OCR Text |
Show UNDERGRADUATE RESEARCH ABSTRACTS SPRING 2007 D. Spencer Mangum Chi-Bin Chien 47 Alternate Splice Forms of the robo2 Axon Guidance Receptor Gene: Evolutionary Conservation and Developmental Regulation D. Spencer Mangum (Chi-Bin Chien) Department of Neurobiology & Anatomy University of Utah Robo2 is an axon guidance receptor found in the growth cones of developing axons. Unpublished work from Dr. Chi-Bin Chien’s laboratory suggested that five different exons are alternatively spliced in robo2. To ascertain if these splice forms are functionally significant, we determined if 1) the alternate splice forms are evolutionary conserved between zebrafish, human, and mouse, 2) the alternate splice forms are expressed at specific times during development, and 3) the alternate splice forms are expressed in specific tissues. First, we decided to confirm that the five putative exons are alternatively spliced. Performing reverse tran-scriptase polymerase chain reactions (RT-PCR) on cDNA isolated from 48 hours post fertilization (hpf) whole zebrafish embryos we found that exons 7, 19, and 23 are indeed alternatively spliced. However, we were not able to confirm as to whether exons 29 and 31 are alternatively spliced. To determine if a given splice form is evolutionary conserved, we used expressed sequence tag (EST) data from the National Center for Biotechnology Information (NCBI) for both human and mouse robo2, and analyzed them using the computer program Sequencher (Ann Arbor, MI). Four human EST’s, and one mouse EST indicated that exon 7 is alternatively spliced, thus satisfying the first criteria for functional significance. However, EST data showed that neither exon 19 nor exon 23 were alternatively spliced. To find out if the splice forms were expressed in a time specific fashion, we isolated mRNA from 24, 36, 48 and 72 hpf whole embryos and performed RT-PCR. Preliminary data suggests that exon 7 is alternatively spliced in a time specific manner. Moreover, we found that Exon 19 and 23 are not expressed in a time specific manner. To determine if a splice form is expressed specifically in the eye, we removed both eyes from approximately 20 different 48 hpf embryos, isolated the mRNA and analyzed it by RT-PCR. We found that exon 23 is always missing from the eye, while results for both exons 7 and 19 were inconclusive. In summary, we have shown that exon 7 of robo2 is alternatively spliced in human and mouse, and have preliminary data suggesting that this exon is expressed in a time specific manner. Additionally, we found that exon 23 is missing from eyes of 48hpf zebrafish embryos, demonstrating that this splice form is required tissue specifically. |