Reporter Fusion Cloning Strategies illustrations
Directory of C. remanei genomic sequences - Cr_seq - find your assigned genomic sequences.
FGENESH - gene prediction program, in case you still need it.
WWWtacg - the web-based version of the restriction analysis program 'tacg' by Harry Mangalam (thanks, Harry!)
| Use site from the top row if available: | WWWtacg v3 - Univ of Victoria, BC | WWWtacg v3 - Univ Mass Medical School |
| Use bottom row sites if others aren't working: | WWWtacg v4.1 - UC Irvine | WWWtacg v4.1 - Cal State Fullerton |
Directory of C. remanei genomic sequences - Cr_seq - find your assigned genomic sequences.
Location of blunt sites in F32G8.6 genomic sequence
Extra credit "pretty" versions of genes:
Using the genetic code printed on the back of your sequence analysis lab handout, translate the DNA sequence on your handout, and determine the reading frame which contains an ORF (open reading frame). You will turn in your manual translation with the rest of the week 1 materials when they are due.
1. Paste your nucleotide sequence into the window.
2. Select an output format, and hit the TRANSLATE button.
3. Examine each of the 3 different ouput forms - which reading frame has the ORF?
4. Confirm your earlier manual translation using your DNA sequence -- check the accuracy of your translation by putting this sequence into the "DNA to Protein translation tool."
Use translations of genes retrieved in exercise 2. [Note that you are provided with accession numbers for the corresponding amino acid sequences.]
1. Select the button: User-entered sequence for each sequence window.
2. Give each sequence a short name
3. Paste each of your two protein sequences into the two windows.
4. Change number of alignments to be computed to: 1 --Leave other default settings unchanged
5. Click SUBMIT button.
1. Go to ENTREZ - Structure
2. Search with one of the following numbers:
New Method using Cn3D
The program Cn3D should be found on your desktop.
4. Download a few mmdb flies for use with Cn3D from those I have already stored locally at
MMDB files.
5. After downloading a file to the desktop, open one by double-clicking or dragging and dropping on the Cn3D icon.
6. Rotate the molecule by clicking and dragging in the window. Under the "Styles" menu, you can alter the look using the "Rendering Shortcuts" and/or "Coloring shortcuts".
7. Examine the structures of some of the molecules we have discussed in class.
Old method using RasMol (or similar)
4. Next to "Reference:" click on the link "PDB:"
5. Under "Summary Information" column at left, click "Download/Display File"
6. Under "Download the Structure File," click on "X" under Compression - None, and file format - PDB. Save the file to the desktop.
7. Under the Netscape file menu, select "Open file" and select the .pdb file on the desktop.
8. Once the structure is loaded (with default 'wireframe' representation), click and hold in the window, and select under "Display" - "Ribbons."
9. Have fun looking at the molecule. Click and drag in the window to rotate the molecule. Hold down the shift key while clicking and dragging to zoom in or out on the molecule
10. To see more features of the protein, try some of the different coloring schemes (under "Color"):
Directory of C. remanei genomic sequences - find your assigned genomic sequences.
Exercise 6
1. Paste your assigned genomic sequence (from the Cr_seq directory) into the sequence window.
2. Select "C. elegans" for Organism.
3. Enter the following into the "advanced options" box:
-pmrna -pexons
This will also show the predicted mRNA and each individual exon from each predicted gene in your sequence. Save this file to use in next week's computer lab. You will use the complete predicted mRNA sequences in exercise 7A below, and the predicted amino acid sequences in exercise 7B below.
4. Click the Search button. You will use this information for exercise 7B.
A. Determine whether each of your predicted genes has an associated cDNA or EST. To qualify, the sequence should have 100% identity (or nearly) over an extended region and a very low E value.
1. Go to BLASTN at NCBI.
2. Paste in the nucleotide sequence from one of your predicted genes (found in the second FGENESH file you created).
3. Alter the settings in the following manner:
a. Under "Choose Database", select "est"
b. Under "Options" in the "Limit by entrez query" text window, type "C. remanei" (no quotes)
c. Under "Format", for both "Descriptions" and "Alignments" select "10".
Click the Search button.
B. Determine whether your predicted proteins have matches in the protein database
1. Go to the NCBI BLAST Server
2. Under Protein, click on Protein-Protein BLAST [blastp].
3. Paste each of the amino acid sequences from your predicted genes from your FGENESH analysis, and
do the BLAST search.
Note also if there are any Conserved Domains found in your search.
See your handout for additional instructions
Miscellaneous resources for getting information on your predicted genes:
Example of further analysis of Bm3859 predicted genes
| Gene | EST? | C. elegans homolog | Conserved Domains | Type of protein encoded |
| Gene 1 | no | cat-1 gene/AAG00026 | KOG6734 | Vesicular monoamine transporter |
| Gene 2 | no | none | none | no significant matches |
| Gene 3 | no | none | KOG0293/WD40 | WD40 repeat protein |
| Gene 4 | no | ZK550.3 | KOG2089 | Oligopeptidase |
| Gene 5 | yes | msh-1/H26D21.2 | KOG0219 | Mismatch repair protein MutS family |