From genome to transcriptome to development: the amphioxus genome - a key to understanding the roles of gene duplication and alternative splicing in chordate development

文昌鱼(Branchiostoma Floridae)是脊椎动物，具有节段性的旁轴肌、脊索、背侧中空神经索和咽鳃裂隙，但结构和基因组更简单，仅有500Mb的基因组，没有成对的眼睛或耳朵。文昌鱼与大多数脊椎动物的基因具有同源性。即便如此，在脊椎动物中交替剪接的基因在文昌鱼中也往往是交替剪接的。文昌鱼缺乏脊椎动物所特有的广泛的基因复制，因此适合于理解选择性剪接和基因复制在增加蛋白质组多样性方面的相对作用。本研究以文昌鱼Pax2/5/8基因为研究对象，分离脊椎动物Pax2、Pax5和Pax8基因。到目前为止，已经确定了文昌鱼2/5/8的四种亚型。这项研究利用了文昌鱼基因组项目和一个大型EST项目，该项目最近由PI的实验室与京都大学的日本同事、联合基因组研究所(JGI)和奥克兰儿童医院的BAC/PAC中心合作完成。所有DNA文库都是公开可用的，基因组的注释正在进行中。EST数据和聚合酶链式反应将用于鉴定文昌鱼Pax2/5/8的选择性剪接异构体。EST数据对于验证基因组分析预测的选择性剪接形式中哪些是真实的mRNAs将是有价值的。此外，选择性剪接形式的验证将通过使用Lambda噬菌体中的cDNA文库作为模板的聚合酶链式反应或从不同发育阶段和成体的RNA开始的RT聚合酶链式反应进行。异构体在发育过程中的相对表达水平将由定量聚合酶链式反应确定。体外研究将测试选定异构体的结合和反式激活特性，体内功能将通过跨越内含子/外显子剪接点的反义吗啉寡核苷酸下调基因功能来测试。了解文昌鱼基因复制和选择性剪接的程度及其在增加蛋白质组多样性中的相对作用，将有助于揭示基因组如何进化来创造蛋白质的基本基础，这些蛋白质反过来又塑造文昌鱼胚胎和包括脊椎动物在内的一般脊索性胚胎。人类基因组大约有25,000个基因。然而，据估计，超过40%或更多的人类基因通过选择性剪接机制编码几种不同的蛋白质，在这种机制中，从给定基因转录的信使RNA的模块以不同的组合拼接在一起，每个组合翻译成不同的蛋白质。本研究的目的是阐明选择性剪接在增加蛋白质多样性中的作用，并阐明基因复制和选择性剪接之间的关系。在这个项目中，研究人员将使用基本的脊索动物文昌鱼，它与脊椎动物的同伴相似，但基因组更简单，因为它缺乏脊椎动物的广泛基因复制特征。所有关于文昌鱼基因的选择性剪接和基因复制的数据都将作为可搜索的数据库公开提供。这项研究将为一名博士后研究员提供培训。与研究人员过去由NSF支持的研究一样，它也将被用于为本科生提供实践培训，其中一些人有望与人共同撰写研究论文。将继续向学生，特别是妇女和来自拉丁美洲的学生提供服务。

The basal chordate amphioxus (Branchiostoma floridae) is vertebrate-like, with segmented paraxial muscles, notochord, dorsal hollow nerve cord and pharyngeal gill slits but structurally and genomically simpler with a genome of only 500 Mb and no paired eyes or ears. Amphioxus has homologs of most vertebrate genes. Even so, genes that are alternatively-spliced in vertebrates also tend to be alternatively-spliced in amphioxus. Amphioxus lacks the extensive gene duplications characteristic of vertebrates and is, thus, appropriate for understanding the relative roles of alternative splicing and gene duplication in increasing diversity of the proteome. This research focuses on the single amphioxus Pax2/5/8 gene, homologous to separate Pax2, Pax5 and Pax8 genes in vertebrates. To date, four isoforms of amphioxus Pax2/5/8 have been determined. This study takes advantage of the amphioxus genome project and a large EST project recently done by the laboratory of the PI in collaboration with Japanese colleagues at Kyoto University, The Joint Genome Institute (JGI) and the BAC/PAC center at the Children's Hospital Oakland. All DNA libraries are publicly available, and annotation of the genome is underway. EST data and PCR will be used to identify alternatively spliced isoforms of amphioxus Pax2/5/8. The EST data will be valuable for verifying which of the alternative-splice forms predicted from genome analysis are true mRNAs. In addition, verification of alternative-splice forms will be done by PCR using cDNA libraries in lambda phage as templates or by rtPCR starting with RNA from various developmental stages and adults. The relative level of expression of isoforms during development will be determined by QPCR. In vitro studies will test binding and transactivation properties of selected isoforms, and function in vivo will be tested by knock-down of gene function with antisense morpholino oligonucleotides that span intron/exon splice sites. Understanding the extent of gene duplication and alternative-splicing in amphioxus and their relative roles in increasing proteome diversity will shed light on the fundamental basis of how the genome has evolved to create proteins that in turn shape the amphioxus embryo in particular and chordate embryos, including vertebrates, in general. The human genome has approximately 25,000 genes. However, it has been estimated that over 40% or more of human genes code for several different proteins by the mechanism of alternative splicing in which the modules of the messenger RNA that is transcribed from a given gene are pieced together in different combinations, each of which translates into a different protein. The goal of this research is both to elucidate the role of alternative splicing in increasing protein diversity and to clarify the relationship between gene duplication and alternative-splicing. For this project the investigator will use the basal chordate amphioxus, which is similar to its fellow chordates the vertebrates, but genomically simpler as it lacks the extensive gene duplications characteristic of vertebrates. All the data concerning alternative splicing and gene duplication of amphioxus genes will be publicly available as searchable databases. This research will provide training for one postdoctoral fellow. As with the investigator's past NSF-supported research, it will also be used to provide hands-on training to undergraduates, some of whom are expected to co-author research papers. Outreach to students, particularly women and those from Latin America, will be continued.