FINDING NOVEL REAGENTS BY BIOINFORMATICS AND BIOCHEMISTRY

描述(由申请人提供)：该项目旨在寻找在分子生物学研究的许多方面都有用的新型限制性内切酶。其中一个特别的焦点将是能够识别修饰的DNA的限制性内切酶，特别是含有5-甲基胞嘧啶和5-羟甲基胞嘧啶的DNA，因为这种酶在表观遗传学研究中有许多潜在的应用。识别这些修饰碱基的酶已知存在于细菌和古细菌菌株中，在过去，许多酶是通过随机生化筛选发现的。在拟议的项目中，潜在新酶的发现将使用生物信息学技术，并利用目前已测序的大量原核生物基因组，包括完全测序的基因组和鸟枪序列。该项目将确定好的候选者，然后使用生化方法来表征他们。除了将从这一筛选中发现的任何有用的酶商业化外，所获得的关于所发现的限制性内切酶的生化功能的信息将对基因组注释具有无价的价值，并将对从事病原生物工作的研究人员具有实际意义。后者将是我们努力描述病原体中的限制性内切酶修饰系统的结果，并识别那些需要在转化之前通过先前的修饰而被敲除或绕过的系统。因此，这项研究的结果将对表观遗传学、发病机制和基因组注释领域产生直接的影响。 公共卫生相关性：通过该项目发现的工具应该会极大地提高和降低表观遗传学研究的成本。它还将增加我们对病原体编码的基因的了解，并促进对这些生物体的研究。

DESCRIPTION (provided by applicant): This project aims to find novel restriction enzymes that will be useful in many aspects of molecular biology research. One particular focus will be on restriction endonucleases that are able to recognize modified DNA, especially DNA containing 5-methylcytosine and 5- hydroxymethycytosine since such enzymes have many potential applications in epigenetics research. Enzymes recognizing these modified bases are known to occur in bacterial and archaea strains and in the past, many have been found by random biochemical screening. In the proposed project, the discovery of potential new enzymes will use bioinformatics techniques and take advantage of the large number of currently sequenced prokaryotic genomes, both completely sequenced genome and shotgun sequences. The project will identify good candidates and then use biochemical methods to characterize them. In addition to commercializing any useful enzymes that are found from this screen, the information gained about the biochemical function of the restriction enzymes discovered will be invaluable for genome annotation and will have practical implications for researchers working with pathogenic organisms. The latter will result from our efforts to characterize the restriction-modification systems in pathogens and identify those systems which need either to be knocked out or circumvented by prior modification prior to transformation. Thus, the results of this research will have immediate implications for the fields of epigenetics, pathogenesis and genome annotation. PUBLIC HEALTH RELEVANCE: The tools discovered through this project should greatly enhance and lower the cost of epigenetic research. It will also add to our knowledge of the genes encoded by pathogenic organisms and facilitate research into those organisms.