Functional Genomics Bioinformatics Instructions and Worksheet
Save this document to your desktop!
Selecting a sequence for gene tagging








Fit this window into the upper left quadrant of your computer screen.
Fit Stanford's FTFLP site into the upper right quadrant of your screen.
On the FTFLP web site click Target Selection.
Scroll down to Table 2.
Find Category 2 and open the tab-delimited list to access a listing of 4000 short-listed A.
thaliana genes of unknown function available for tagging.
Study the information for the genes and select one for further analysis:
o _________________________________________________________________
o _________________________________________________________________
o _________________________________________________________________
o _________________________________________________________________
Contact CSHL for further instructions on how to order sequences and how to avoid using
sequences that are already being tagged by other labs.
To learn how to find more information about a gene go to the next slide.
Find out where in the A. thaliana genome the sequence is located










Fit this window into the upper left quadrant of your computer screen.
Highlight and copy the Locus name (AT#) for a gene.
Fit NCBI's Map Viewer into the lower right quadrant of your screen.
In Map Viewer click A. thaliana.
Paste the Locus name into the Search for window, click Find.
The sequence locus is flagged with a red tag.
To zoom in click the number underneath the chromosome.
Zoom in and determine what genes are located in the same region.
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
Increase the resolution until you can identify the structure of the gene.
Click sv to view the gene sequence. Discern introns and exons.
Find sequence information for a gene



Fit this window into the upper left quadrant of your computer screen.
Highlight and copy the Locus name (AT#) for a gene.
Fit the TAIR site into the lower left quadrant of your screen.
Worksheet Functional Genomics, Page 1 of 4





Under Advanced Search click Genes.
Paste the Locus name into the Search by name window, click submit query.
Click the link underneath Locus.
You can access the sequence information for the coding sequence (CDS), the genomic
DNA, and for the deduced protein sequence.
To examine the protein go to the next slide.
Find information about the protein product of a gene









Fit this window into the upper left quadrant of your computer screen.
Highlight and copy the Locus name (AT#) for a gene.
Fit the TAIR site into the lower left quadrant of your screen.
On the TAIR-site find Advanced Search click Proteins.
Paste the Locus name into the Search by name window, click submit query.
Click the link underneath Name.
The result page contains links to SwissProt, GenPept, and NCBI BLink (3D-structures).
To analyze the protein's domains highlight the amino acid sequence and analyze it at the
SMART website.
Also follow the link to The Arabidopsis Subcellular Proteomics Database.
Find expression data for a tagged gene
Tagged genes are being transferred into A. thaliana plants using Agrobacterium tumefaciens
and transformed plantlets assayed for gene expression.





To view the results click Browse Images on the FTFLP website.
Highlight All_localization and click Go.
Click a Gene Name for the sequence of the tagged locus.
Click the small green camera icon for a clickable fluorescence microscopy image of a
transformant; an Image-Description explains what you see.
Match the results (columns) for a tagged gene and associate it with the biology of the
protein an /or the organelle it is localized in.
Find the gene model for a tagged gene
Tagged genes are being transferred into A. thaliana plants viaAgrobacterium-mediated
transformation and transformed plants assayed for gene expression.





To view the results click Browse Images on the FTFLP website.
Highlight All_localization and click Go.
Click a Gene Name for the sequence of the tagged locus.
What do black, blue, and red letters denote?
Can you discern any pattern that could be used to detect splice sites (exon/intron
borders)?
Worksheet Functional Genomics, Page 2 of 4
Search databases for proteins that confer the ability to alter light by searching for proteins
with similar composition.
Search for proteins with amino acid sequences similar to GFP






Highlight and copy this aminoacid sequence.
Open the NCBI Internet site, click BLAST, find Protein, click Protein-protein BLAST
(blastp).
Paste the sequence into the window, and click BLAST.
Record the request ID ________________________________ .
Click Format!
Examine the titles for some of the search hits and the related GenBank database entries
using the links labeled gi ... determine what proteins the search yielded.
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Search databases for proteins that confer the ability to alter light by searching for proteins
with similar domains.
 Highlight and copy this aminoacid sequence.
 Open the NCBI Internet site, click BLAST, find Protein, click Search by domain
architecture (cdart).
 Paste the sequence into the window, and click Search.
 Click the red bar labeled GFP.
 Click of the most diverse members and change it to select from list.
 Search the column titled Definition for proteins which alter the color of the light to
something else than green. Which other colors are listed?
 Click on the PDB-ID/gi entry for a representative of each of the different types of
flourescent proteins (FPs) and try to view the structures by clicking View 3D Structure.
How do the structures differ for FPs which emit different light?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Search databases for proteins that confer the ability to alter light by searching for proteins
with similar structures.






Open the NCBI Internet site.
Click Entrez and change it to Structure.
Search Structure for 1C4F, click 1C4F.
Click the pink bar labeled Chain A.
Click Medium redundancy and change it to All sequences.
Change Graphics to Table. Click List.
Worksheet Functional Genomics, Page 3 of 4



Check one representatives from each: cyan, red, green, and yellow fluorescent protein,
click View 3D Structure.
Click Style, click Coloring Shortcuts, select Aligned.
Are the capabilities of the proteins to differentially alter light reflected in structure
differences?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Worksheet Functional Genomics, Page 4 of 4