Download You have worked for 2 years to isolate a gene involved in axon

You have worked for 2 years to
isolate a gene involved in axon
guidance. You sequence the
cDNA clone that contains axon
guidance activity. The sequence
gets emailed to your account.
What do you do next?
BLAST
BLAST WHAT?
Amino vs Nucleic??
-Which is more likely to be the same: a match of 10 amino
acids or a match of 10 nucleotides?
-4 bases vs 20 amino acids.
-amino acids more have more degeneracy. If see
similar amino acid, we assume that it did not occur
through chance.
What steps would you take to
Blast the Amino Acid sequence
if you start with the nucleic acid
sequence?
-tblastn vs blastx
-Look for ORF …how?
When might need to blast nucleic
acid?
-paralogs or orthologs exist where amino acid is highly
similar. I.e… Mouse smad2 and frog smad2 are 98%
identical. Activin Receptor has many isoforms IA, IB IIA
IIB, etc. That are very similar at the protein level.
-blat searches in genome for cis regulatory elements.
- your impatient and the protein blast searches are slow.
You blast the protein sequence..…
There is nothing like it in the
database. Now what?
-motifs/domains
You blast the protein. There is similarity over
certain regions to several molecules
containing kinase domains. What does this
tell you?
- it’s a kinase!
- location in the cell?
Furthermore, it has great similarity to the Erk
family of kinases, meaning similarity
outside the kinase domains. Does this help?
Why is it useful to ask if your unknown is like
something else???
Clues to function!!!!! In a huge and broad
context that might mean…
-what it interacts with
-what its biology is known for..
-pathway
- is it like a molecule in a more tractable
and studied system (yeast that doesn’t even
have neurons!)
SIMILARITY…. What it gets you
looking for new proteins with similarities to known
proteins with interesting activities. Seratonin
receptors, Tyrosine Kinases, Hedgehogs, TGFbs,
etc, etc…..
Domain similarity tells you tons… RING fingers (E3
ligases), Kinase domains, TM domains, signal
sequences, HLH dna binding domains….etc…etc..
Methods to look for similarity
- blast/blat
- Zoo blots
- Function…. Can it complement? (h ras into
yeast)
- Protein structure
- degenerate PCR over similarity regions.
Ortholog - gene copies derived from
speciation. Usually functional orthologs.
Paralogs - gene copies derived from
duplication. Within same species…. Ie.
Members of a human gene family share
sequence similarity, but may have distinct
functions.
Is sequence similarity relevant??
DOMAINS
Whats a domain?
A contiguous segment of the primary
sequence of a molecule that - in isolationdisplays a significant property of the intact
molecule. The most stringent use of the
term requires the domain to be structurally
stable and display some function.
It is usually associated with a function,
including providing a structural element to
the protein…, but can also be a folding
domain.