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

研究的智力价值研究项目是对185/333基因和表达的蛋白质的调查，这是一个高度多样化的大家庭，推测具有先天免疫功能。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。在真核宿主和病原体之间的军备竞赛中，免疫多样化是必不可少的，对于长寿的宿主来说，这需要基因多样化的分子机制。脊椎动物使用免疫基因的体细胞重排或组装，而无脊椎动物使用了几种新的机制。了解185/333系统的多样性和多样性是该项目的中心目标。这些基因(有50个紧密聚集的座位)编码了两个外显子中的第二个内含子内的大部分序列多样性。海胆之间没有共享的基因序列，只有几个基因与单个动物的mRNAs序列匹配。根据序列变异，有三种类型的5‘侧翼区(推定的顺式启动子)。它们可能调节不同病原体相关分子模式(PAMPs)所观察到的差异基因表达。同样，185/333蛋白的不同组合在不同的海胆中表达，并对不同的PAMP做出反应。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。这个系统的多样化可能在几个层面上起作用：受调控的基因表达，显著的基因重组，增加基因多样性；转录和/或转录后对mRNAs的低保真度修饰，以及对蛋白质的翻译后修饰。史密斯将调查185/333系统的多样化。~280kB水平的基因组序列编码多达55个平行的185/333等位基因，这将为提出作用于该基因家族的多样化和/或重组机制的假说提供数据。用单细胞聚合酶链式反应研究185/333基因在单细胞体腔细胞中的表达是否受到限制。将测试185/333蛋白的抗菌活性以及参与细胞聚集和包裹的能力，并将建立海胆免疫细胞的基因敲除系统。由于棘皮动物和脊索动物之间的密切关系，海胆的免疫系统对于推断动物后肢动物谱系内的免疫进化将是重要的。研究产生的更广泛的影响无脊椎动物免疫学领域的新范式表明，无脊椎动物的免疫反应既不简单也不静态。这一变化是由先天免疫反应中复杂的多样化机制的确定推动的，包括在前一个获奖期间发表的关于185/333系统多样性的结果。未来对该系统的研究将为博士后研究员、研究生、本科生、技术员和当地高中生提供项目。妇女和来自代表性不足群体的妇女将继续被招募到实验室，参与该项目的各个方面。学生将接受多学科的培训，要求熟悉免疫学、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、进化论、系统学和无脊椎动物动物学，包括海胆发育、解剖学和生态学。研究与教学的结合将包括一个生物信息学项目，供招收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。学生专题的结果将被上传到注释网站，并将向公众开放。

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.