Transcript Genomics of sensory systems - University of Maryland

TRP channels
10/19/09
Plan

Lecture on Wed - Richard Payne
The problem
Brainstorm a plan

One week to gather sequences
Each of you will do a different species
I am away Wed pm to Fri am. I’ll be in my office all
afternoon and all Monday - bring questions to
class!
We will then compile all the data

One week to analyze data
Present results to Dr. Payne
Drosophila mutant screen
 Forward
genetics
Screen for mutants which have visual defect
Find gene causing defect
Piece together parts of visual pathway
Screen by electroretinograms,
ERGs
 Send
light pulse to eye
 Wire
outside of retina to
record electrical signal
coming from whole eye
response
Sum of all neural
responses in eye
Light coincident receptor
potential
Light coincident receptor
potential
Drosophila mutants
Norp - no receptor potential (phospholipase C)
Trp - transient receptor potential
Ion channel mutants
Trp = Transient receptor potential - channels close sooner
Trp is …
…an ion channel
Textbook example - nompC
7 families of TRPs
Trp
NompC
TRP families
TRPC Classic - like Ca+2
Drosophila
DAG
TRPV Vanilloid
receptors
Light, pheremone
Heat, cold, capsaicin
TRPM Melastatin
receptor
Ca+2
Taste, menthol, cool
TRPN NOMPC
Ca+2
Mechanosensation
(none in mammals)
Cold, hearing
TRPA ANK repeats
How is Trp activated - DAG?
How is Trp activated IP3 release of Ca+2?
Mammalian TRPCs
TRP domain - highly conserved
TRP domain
Seems to be highly
conserved within a
family, particularly
in TRPCs
#s of copies
TRPC and TRPN
most closely related
C. elegans has TRP3
which is like TRPC
Dr. Payne is world expert on
limulus vision!
Response to IP3
Causes
depolarization
 Causes release of
Ca2+

Adaptation
Can’t respond
immediately - takes
recovery time to
rebuild Ca stores
Payne et al 1990
Vertebrate divergence times
Human
92
Cow
41
71
Mouse
Rat
276
Reptiles
310
Birds
360
Amphibians
Tilapia
120
200
Fugu
250
Zebrafish
528
Cartilagenous fish
564
600
500
400
Bony
fish
Agnatha
300
Time (Myr)
200
100
0
Kumar and
Hedges 1998
Comparative genomics
 Dr.
Payne is interested in invertebrates
 Invertebrates
Molting animals
Trochozoa
Lophophorata
Genome projects for inverts

Model systems
C. elegans, Drosophila
Sea urchins, amphioxus
Genome projects for inverts
 Vectors
for disease
Mosquitos, tick, louse, leech
Other insects
 Bee,
pea aphids, flour beetle, wasp
Miscellaneous
 Polychaete
 Limpet
 Daphnia
worm
BLAST-able invertebrate genomes
Deuterostomes
Craniate
Cephalochodates
Tunicate
Echinoderm
Human
Amphioxus
Sea squirt
Sea urchin
Protostome - annelid
Polychaete
Clitellate
Capitella
Leech
Protostome - Mollusc
Gastropod
Limpet
Protostome - Arthropod
Crustacean
Water flea
Protostome - Arthropod
Insect - Coleoptera
- Diptera
Flour beetle
Fruit fly (1 of 12)
Mosquito (3)
Honey bee, wasp
Pea aphid
Louse
- Hymenoptera
- Hemiptera
- Phthiraptera
Horseshoe crab is closest to tick!!
How do we get sequences from
these genomes
 Can’t
use ortholog links in Ensembl
Haven’t made links for inverts!
 We
have to BLAST our way in
NCBI has links to all genome projects Not all projects are BLASTable
Bee genome page with BLAST link
What kind of blast should we
use?
MegaBLAST - highly
similar
BLASTN - nucleotide
BLASTP - protein
Can blast to different databases
Ab initio = known + predicted
genes
Use Drosophila Trp gene as bait
BLAST interim screen
Blast result
Download sequence
TRP domain
Seems to be highly
conserved within a
family, particularly
in TRPCs
Alignment = find Trp box
TRP domain - highly conserved
Lots of variation before ion channel - we may need to deal just with channel part
ANK
ANK
Sometimes gene is poorly
predicted
Human_rho
Chimp_rho
Dog_rho
Mouse_rho
Rat_rho
Cow_rho
Zfish_rho
Chick_rho
Human_rho
Chimp_rho
Dog_rho
Mouse_rho
Rat_rho
Cow_rho
Zfish_rho
Chick_rho
Human_rho
Chimp_rho
Dog_rho
Mouse_rho
Rat_rho
Cow_rho
Zfish_rho
Chick_rho
Human_rho
Chimp_rho
Dog_rho
Mouse_rho
Rat_rho
Cow_rho
Zfish_rho
Chick_rho
MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
MNGTEGPNFYVPFSNKTGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
MNGTEGPNFYVPFSNVTGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
MNGTEGPNFYVPFSNITGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLY
MNGTEGPAFYVPMSNATGVVRSPYEYPQYYLVAPWAYGFVAAYMFFLIITGFPVNFLTLY
MNGTEGINFYVPMSNKTGVVRSPFEYPQYYLAEPWKYRLVCCYIFFLISTGLPINLLTLL
****** ****:** *******:* *****. ** : ::..*:*:** *:*:*:***
23 variable
VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLG
VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLG
VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNVEGFFATLG
VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
VTIEHKKLRTPLNYILLNLAIADLFMVFGGFTTTMYTSLHGYFVFGRLGCNLEGFFATLG
VTFKHKKLRQPLNYILVNLAVADLFMACFGFTVTFYTAWNGYFVFGPVGCAVEGFFATLG
**.:***** ******:***:*****. *** *:**: :****** ** :********
17
GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS---GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS---GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSSLLS
GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWS---GEIGLWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS---GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS---GEMGLKSLVVLAIERWMVVCKPVSNFRFGENHAIMGVAFTWVMACSCAVPPLVGWS---GQVALWSLVVLAIERYIVVCKPMGNFRFSATHAMMGIAFTWVMAFSCAAPPLFGWS---*::.* *********::*****:.****. .**:**:.***:** :**.*** ***
23
------RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTV
------RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTV
HSPLVLRYIPEGMQCSCGIDYYTLKPEINNESFVIYMFVVHFAIPMIVIFFCYGQLVFTV
------RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTV
------RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTV
------RYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTV
------RYIPEGMQCSCGVDYYTRTPGVNNESFVIYMFIVHFFIPLIVIFFCYGRLVCTV
------RYMPEGMQCSCGPDYYTHNPDYHNESYVLYMFVIHFIIPVVVIFFSYGRLICKV
**:***:***** ****
:***:*:***::** **:::***.**:*: .*
28
Wise2 - can predict mRNA from
gDNA using related protein
Predict dog RH using cow RH AA
Cow AA
Dog seq
Finds introns so know where they
are
Fixes intron prediction to get rid
of extra stuff