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.
DOMAINS Whats a domain? A contiguous segment of the primary sequence of a molecule that - in isolation- displays 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.