Preferential modification of GC boxes by benzo[a]pyrene‐7,8‐diol‐9,10‐epoxide

Abstract

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.

Effect of excision repair by diploid human fibroblasts on the kinds and locations of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene in the coding region of the HPRT gene.

“…2 and Tables 1 and 2). Although it has been reported that certain poly(dG) sequences are the preferred sites for BPDE binding (31,32), this cannot explain the cell-cycle dependent feature of the very prominent hot spot region that we observed. It is more likely that this hot spot region seen in the G1 cells resulted from inefficient repair of BPDE adducts in the run of six consecutive guanine bases so that after excision repair had occurred for a certain period of time, the adducts remaining in this region represented a higher fraction than originally present.…”

High-Frequency Illegitimate Integration of Transfected DNA at Preintegrated Target Sites in a Mammalian Genome

“…The 5Ј APRT sequence includes the upstream portion (600 nucleotides) of intron 2. Plasmid pGAL contains the full-length APRT sequence and is described elsewhere (29). Plasmid pMC1gptPolA (kindly provided by Geoff Sargent) is a derivative of pMC1neoPolyA (73) in which the neo gene has been replaced by the bacterial gpt cassette from pSV2gpt (40).…”

Protection from UV-B-induced DNA damage by flavonoids

“…The T 7 RNA polymerase promoter site was orientated at the 5' end of the aprt sequence and in vitro transcription of the linearized plasmid pGAL resulted in run off transcripts that were 1836 nucleotides long [15]. In all experiments Xho I-linearized plasmid DNA was used as the template in T7 RNA polymerase runoff transcription assays.…”

“…The samples were gently mixed, microfuged, and run-off transcription was carried out at 37 ° C for 60 rain. S ample application buffer (10 #1) was added [15], mixed and 20 #1 were loaded on a 1 7o agarose gel. Electrophoresis was carried out in 1 x TBE buffer in the presence of ethidium bromide for 60 min at 100 V and transcripts were visualized and photographed.…”