RNases for understanding the origin of new gene function

描述（由申请人提供）：我实验室的长期目标是了解具有新功能的新基因是如何产生的，以及这些分子创新如何促进生物体的生存、适应和进化。虽然我们都知道去复制在新的去复制功能的进化中起着重要的作用，但对去复制基因功能变化的进化力量和分子机制仍然知之甚少。本文以哺乳动物的核糖核酸酶（RNase） A基因超家族作为模型系统来解决上述问题，因为(1)该超家族包括许多最近重复的具有不同功能的基因，(2)这些功能可以相对容易地在体外进行分析，具有分析由位点定向诱变产生的突变蛋白的可行性，(3)该超家族成员参与免疫和癌症，并与人类健康有关，(4)大量的生化，在超族中可以获得结构和功能信息。人类已知的超家族有8个成员，分别是胰腺RNase（或RNase 1）、嗜酸性粒细胞衍生神经毒素（EDN或RNase 2）、嗜酸性粒细胞阳离子蛋白（ECP或RNase 3）、RNase 4、血管生成素（或RNase 5）、RNase k6（或RNase 6）、RNase 7和RNase 8。作为酶，它们都可以切割RNA中的磷酸二酯键。但它们也进化出其他功能，并参与各种生理过程，包括消化膳食rna、血管生成和宿主防御。我们的具体目标是：(1)通过对高等灵长类动物分别具有抗病毒和抗菌活性的EDN和ECP重复基因重建的共同祖先基因的功能表征，验证两种相互竞争的新基因功能起源理论；(2)鉴定ECP中负责抗菌功能的氨基酸取代；(3)鉴定导致EDN具有强效核糖核酸酶活性和抗病毒活性的氨基酸取代；(3)研究最近在人类中出现的RNase 7基因的杀菌等位基因形式，并测试其在人类健康和进化中的潜在作用；(5)研究类人猿RNase 8中二硫键桥接的演化及二硫键改变对活性的功能影响；(6)研究灵长类动物血管生成快速进化的原因，确定血管生成素中血管生成活性所需的关键氨基酸残基。

DESCRIPTION (provided by applicant): The long-term objective of my laboratory is to understand how new genes with novel functions originate and how these molecular innovations contribute to the survival, adaptation, and evolution of organisms. Although it is well known that gone duplication plays an important role in the evolution of novel gone functions, the evolutionary forces and molecular mechanisms underlying functional changes of duplicated genes remain poorly understood. Here the ribonuclease (RNase) A gene superfamily of mammals is used as a model system to address the above questions, because (1) the superfamily includes many recently duplicated genes with distinct functions, (2) these functions can be assayed relatively easily in vitro, with the feasibility of analyzing mutant proteins generated by site-directed mutagenesis, (3) members of the superfamily are involved in immunity and cancer and are related to human health, and (4) substantial biochemical, structural, and functional information is available on the superfamily. Eight members of the superfamily are known in humans, and they are pancreatic RNase (or RNase 1), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil cationic protein (ECP or RNase 3), RNase 4, angiogenin (or RNase 5), RNase k6 (or RNase 6), RNase 7, and RNase 8. As enzymes, all of them can cleave phosphodiester bonds in RNA. But they have also evolved other functions and are involved in various physiological processes including digestion of dietary RNAs, angiogenesis, and host defenses. Our specific aims are (1) to test two competing theories on the origin of new gene function by functional characterization of the reconstructed common ancestral gene of the duplicated EDN and ECP genes of higher primates, which have antiviral and antibacterial activities, respectively; (2) to identify amino acid substitutions responsible for the antibacterial function of ECP; (3) to identify amino acid substitutions that led to the potent ribonuclease activity and antiviral activity in EDN; (3) to investigate the recent emergence of a bactericidal allelic form of the RNase 7 gene in humans and to test its potential role in human health and evolution; (5) to study the evolution of disulfide-bridging in RNase 8 among hominoids and the functional consequences caused activity by changes in disulfide bonds; and (6) to study the cause of the rapid evolution of primate angiogenic and to identify key amino acid residues necessary for the angiogenic activity in angiogenin.