Characterization of Two Novel Related Drosophila Nucleases

描述（由申请人提供）：我们小组和其他人的工作表明，DNase II酶对吞噬后摄入细胞DNA的降解至关重要。我的研究小组克隆了果蝇DNA酶II基因，这使我们能够进一步研究与这种酶的生物学功能有关的问题。使用RNA干扰和可用的DNase II缺陷系，我们已经证明，DNase II缺陷导致对细菌感染的易感性增加。为了确定DNA酶II缺陷型苍蝇在其他过程（例如吞噬、抗菌反应等）中是否存在缺陷，我们对感染和未感染的对照和DNase II缺陷果蝇进行了一系列全基因组表达微阵列分析。这些阵列分析的一个意想不到的结果是在细菌感染后检测到正常的抗菌肽（AMP）反应，该反应应该保护受感染的苍蝇免受感染。由于DNase II缺陷果蝇的体液和细胞反应明显正常，因此分析了其他基因的失调。我们的微阵列结果显示，大量基因（n=42个基因，p=0.05）的表达受到感染前DNase II耗竭的影响。有趣的是，在DNase II缺陷的果蝇中，发现一个最高上调的开放阅读框（CG 33346）编码一种新的非特异性核酸内切酶。我们将这种核酸内切酶命名为DNase III，属于进化上保守的核酸酶家族，其包括参与非胱天蛋白酶依赖性DNA降解的哺乳动物Endo-G蛋白。DNA酶III基因随后被克隆并引入原核表达载体，发现其编码真正的阳离子依赖性内切核酸酶。有趣的是，第二个高度同源的ORF，CG 9989（DNA酶III），被发现相邻的CG 33346基因，在先前的微阵列分析中检测到的基因上调细菌感染和寄生蜂入侵。我们最近的阵列分析显示，CG 9989的表达仅在革兰氏阳性细菌感染后上调2-3倍，而在革兰氏阴性细菌感染后没有上调。我们的数据表明，新的DNase基因的差异上调感染。在这个提议中，我们打算主要通过RNAi介导的基因缺失来确定DNase III酶的功能。这些实验也被设计来确定DNA酶III是否被上调以补偿DNA酶II表达的损失。 公共卫生相关性：发现了两种新型相关DNA降解酶，其基因被不同的刺激激活。其中一个基因在另一种核酸酶缺失时被激活，另一个基因在苍蝇被细菌感染时被激活。除了对这两种新型核酸酶的表征外，这项工作还可能导致在果蝇和其他生物体中发现一种新的DNA传感机制。

DESCRIPTION (provided by applicant): Work from our group and others has demonstrated that DNase II enzymes are essential for degradation of ingested cellular DNA after phagocytosis. The cloning of the Drosophila dnase II gene by my group has allowed us to pursue additional questions related to the biological function of this enzyme. Using RNA interference and available DNase II-deficient lines, we have demonstrated that DNase II-deficiency results in increased susceptibility to bacterial infection. To determine if DNase II- deficient flies were defective in other processes such as engulfment, antimicrobial response, etc., we performed a series of genome-wide expression microarray analyses of infected and uninfected control and DNase II-deficient flies. One unexpected result of these array analyses was the detection of a normal antimicrobial peptide (AMP) response after bacterial infection that should have protected the infected flies from infection. Due to the apparently normal humoral and cellular response of DNase II-deficient flies, the misregulation of other genes was analyzed. Our microarray results revealed that the expression of a significant number of genes (n=42 genes, p=0.05) was affected by the DNase II-depletion prior to infection. Interestingly, one of the highest up-regulated open reading frames (CG33346) in DNase II-deficient flies was found to encode a novel non-specific endonuclease. This endonuclease, which we have named DNase III, belongs to an evolutionarily conserved family of nucleases that includes the mammalian Endo-G protein involved in Caspase- independent DNA degradation. The DNase III gene was subsequently cloned and introduced into a prokaryotic expression vector and found to encode a bona fide cation-dependent endonuclease. Interestingly, a second highly homologous ORF, CG9989 (DNase III), was found adjacent to the CG33346 gene that was detected in prior microarray analyses as a gene up-regulated by bacterial infection and parasitic wasp invasion. Our recent array analyses have revealed that expression of CG9989 is up-regulated by 2-3 fold only after infection with Gram positive but not by Gram negative bacteria. Our data indicate that the novel DNase genes are differentially up-regulated by infection. In this proposal, we intend to determine the function of DNase III enzymes primarily via RNAi- mediated gene depletion. These experiments are also designed to determine if DNase III is up- regulated to compensate for the loss of DNase II expression. PUBLIC HEALTH RELEVANCE: Two novel related DNA degradation enzymes were discovered whose genes are activated by distinct stimuli. One of these genes is activated when another nuclease is absent and the other when flies are infected by bacteria. Apart from the characterization of the two novel nucleases, the proposed work can also lead to the discovery of a novel DNA sensing mechanism in fruit flies and perhaps other organisms.