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

从基因组到转录组再到发育：文昌鱼基因组 - 了解基因复制和选择性剪接在脊索动物发育中的作用的关键

• 批准号: 0620019
• 负责人: Linda Holland
• 金额: $ 32.3万
• 依托单位: University of California-San Diego Scripps Inst of Oceanography
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-10-01 至 2009-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0620019&HistoricalAwards=false
• 关键词: genome; transcriptome; development; amphioxus; key

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.

基底脊椎动物（佛罗里达山枝）是脊椎动物样的，具有分割的近视肌肉，脊索，背部空心神经索和咽g的缝隙，但在结构和基因组上较简单，基因组只有500 mb，没有配对的眼睛或耳朵。两栖动物具有大多数脊椎动物基因的同源物。即便如此，在脊椎动物中被缩放的基因也倾向于在两栖焦油中被缩放。 Amphioxus缺乏脊椎动物的广泛基因重复特征，因此适合理解替代剪接和基因重复在增加蛋白质组多样性中的相对作用。这项研究的重点是脊椎动物中与单独的PAX2，PAX5和PAX8基因同源的单一二苯二氧化基因。迄今为止，已经确定了四个Amphioxus Pax2/5/8的同工型。这项研究利用了PI实验室与京都大学的日本同事，联合基因组研究所（JGI）和Oakland儿童医院的BAC/PAC中心合作，利用了PI实验室最近进行的大型EST项目。所有DNA库都可以公开使用，并且基因组的注释正在进行中。 EST数据和PCR将用于识别Amphioxus Pax2/5/8的剪接的同工型。 EST数据对于验证基因组分析预测的哪些替代剪接形式是真正的mRNA。 此外，PCR将使用Lambda噬菌体中的cDNA文库作为模板或从各种发育阶段和成人的RNA开始对替代切割形式进行验证。发育过程中同工型的相对表达水平将由qPCR确定。体外研究将测试所选同工型的结合和反式激活特性，并将通过用反义的莫菲利诺寡核苷酸敲入基因功能来测试体内的功能，该功能跨越了内含子/外显子剪接位点。了解两栖动物中基因复制和替代性的程度及其在增加蛋白质组多样性中的相对作用将以基本的基础来阐明基因组如何演化以创建蛋白质，从而创建蛋白质，从而构成尤其是chordate胚胎的蛋白质，尤其是脊椎动物，包括脊椎动物，包括脊椎动物，一般而言。 人基因组有大约25,000个基因。然而，据估计，通过替代剪接的机制，超过40％或更多的人类基因代码为几种不同的蛋白质代码，其中从给定基因转录的使者RNA的模块以不同的组合拼合在一起，每种组合都会转化为不同的蛋白质。这项研究的目的是阐明替代剪接在增加蛋白质多样性中的作用，又阐明了基因复制与替代替代性之间的关系。对于这个项目，研究者将使用基底弦舌二氧化碳，这与脊椎动物的弦弦相似，但在基因组上更简单，因为它缺乏脊椎动物的广泛基因重复特征。有关替代剪接和两栖基因的基因重复的所有数据将作为可搜索的数据库公开获得。这项研究将为一个博士后研究员提供培训。 与研究者过去的NSF支持研究一样，它也将用于为本科生提供动手培训，其中一些人有望共同撰写研究论文。 将继续向学生，尤其是女性和拉丁美洲的女性推广。