Transcript 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.
What do you do next?
BLAST
Methods to look for
similarity
- Blast (and derivatives)/Blat
- Zoo blots
- Degenerate PCR against conserved
regions
- Functional complementation
- Protein structure
http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/tut1.html
BLAST WHAT?
Proteins or DNA?
-Which is more likely to be the same: a match of 10
amino acids or a match of 10 nucleotides?
- 4 possible bases vs. 20 amino acids.
- amino acids more have more degeneracy. If we
see a run of similar amino acids, it is less likely
to have occurred by chance.
What steps would you take to
blast the amino acid
sequence if you start with the
nucleic acid sequence?
-tblastn vs blastx?
Program Description
BLASTP
amino acid query sequence against a protein sequence database.
BLASTN
nucleotide query sequence against a nucleotide sequence database.
BLASTX
nucleotide query sequence translated in all reading frames against a
protein sequence database.
TBLASTN
Compares a protein query sequence against a nucleotide sequence
database dynamically translated in all reading frames.
TBLASTX
Compares the six-frame translations of a nucleotide query sequence
against the six-frame translations of a nucleotide sequence database.
Computationally intensive.
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 you need to
blast nucleic acid?
DEFINITIONS
Homologs - sequences that have shared
ancestry. Note homology is either true or not.
There is no such thing as high homology or
low homology. Usually inferred from
significant similarity.
Orthologs - homologs separated by a speciation
event. May be functionally equivalent.
Paralogs - homologs derived from a duplication
event. Within same species…. I.e. Members of a
human gene family share sequence similarity,
but may have distinct functions.
- paralogy or orthology is inferred from significant
sequence similarity.
Examples:
Mouse smad2 and frog smad2 are 98% identical
(Orthologues)
The Activin Receptor has many isoforms IA, IB IIA IIB, etc.,
that are very similar at the protein level.
(Paralogues)
You blast the protein
sequence..… and there is
nothing like it in the database.
Now what?
motifs/domains
DOMAINS
Definition:
A contiguous segment of the primary
sequence of a molecule that - in isolationdisplays a significant property of the
intact molecule.
It is usually structurally stable and
associated with a function, including
providing a structural element to the
protein.
http://www.sanger.ac.uk/Software/Pfam/
There is similarity over certain regions
to several molecules containing
kinase domains. What does this tell
you?
- it’s a kinase!
- location?
What knowing domains gives:
Clues to function:
-what it interacts with
-what its biology is known for..
-signaling pathway
-is it like a molecule in a more
tractable and studied system?
Use of similarities
Looking for new proteins with
similarities to known proteins with
interesting activities. Serotonin
receptors, Tyrosine Kinases,
Hedgehogs, TGFs, …..
Domain similarity RING fingers (E3 ligases),
Kinase domains, DNA binding domains (bHLH
Homeobox)
Localization (TM domains, signal sequences,
NLS, NES, signal peptide)
Localization motifs
nucleus
Nuclear localization signal (NLS)
Stretch of basic residue: P-P-K-K-K-R-K-V
http://cubic.bioc.columbia.edu/predictNLS/
cytoplasm
Nuclear Export Sequence (NES)
Hydrophobic helix: L-X(2,3)-[LIVFM]-X(2,3)-L-X-[LI]
http://www.cbs.dtu.dk/databases/NESbase/
Transmembrane or secreted
Signal peptide: Hydrophobic helix in the N-terminal region
Often followed by a cleavage site for a protease
http://www.bioinformatics.leeds.ac.uk/prot_analysis/Signal.html
http://www.cbs.dtu.dk/services/SignalP/
http://ca.expasy.org/prosite/
Enter your favorite protein here
Results
http://scansite.mit.edu/