| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 97 === DNase II, an acidic DNase, is known to play a prominent role in digestion of DNA from apoptotic cells across a wide spectrum of animals, including invertebrates and mammals. In contrast to two DNase II α and β, in human, there are three homologous DNase II genes, nuc-1, crn-6 and crn-7, in C. elegans. By using the metachromatic agar-diffusion assay (MADA), I first characterized embryonic extracts from N2, and three single mutants (nuc-1, crn-6, or crn-7) that exhibited the optimal pH at 4.5 and temperature at 25℃ for DNA digestion, indicating that NUC-1, CRN-6, and CRN-7 are likely the acid DNase II in worms. This observation was further supported by the result of a triple mutant (crn-7; crn-6; nuc-1) which showed no DNA digestion activity. MADA results of the L4 larvae and embryonic extracts’ DNase II activity from three single mutants and three double mutants (crn-7; nuc-1, crn-6; nuc-1 and crn-7; crn-6) indirectly showed that NUC-1 had the highest DNase II activity while CRN-6 and CRN-7 exhibited 10 fold less activity. To define NUC-1 expression pattern in embryos, transgenic worms expressing NUC-1::GFP were observed under a fluorescence microscope and did not show GFP signals, suggesting that NUC-1::GFP was not accumulated in lysosomes but secreted. To test this hypothesis, I fused NUC-1 with the C-terminus of LMP, which has a membrane domain and remains in lysosomes, followed by GFP. Transgenic worms bearing this construct showed GFP signals in early embryos, supporting the idea that majority of functional NUC-1 is in secreted form. Unexpectedly, in a rescue assay to examine ventral cords DNA staining, the lysosome-bound version of NUC-1 almost digests DNA effectively as wild-type one. Accordingly, NUC-1 appears to also function in non-secreted fashion as executing DNA degradation in ventral cords. This result raises a scenario that NUC-1 expresses autonomously in dying cells as well as being secreted by the engulfing cells during the apoptotic DNA degradation process.
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