Indexed on: 26 May '04Published on: 26 May '04Published in: Insect Molecular Biology
Abstract Using RT-PCR, we examined expression of the ribonucleotide reductase R2 subunit (RNR-R2) in Aedes albopictus mosquito cells after treatment with ultraviolet light (UV). In control cells, a predominant band at 1.2 kb corresponded to the full-length cDNA. A smaller 650 bp band was unique to UV-treated cells. Sequence analysis showed that the 650 bp band encoded a protein with an internal deletion of 179 amino acids, relative to Ae. albopictus RNR-R2. The N-terminal twenty amino acids were identical between AalRNR-R2 and AalDeltaR2; downstream of the deletion, the proteins differed at only four residues. In AalDeltaR2, the internal deletion spanned five residues critical to RNR-R2 enzymatic activity, including a key tyrosine residue that generates an essential free radical. The full-length 46 kDa and truncated 25 kDa RNR-R2 proteins were shown to be expressed on Western blots, and to differ in their subcellular localization. Similarly, expression of the two proteins was differentially regulated during the cell cycle, and expression of AalDeltaR2 predominated after UV treatment. AalDeltaR2 resembled a human RNR-R2 variant called p53R2, which was induced by agents that damage DNA. As was the case with p53R2 and its antisense RNA, levels of AalDeltaR2 were diminished after treatment of mosquito cells with RNAi corresponding to p53 from Drosophila melanogaster. Examination of the AalRNR-R2 homologue in the Anopheles gambiae genome suggested that AalDeltaR2 resulted from precise splicing between Exons 1, 4 and 5, eliminating Exons 2 and 3. The likelihood that AalDeltaR2 is a non-enzymatic, functional participant in DNA metabolism is suggested by enhancement of DNA repair in an in vitro system and by the presence of a similar gene (rnr4) in yeast.