Kamiya Hiroyuki, Suzuki Akihiro, Kawai Kazuaki, Kasai Hiroshi, Harashima Hideyoshi
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Free Radic Biol Med. 2007 Sep 1;43(5):837-43. doi: 10.1016/j.freeradbiomed.2007.05.034. Epub 2007 Jun 6.
Oxidation of RNA precursors may disturb genetic information. In this study, the effects of oxidized RNA precursors on in vitro transcription were examined. Two oxidized ribonucleoside triphosphates, 8-hydroxyguanosine 5'-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP), were added to in vitro transcription reactions. The addition of 8-OH-GTP and 2-OH-ATP reduced the amount of RNA synthesized in vitro. Moreover, to examine qualitative alteration of the mRNA, it was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA, and the DNA sequence was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting the disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T-->G plus T-->C mutations, and 2-OH-ATP caused T-->C mutations. These results indicate that the formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively.
RNA前体的氧化可能会干扰遗传信息。在本研究中，检测了氧化的RNA前体对体外转录的影响。将两种氧化的核糖核苷三磷酸，即8-羟基鸟苷5'-三磷酸（8-OH-GTP）和2-羟基腺苷5'-三磷酸（2-OH-ATP）添加到体外转录反应中。添加8-OH-GTP和2-OH-ATP会减少体外合成的RNA量。此外，为了检测mRNA的定性改变，通过逆转录酶将其转化为cDNA，然后通过PCR扩增该cDNA。随后将PCR产物克隆到质粒DNA中，并对每个细菌菌落的DNA序列进行分析。这两种氧化的核糖核苷酸在cDNA中诱导了突变，表明在转录和/或逆转录过程中遗传信息受到了干扰。8-OH-GTP诱导T→G加T→C突变，而2-OH-ATP导致T→C突变。这些结果表明，细胞中这些氧化的RNA前体的形成在数量和质量上都会影响转录。
