Wow — it was so easy to find the beginning of exon 1 in D. eugracilis using a blastn alignment. Why did that work so well, and will this approach work with all genes? Let’s look at the D. melanogaster sequence again, using a new tool in the Genome Browser to answer this question. 1. Open a new web browser window and go to the GEP UCSC Genome Browser Mirror at . 2. To navigate to the genomic region surrounding the sev gene in Drosophila melanogaster, select “D. melanogaster” under “Represented Species”, select “Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6)” under “D. melanogaster Assembly”, and then enter “sev” under the “Position/Search Term” field. Click on the “GO” button (Figure 4). Figure 4 Change the settings on the Genome Browser Gateway page to navigate to the sev gene in the D. melanogaster Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6) assembly. 3. This region on the X chromosome, denoted “chrX:11,071,441-11,086,299” contains the entire sev gene. The suffix “-RA” corresponds to the name of the isoform that is associated with the gene. Hence sev-RA corresponds to the A isoform of the sev gene. This gene only has one isoform. 4. The direction of the arrows in the intron indicates that the sev gene is on the minus strand. Click on the “reverse” button below the Genome Browser image to reverse complement the chrX sequence (Figure 5). Figure 5 Click on the “reverse” button to reverse complement the entire chrX sequence (red arrow). 5. To reset the display settings, click on the “hide all” button. Then, configure the display modes as follows: Under “Mapping and Sequencing” Tracks Base Position: full Click on the blue “Short Match” link Enter “TCAKTY” (i.e., the Initiator motif) into the “Short (2-30 base) sequence” text box Change the “Display mode” to “pack” Click on the “Submit” button Under “Gene and Gene Prediction” Tracks
Go back to the web browser tab with the GEP UCSC Genome Browser to examine the D. eugracilis sequence further. Enter “KB465191:113,830-114,429” into the “enter position or search terms” text box, and then click on the “go” button to navigate to the region surrounding the TSS of Sev Ensure that your browser is still configured as follows: Base Position: full, D. mel Transcripts: pack, modENCODE RNA-Seq Coverage: full, Match: pack Under “Mapping and Sequencing Tracks”, click on the blue “Short Match” link Enter the BREd consensus sequence RTDKKKK in the “Short (2-30 base) sequence” text box Click “Submit” Figure 8 UCSC Genome Browser view of the Short Match search results to the BREd motif consensus sequence The “Short Match” track shows there are multiple matches to the RTDKKKK sequence on both the plus and minus strands in this genomic region (Figure 8). Note that we expect the core promoter motifs to be in the same orientation as the direction of transcription. In addition, given the information in Table 2, we can see that BREd motifs are usually found at position -23 relative to the TSS in Drosophila. The location of the Inr motif and alignment with D. melanogaster sequence allow us to predict the TSS position. Let’s zoom into this region to investigate further. Enter “KB465191:114,064-114,196” into the “enter position or search terms” text box, and then click on the “go” button to zoom in closer to the beginning of exon 1. Q12. Analyze the BREd motif instance on the Short Match track (Figure 9). Is the BREd motif at -23 relative to the start of transcription (+1)? Note that the core promoter motif should be in the same orientation as the direction of transcription. Based on the information about the characteristics of core promoter motifs above, is there a canonical BREd motif for the core promoter of sev? Figure 9 BREd instance on the Short Match track Q13. Perform Short Match searches for the rest of the core promoter motifs in