摘要：

DNase II is an acidic DNase which plays an important role in the degradation of apoptotic cell DNA from a wide spectrum of animals. In C. elegans, three DNase II genes, nuc-1, crn-6 and crn-7, are identified, in contrast two DNase II (α and β) are found in mammals. There are three waves of cell apoptosis, embryonic, larval stage and oogenesis during C. elegans development. The role of nuc-1 and crn-6 in embryonic and larval development have been demonstrated; however enzymatic activities of three DNase II remain obscure. Here, we report three methods for detecting NUC-1, CRN-6 and CNR-7 activities in vitro and in vivo. First, using metachromatic agar-diffusion assay (MADA), DNase II activity in embryonic extracts from wild-type and mutant animals were examined. Results showed that NUC-1 had the highest DNase II activity while CRN-6 and CRN-7 exhibited 10 fold lower activity than NUC-1. Second, recombinant proteins of GST fusion with NUC-1, CRN-6 and CRN-7 were generated in E. coli. The enzymatic activity of purified DNase II fusion proteins were analyzed using non-denaturing PAGE. DNA digestion by NUC-1 and CRN-7 was demonstrated in gels, but little or no DNA digestion by CRN-6. Thirdly, a technique which directly labels the DNase II-type breaks with a fluorescent probe was applied to detect the DNA fragmentation in embryos. Results showed that the fluorescent signals in nuc-1 mutant are twice less than those in wild-type, crn-6 and crn-7 embryos. This result is consistent to the TUNEL assay in embryo as demonstrated before. In the future, the technique of direct libeling DNase II-type breaks can be employed to study roles of three DNase II in germ-line apoptosis. In preliminary data, DNase II-type breaks can not be generated in gonad in crn-7mutant, it implies that crn-7 palys a role in germ-line apoptosis.

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