fulltext.pdf
Author Zhang, Bo| Watanabe, Sekiko| Akiyama, Kosuke| Li, Ting| Fukushima, Keisuke| Tsutsui, Ken| Seki, Shuji|
Abstract <p>DNA repair synthesis induced in permeable mouse ascites sarcoma cells by peplomycin, an antitumor antibiotic, was studied. Mouse ascites sarcoma (SR-C3H/He) cells were permeabilized with a low concentration of Triton X-100 in an isotonic condition. Permeable cells were treated with an appropriate concentration of peplomycin to introduce single-strand breaks in permeable cell DNA. DNA repair synthesis in peplomycin-treated permeable cells was measured by incubating the cells with four deoxynucleoside triphosphates in an appropriate buffer system. The DNA repair synthesis was enhanced by ATP and NaCl at near physiological concentrations. More than 90% of DNA synthesis in the present system depended on the peplomycin-treatment. The repair nature of the DNA synthesis was confirmed by a BrdUMP density shift technique. The repair patches were largely completed and ligated in the presence of ATP. Analyses using selective inhibitors for DNA polymerases showed that both DNA polymerase Beta and aphidicolin-sensitive DNA polymerases (DNA polymerase alpha and/or delta) were involved in the repair DNA synthesis.&#60;/P&#62;</p>
Keywords DNA repair peplomycin DNA polymerases permeable mouse cells
Amo Type Article
Published Date 1991-04
Publication Title Acta Medica Okayama
Volume volume45
Issue issue2
Publisher Okayama University Medical School
Start Page 89
End Page 94
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 1714230
Web of Sience KeyUT A1991FL60800004
JaLCDOI 10.18926/AMO/30857
FullText URL fulltext.pdf
Author Ikeda, Shogo| Yamamoto, Mihoko| Nagao, Kazutaka| Zhang, Bo| Watanabe, Sekiko| Oda, Takuzo|
Abstract <p>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.</p>
Keywords non-rradioctive probe biotin nucleotide M13 phage DNA universal sequencing primer Southern hybridization
Amo Type Article
Published Date 1989-08
Publication Title Acta Medica Okayama
Volume volume43
Issue issue4
Publisher Okayama University Medical School
Start Page 197
End Page 202
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 2678902
Web of Sience KeyUT A1989AP79100001