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

研究的智力价值该研究项目是对185/333基因和表达蛋白的调查，这是一个庞大且高度多样化的家族，具有假定的先天免疫功能。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。免疫多样化在真核宿主及其病原体之间的军备竞赛中是必不可少的，对于长寿宿主来说，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免疫基因组装，而无脊椎动物采用了几种新的机制。了解185/333系统的多样性和多样化是这个项目的中心目标。这些基因（约有50个紧密聚集的基因座）在两个外显子中的第二个外显子内编码了大部分序列多样性。海胆之间没有共享的基因序列，个体动物的基因和mrna之间只有少数序列匹配。根据序列的变化，有三种类型的5'侧翼区域（假定的顺式启动子）。据推测，它们调节不同病原体相关分子模式（PAMPs）反应中观察到的差异基因表达。同样，在不同的海胆中表达不同的185/333蛋白组，并对不同的PAMPs做出反应。分子量和等电点范围比推导出的氨基酸序列预测的要宽得多。这一系统的多样化可以在几个层面上起作用；基因表达受到调控，显著的基因重组增加了基因多样性；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。史密斯将研究185/333体系的多样化。“完成”水平的基因组序列约280kB，编码多达55个平行的185/333等位基因，将为制定作用于该基因家族的多样化和/或重组机制的假设提供数据。185/333基因在单个体腔细胞中是否存在表达限制，我们将使用单细胞PCR进行研究。将测试185/333蛋白的抗菌活性和参与细胞聚集和封装的功能，并建立海胆免疫细胞的基因敲低系统。由于棘皮动物和脊索动物之间的密切关系，海胆免疫系统将对推断动物后口动物谱系的免疫进化具有重要意义。研究产生的广泛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是静态的。这一变化是由先天免疫反应中复杂多样化机制的确定所推动的，包括在前一个奖励期间发表的185/333系统多样性的结果。该系统的未来研究将为博士后、研究生、本科生、技术人员和当地高中生提供项目。妇女和来自代表性不足群体的人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。研究与教学的整合将包括一个生物信息学项目，让细胞生物学专业的本科生参与对海胆基因组的注释，其中56%的基因模型尚未被注释。学生专题的结果将上传到注释网站，并将公开提供。

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.