Header

UZH-Logo

Maintenance Infos

Detection by 32P-postlabeling of thymidine glycol in γ-irradiated DNA


Hegi, Monika E; Sagelsdorff, Peter; Lutz, Werner K (1989). Detection by 32P-postlabeling of thymidine glycol in γ-irradiated DNA. Carcinogenesis, 10(1):43-47.

Abstract

The 32P-postlabeling method has been adapted for the analysis of thymidine-cis-glycol-3′-phosphate (cis-dTGp, cis-5,6-dihydroxy-5,6-dihydrothymidine-3′-phosphate). Cis-dTGp was isolated and purified from normal nucleotides by phenylboronate affinity chromatography and phosphorylated by T4 polynucleotide kinase in presence of 1 mM BeCl2 at pH 7.5. These modifications of the postlabeling method resulted in a 5′-phosphorylation of dTGp with a labeling efficiency of up to 20% whereas the natural nucleotides were almost completely dephosphorylated at the 3′ position under these conditions. The reaction products, containing radio-labeled thymidine-cis-glycol-3′ ,5′-bis-[5′-32P]phosphate (cis-*pdTGp), were separated by two-dimensional anion-exchange TLC on polyethyleneimine cellulose sheets. Boric acid was added in the second dimension in order to selectively retard cis-glycols. The method was applied to γ-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330-99 000 thymine glycol (TG) moieties were detected per 106 thymines (T) in a dose range of 14-1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/106 T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 1OTG/106 T. This practical limit of detection was higher than can be achieved with the postlabeling technique, indicating that the present method might be a sensitive alternative for a determination of oxidative DNA damage

Abstract

The 32P-postlabeling method has been adapted for the analysis of thymidine-cis-glycol-3′-phosphate (cis-dTGp, cis-5,6-dihydroxy-5,6-dihydrothymidine-3′-phosphate). Cis-dTGp was isolated and purified from normal nucleotides by phenylboronate affinity chromatography and phosphorylated by T4 polynucleotide kinase in presence of 1 mM BeCl2 at pH 7.5. These modifications of the postlabeling method resulted in a 5′-phosphorylation of dTGp with a labeling efficiency of up to 20% whereas the natural nucleotides were almost completely dephosphorylated at the 3′ position under these conditions. The reaction products, containing radio-labeled thymidine-cis-glycol-3′ ,5′-bis-[5′-32P]phosphate (cis-*pdTGp), were separated by two-dimensional anion-exchange TLC on polyethyleneimine cellulose sheets. Boric acid was added in the second dimension in order to selectively retard cis-glycols. The method was applied to γ-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330-99 000 thymine glycol (TG) moieties were detected per 106 thymines (T) in a dose range of 14-1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/106 T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 1OTG/106 T. This practical limit of detection was higher than can be achieved with the postlabeling technique, indicating that the present method might be a sensitive alternative for a determination of oxidative DNA damage

Statistics

Citations

Dimensions.ai Metrics
48 citations in Web of Science®
46 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

14 downloads since deposited on 17 Oct 2018
9 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:National licences > 142-005
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Life Sciences > Cancer Research
Language:English
Date:1 January 1989
Deposited On:17 Oct 2018 14:42
Last Modified:31 Jul 2020 02:22
Publisher:Oxford University Press
ISSN:0143-3334
OA Status:Green
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1093/carcin/10.1.43
Related URLs:https://www.swissbib.ch/Search/Results?lookfor=nationallicenceoxford101093carcin10143 (Library Catalogue)

Download

Green Open Access

Download PDF  'Detection by 32P-postlabeling of thymidine glycol in γ-irradiated DNA'.
Preview
Content: Published Version
Language: English
Filetype: PDF (Nationallizenz 142-005)
Size: 3MB
View at publisher