Wilen 1 Craig Wilen Professor Elgin Bio 4342 May 2, 2006 Annotation of 7G17 Overview Figure 1. Map of 7G17 Fosmid 7G17 contains the partial sequence of only one gene, unc-13. This protein, critical in neurotransmitter release, is highly conserved between drosophila and mammals suggesting its important role in the nervous system. 37,937bp of the 39,270bp gene representing 20 of 21 exons are located on 7G17. The 40,389bp fosmid contains 45 repetitive elements comprising 27.5% of the entire fosmid including 4 putative novel repeats. Retroelement family repeats and DNA element repeats make up 14.1% and 4.5% of the fosmid, respectively.Wilen 2 Gene I began the annotation of 7G17 by analyzing the Genscan output (Figure 2). Figure 2. Genscan output of 7G17 The Genscan data predicts that 7G17 contains three genes with the second gene in opposite orientation. All predicted gene features were then compared to D. melanogaster by using Blat on the UCSC Genome Browser. Predicted gene feature 1 corresponds to part of the N-terminus of unc-13. The output showing the position of unc-13 on chromosome 4 of D. melanogaster as well as its alignment with 7G17 is shown below in Figure 3.Wilen 3 Figure 3. Predicted gene region 1. Predicted gene region 2 was similarly analyzed. The results shown below in Figure 4 show that predicted gene region 2 matches to an unordered chromosome comprised of all unassembled D. melanogaster contigs. Thus, it was determined the predicted gene region 2 was not a gene. Figure 4. Blat hits of predicted gene region 2 Predicted gene region 3 also aligned to unc-13 in D. melanogaster. The results are shown below in Figure 5. Figure 5. Blat results from predicted gene region 3 against D. melanogaster Thus, the Blat data suggests that only one gene is present in 7G17 and that the genes and exons predicted by Genscan are incorrect. This was confirmed by using Blastn to compare the predicted gene regions on 7G17 with all known sequences. PredictedWilen 4 gene regions 1 and 3 had highly significant hits to D. melanogaster unc-13 (e-value less than 10^-5). Predicted gene region 2 had no significant hits (e-value greater than 10^-5). Thus, both the Blat and Blast data confirm that 7G17 contains only one gene. Fosmid 7G17 contains the partial sequence of only unc-13. This highly conserved and exclusively neural 200kD protein participates in the regulation of neurotransmitter release. The chemical messengers that transmit information from the pre-synaptic neuron to the post-synaptic neuron are neurotransmitters. The exocytotic release of neurotransmitters from the pre-synaptic neuron is tightly regulated, and the cellular machinery, including the SNARE complex and unc-13, needed for such release is extraordinarily conserved. Unc-13, short for uncoordinated mutant 13, was first discovered by classical mutagenesis studies that screened for uncoordinated movements in C. elegans. Despite the viability of unc-13 knockouts in C. elegans, the gene is essential in both drosophila and mice. It was later learned that unc-13 was essential for proper synaptic vesicle fusion. Although the precise function of unc-13 remains elusive, the C-terminus of the protein is known to interact directly with syntaxin, a key component of the SNARE complex. It is thought that unc-13 may bind calcium and the internal messenger diacylglycerol (DAG) as part of its role in the neuron, but more research is needed to identify its precise mechanism of action. Three unc-13 isoforms have been detected in D. melanogaster; however, only isoform A, the largest of the three, was annotated here. The variations between isoforms can be seen below in Figure 6. The differences between isoforms of the large N-terminalWilen 5 exons at 910kb and 920kb are irrelevant to the annotation of 7G17 because the sequence of these exons is not fully contained in 7G17. Figure 6. The three isoforms of unc-13 from D. melanogaster To annotate unc-13, Blastx (filter off) was used to compare the entire 40kb of 7G17 with the amino acid sequence of each of the 21 unc-13 isoform-a exons from D. melanogaster. Only 1,060bp of exon 1 are contained in 7G17. The initial 1,333bp of the exon extend beyond my fosmid. However, since the complete exon 1 was annotated last year, we have now completely annotated unc-13. Exon 2 from D. melanogaster is the first complete exon contained in 7G17. A detailed description of its annotation, which follows, serves as a representative example for each of the 20 other exons annotated. Ensembl was used to extract the peptide sequence of exon 2 from D. melanogaster (NP_726614). This sequence was then compared to 7G17 by Blastx. The output is below in Figure 7. Figure 7. Blastx output of 7G17 and exon 2.Wilen 6 Next, to define the exact intron/exon boundaries for exon 2, the DNA sequence immediately adjacent to 24,029bp and 24,160bp was extracted by using “Get DNA” from 7G17 on the goose.wustl.edu site. The AG end of intron 1 was located at 24,027-8bp and the GT beginning of intron 2 was located at 24,161-2bp. Exon 2 has no phase shift, and its boundaries are 24,029 and 24,160. This method was successfully used to identify the exon boundaries of all 21 exons. The high conservation and limited number of insertions and deletions in unc-13 facilitated this analysis by reducing the search time for AG and GT splice sites. The same analysis on a less conserved protein may have been significantly more challenging and less accurate. Finally, the 5’ UTR extends beyond 7G17 and the conservation of the 3’ UTR was too low to produce significant Blast hits; thus, neither can be reported on here. Clustal Analysis1 ClustalW was used to align the region of unc-13 contained in 7G17 from D. virilis, with D. mojavensis, D. melanogaster, Homo sapien (human), Canis familiaris (dog), and Mus musculus (mouse). Blast was used to align the translated partial D. virilis sequence with the genbank nr database. The D. melanogaster, human, dog, and mouse sequences were then extracted. The D. mojavensis sequence was found on the UCSC genome browser under gene mapper predictions (NM_143692). Unc-13 is highly conserved across all analyzed organisms as expected due to its important role in neurotransmitter release. The most highly conserved region lies between D. virilis residues 14 and 836. Although 7G17 does not contain the N-terminus 1 No promoter region was present on 7G17, so