Problem Summary:
Find the location of a gene, and download the sequence for the gene plus the promoter region |
| Sample User Question |
 |
| |
|
I want to examine how cycles of night and day regulate the expression of the per2 gene. I need the sequence of the per promoter so that I can create a promoter/reporter construct.
|
|
|
| Analysis/Comments |
|
The promoter sequence that lies upstream, or 5', to the gene sequence is critical in regulating when and how the gene is turned on. To find the promoter region, use Map Viewer to locate the gene within a chromosomal context. Then increase the value of the coordinates that surround the gene to a larger sequence that includes the promoter.
IMPORTANT: The chromosomal coordinates shown in the answer below might not correspond exactly to what you will see. Additional or revised sequence data continues to be generated, so the human genome is rebuilt periodially. Therefore, the chromosomal location of a gene on one build might be different from the the location on another build. The numbers below correspond to human genome build 32.
|
| Flow Chart |
|
- Human Map Viewer - Search for the per2 gene.
- Chromosome schematic - Click on "Genes_seq" link near bottom of page.
- MapViewer, Genes_seq map - At PER2 entry (highlighted in pink), click "dl" link.
- "Strand" box - Select "minus" strand from the pulldown menu because PER2 gene is on the
minus strand (note arrow next to PER2 on Map Viewer points up).
- "adjust by" box in the "to" line - Increase sequence information displayed by inserting
"+3K", for example. Click Change Region/Strand (note how coordinates change and strand
designation becomes "-"). Click Display. [See also Additional Notes].
- BLAST the extended sequence against the human genome - Confirm that the extended region
lies at 5' end of the PER2 sequence. Copy and paste sequence display above into a BLAST search
against the human genome. On BLAST results page, click on "Genome View", then click on
chromosome 2 to go directly to Map Viewer to see results (hit is a red line). Adjust "Maps and
Options" so that "Gene" (Genes_seq) is master map. If necessary to see all of PER2 gene,
adjust "Region Shown" to 238.67M to 238.74M, for example. The BLAST hit (shown in red) should
extend 3K past the 5' end of the gene (shown in blue). Confirm that BLAST results have
retrieved a hit on the minus strand by noting the numbering of the hit ("47407..1" on the
Genome View) and by noting "Strand = Plus/Minus" on the "Results of BLAST" page.
|
| Additional Notes |
|
Coordinates in "seq" link display screen of Map Viewer:
Two types of coordinates are shown in this display. The first set, at the top of the display, shows the location of the gene on the overall chromosome. The second set of coordinates, in the gray shaded bar, shows the location of the gene on the individual contig, or chromosome fragment.
To obtain 5' sequence of a gene, first the directionality of the gene must be determined.
In other words, is the gene on the plus strand, and therefore read in a direction that moves from top to bottom of the chromosome? Or is the gene on the minus strand, and therefore read in a direction that moves from bottom to top of the chromosome? Knowing the directionality of the gene is essential to determining the location of the promoter region.
There are several ways to determine the directionality of the PER2 gene on the assembled chromosome sequence:
- a small black arrow beside the gene name points up or down. For PER2, the arrow points up, meaning the gene is on the minus strand, and that the sequence of the chromosome in that region should be read in the direction of the arrow (5' end of the gene, to the 3' end of the gene).
- The chromosome sequence is always labeled with coordinates in
which the top (p telomere) of the chromosome has the lowest number while the
bottom (q telomere) has the highest number. The 5' end of the gene has the higher chromosome coordinate value relative to the 3' end when that gene is on the minus strand. Conversely, if the gene is on the plus strand, the 5' end has the lower chromosome coordinate value relative to the 3' end.
- A gene on the minus strand is shown by a blue line to the left of the gray line that represents the
chromosome (while a gene on the plus strand is shown to the right of the vertical gray line).
In the "seq" link display screen, the plus or minus strand can be selected from the
pulldown menu. The "from" and "to" values show the position of the gene on the
chromosome. For example, in human genome build 32, the gene is located "from" base
pair 238685898 on the chromosome "to" base pair 238730304. (These chromosome
coordinates might change in subsequent builds, due to the addition of more sequence data, or
to changes in the assembly/annotation process.)
|
|
