Diversity of the 185/333 System and Innate Immunity in the Purple Sea Urchin

该研究项目是对185/333个基因和表达蛋白的研究，这是一个具有假定先天免疫功能的大而高度多样化的家族。海胆基因组注释的一个令人惊讶的结果是发现免疫系统非常复杂。免疫多样化是真核宿主与其病原体之间的军备竞赛所必需的，对于长寿宿主，这需要基因多样化的分子机制。脊椎动物采用体细胞重排或组装免疫基因，而无脊椎动物采用几种新的机制。了解185/333系统的多样性和多样化是本项目的中心目标。基因（有~50个紧密聚集的基因座）编码两个外显子中第二个内的大部分序列多样性。海胆之间没有共同的基因序列，只有少数基因和来自个体动物的mRNA之间的序列匹配。基于序列变异，存在三种类型的5 '侧翼区（推定的顺式启动子）。它们调节不同病原体相关分子模式（PAMP）引起的差异基因表达。类似地，不同的185/333蛋白质组在不同的海胆中表达，并响应于不同的PAMP。分子量和等电点范围比从推导的氨基酸序列预测的要宽得多。该系统的多样化可能在几个层面上起作用;调节基因表达，增加基因多样性的显著基因重组;转录和/或mRNA转录后修饰的低保真度，以及蛋白质的翻译后修饰。Smith博士将研究185/333系统的多样化。编码多达55个旁系同源185/333等位基因的~280kB的"精加工"水平基因组序列将提供数据以制定关于作用于该基因家族的多样化和/或重组机制的假设。将研究使用单细胞PCR是否185/333基因在单个体腔细胞中显示任何表达限制。将测试185/333蛋白质的抗微生物活性和参与细胞聚集和封装的功能，并将为海胆免疫细胞建立基因敲低系统。由于棘皮动物和脊索动物之间的密切关系，海胆免疫系统将是重要的推断免疫进化的动物的后口谱系。更广泛的影响，从研究无脊椎动物免疫学领域的新范式指出，无脊椎动物的免疫反应既不是简单的，也不是静态的。这一变化是由先天免疫反应中复杂多样性机制的鉴定所驱动的，包括在前一个奖项期间发表的185/333系统多样性的结果。未来的研究，这个系统将提供项目的博士后研究员，研究生，本科生，技术人员，和当地的高中生。妇女和代表性不足的群体将继续被招募到实验室参与项目的各个方面。学生将接受多学科方法的培训，需要熟悉免疫学，分子生物学，细胞生物学，生物化学，微生物学，基因组学，蛋白质组学，生物信息学，进化，系统学和无脊椎动物学，包括海胆发育，解剖学和生态学。研究和教学的整合将包括一个生物信息学项目，供就读于细胞生物学的本科生参与注释海胆基因组，其中56%的基因模型尚未注释。学生项目的结果将被上传到注释网站，并将成为可访问的。

Intellectual merit of the researchThe research project is an investigation of the 185/333 genes and expressed proteins, which is a large and highly diverse family with putative innate immune functions. One of the surprising results from the sea urchin genome annotation was the discovery that the immune system is very complex. Immune diversification is essential in the arms race between eukaryotic hosts and their pathogens, which, for long-lived hosts, requires molecular mechanisms for gene diversification. Vertebrates employ somatic rearrangement or assembly of immune genes, whereas several novel mechanisms are employed by invertebrates. Understanding the diversity and diversification of the 185/333 system is the central aim of this project. The genes (there are ~50 tightly clustered loci) encode most of the sequence diversity within the second of two exons. No gene sequence is shared among sea urchins, and there are only a few sequence matches between genes and mRNAs from individual animals. There are three types of 5' flanking regions (the putative cis promoters) based on sequence variations. They putatively regulate differential gene expression observed in response to different pathogen associated molecular patterns (PAMPs). Similarly, different suites of 185/333 proteins are expressed in different sea urchins and in response to different PAMPs. Molecular weight and iso-electric point ranges are much broader than predicted from deduced amino acid sequences. Diversification of this system may act on several levels; regulated gene expression, significant gene recombination that increases gene diversity; low fidelity of transcription and/or post transcriptional modifications to the mRNAs, and post translational modifications to the proteins.Dr. Smith will investigate the diversification of the 185/333 system. "Finishing" level genomic sequence of ~280kB encoding up to 55 paralogous 185/333 alleles will provide data to formulate hypotheses on the diversification and/or recombination mechanisms that act on this gene family. Whether the 185/333 genes show any restriction in expression in single coelomocytes using single cell PCR will be investigated. 185/333 protein function will be tested for antimicrobial activity and involvement in cell aggregation and encapsulation, and a gene knockdown system will be established for sea urchin immune cells. Because of the close relationship between echinoderms and chordates, the sea urchin immune system will be important for deducing the evolution of immunity within the deuterostome lineage of animals.Broader impacts resulting from the researchThe new paradigm in the field of invertebrate immunology states that invertebrate immune responses are neither simple nor static. This change has been driven by the identification of complex diversification mechanisms in innate immune responses, including results on the diversity of the 185/333 system published during the prior award period. Future research on this system will provide projects for postdocdtoral fellows, graduate students, undergraduates, technicians, and local high school students. Women and those from underrepresented groups will continue to be recruited to the lab to participate in various aspects of the project. Students will be trained in a multidisciplinary approach that requires familiarity with immunology, molecular biology, cell biology, biochemistry, microbiology, genomics, proteomics, bioinformatics, evolution, systematics and invertebrate zoology including sea urchin development, anatomy and ecology. Integration of research and teaching will include a bioinformatics project for undergraduates enrolled in Cell Biology to participate in annotating the sea urchin genome, of which 56% of the gene models have not been annotated. Results from student projects will be uploaded into the annotation website and will become publically available.