11. 1,N6-ethenoadenine is not a block to mammalian RNA polymerase II transcription
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Abstract: The aim of this work was to elucidate mechanisms of repair of eA in either a transcriptionally active or inactive context in embryonic fibroblasts from normal, or alkylpurine-DNA-N-glycosylase deficient mice. Non-replicating variants of a shuttle vector (pS189) containing a single eA adduct were constructed: pSDori for the study of transcription-coupled repair (deletion of SVori) and pSDT3 for transcription independent repair (both pSVori and promoter regions deleted). However, although pSDori and pSDT3 were successfully transfected into APNG proficient and deficient cells, highly variable recovery of the vector prevented us from using this method to measure eA repair in vivo.
However, we were able to show that eA does not block RNA polymerase II directed transcription: RNA was recovered from pSDori and pSDT3 transfected cells and RT-PCR carried out using primers designed to amplify a region, either spanning (251bp), or upstream (300bp) of the eA adduct present in the shuttle vector. As expected, no specific signal was obtained from cells transfected with pSDT3, which lacks the TAg promoter. However, RT-PCR fragments were obtained from both APNG proficient and deficient cells transfected with the transcription competent pSDori. Thus, we can conclude that eA does not block RNA Pol II dependent transcription.
Subject Descriptors: Cell biology
Subject Index Codes: Biotechnology
Stage of Development: Scientific and/or technical knowledge (basic research)
Collaboration Sought: Information exchange/Training