Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene

John D. Bartlett, David A. Scicchitano, Steven H. Robison

Research output: Contribution to journalArticle

Abstract

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0,6, and 24 h), T-lymphocytes DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliqout provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt > dhfr > dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discusses in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.

Original languageEnglish (US)
Pages (from-to)247-256
Number of pages10
JournalMutation Research-DNA Repair
Volume255
Issue number3
DOIs
StatePublished - Nov 1991

Keywords

  • Chromatin
  • Human T-lymphocytes
  • Methoxyamine
  • N-Methylpurines
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Toxicology
  • Genetics

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