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, print the FIRST PAGE ONLY from the final ("with nucleotide seq") format.
4. Check your earlier manual translation using your DNA sequence.
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:
Extras
6. Compare structures of Lambda Cro and Lambda Repressor proteins bound to DNA
7. Compare structures of Ubx homeodomain and Lambda Cro helix-turn helix proteins bound to DNA
8. Compare structures of a bHLH protein (e.g., GCN) and a bZIP protein (e.g., Fos-Jun heterodimer) bound to DNA
PDB files of noteworthy molecules
Directory of C. remanei genomic sequences - find your assigned genomic sequences.
Exercise 6
FGENESH - a gene prediction program
1. Paste your assigned genomic sequence into the sequence window (or browse the appropriate file if you have downloaded it).
2. Select "C. elegans" for Organism.
3. Select the following "advanced options" from the list below:
- print mRNA sequences for predicted genes.
- print exon sequences for predicted genes.
4. Click the "Search" button.
5. Save the resulting file to use in next week's computer lab as well. Beside the basic information on the predicted genes found in your genomic sequence, you will use the predicted mRNA sequences in exercise 7A below, and the predicted protein sequences in exercise 7B below for each of your predicted genes.
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 the NCBI BLAST Server
2. Under Basic BLAST, click on nucleotide blast.
3. Paste your nucleotide sequence into the large window under "Enter Query Sequence"
4. Alter the settings under "Choose Search Set" in the following manner:
a. For "Database", click "Others"; then on the pull-down menu select "Expressed sequence tags (est)"
b. For "Organism" type "Caenorhabditis" (no quotes) into the text window,
5. Select the "Show results in a new window" checkbox next to the BLAST button.
6. Click "Algorithm parameters"
For "Max target sequences" select "10"
7. Click the BLAST button.
B. Determine whether your predicted proteins have matches in the protein database.
1. Go to the NCBI BLAST Server
2. Under Basic BLAST, click on protein blast (blastp).
3. Paste each predicted amino acid sequence from your FGENESH analysis into the large window under "Enter Query Sequence"
All the other parameters can be left at their default settings.
5. Select the "Show results in a new window" checkbox, then click the BLAST button.
Note also if there are any Conserved Domains found in your search. (This information will come up immediately after initiating the search in the 'Format' window.)
Miscellaneous resources for getting information on your predicted genes:
Example of further analysis of Cr0062a.seq 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 |