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.
