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已经证明，DNase II酶对于吞噬后摄入的细胞DNA的降解是必不可少的。我的小组克隆果蝇dna酶II基因，使我们能够追求与这种酶的生物学功能相关的其他问题。利用RNA干扰和现有的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DNase II缺陷果蝇是否在吞噬、抗菌反应等其他过程中存在缺陷，我们对感染和未感染的对照以及DNase II缺陷果蝇进行了一系列全基因组表达芯片分析。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，这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，感染前DNase ii缺失影响了大量基因的表达（n=42个基因，p=0.05）。有趣的是，在dna酶ii缺陷的果蝇中，发现一个最高上调的开放阅读框（CG33346）编码一种新的非特异性内切酶。这种内切酶，我们将其命名为DNase III，属于进化上保守的核酸酶家族，包括参与Caspase非依赖性DNA降解的哺乳动物Endo-G蛋白。随后克隆了DNase III基因并将其导入原核表达载体中，发现该基因编码一个真正的阳离子依赖性核酸内切酶。有趣的是，第二个高度同源的ORF， CG9989 (DNase III)，被发现与CG33346基因相邻，CG33346基因在先前的微阵列分析中被检测到，是一个受细菌感染和寄生蜂入侵上调的基因。我们最近的阵列分析显示，只有革兰氏阳性菌感染后，CG9989的表达才会上调2-3倍，而革兰氏阴性菌则不会上调。我们的数据表明，新的dna酶基因受到感染的差异上调。在这个提议中，我们打算主要通过RNAi介导的基因耗竭来确定DNase III酶的功能。这些实验还旨在确定DNase III是否上调以补偿DNase II表达的缺失。

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.