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Lecture 22 : Taste II. 11/18/09. Today or Monday. Final project: 1 pg of background. Each taste modality relies on different receptors. pH sensing. T1R2+T1R3. Cats don’t taste sweet. Search for T1R2 and T1R3. Screen cat BAC library Sequence positive clones. Find complete T1R3 gene.

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Today or monday
Today or Monday

  • Final project:

    • 1 pg of background




Search for t1r2 and t1r3
Search for T1R2 and T1R3

  • Screen cat BAC library

  • Sequence positive clones






Pseudogene common to several cat lineages

X

X

X

Pseudogene common to several cat lineages




Human

Mouse

Dog

Cow

Opossum

Chicken





T2r genes in mouse and human
T2R genes in mouse and human

  • Mouse

    • 35 functional 6 pseudogenes

  • Humans

    • 25 functional 11 pseudogenes

  • Other primates


Human

OWM

NWM

Prosimians

Mouse

X marks pseudogenes

Amany : one

Human:mouse

Bone : many

Human:mouse

Cone : one

Human:mouse


Human variation in pseudogenes
Human variation in pseudogenes

T2R62

T2R2

Functional

Hosa - Homo sapiens Hyag - Hylobates agilis gibbon

Patr - Pan troglodytes chimp Mamu - Macaca mulatta rhesus

Gogo - Gorilla gorilla Caja - Callithrix jacchus marmoset

Popy - Pongo pygmaeus orangutan Ceap - Cebus apella capuchin


Convergence of pseudogenes
Convergence of pseudogenes

T2R64

T2R55

Hosa - Homo sapiens Hyag - Hylobates agilis gibbon

Patr - Pan troglodytes chimp Mamu - Macaca mulatta rhesus

Gogo - Gorilla gorilla Caja - Callithrix jacchus marmoset

Popy - Pongo pygmaeus orangutan Ceap - Cebus apella capuchin




PTC

  • 75% of humans taste as bitter

    • Discovered in 1930’s

  • “Single” gene trait

  • Originally thought was Mendelian recessive

    • AA, Aa = sensitive

    • aa = not sensitive

  • Probably 2 loci


Give people increasing concentrations of ptc till they tasted bitter
Give people increasing concentrations of PTC till they tasted bitter

x2

x2

x2

x2

x2

x2

x2

1 2 3 4 5 6 7

x2

x2

x2

x2

x2

x2

8 9 10 11 12 13 14

1M

Increasing concentration


Quantitative trait locus qtl study
Quantitative trait locus (QTL) study tasted bitter

  • Link phenotype (sensitivity to PTC) to genotype in Utah pedigree

  • Found link to 4 Mb region on chromosome 7

    • Are 9 T2R and 7 OR genes in this region

  • Finer scale

    • Genotyped 50 more SNPs in this region

    • Added broader diversity of taster and non-taster individuals



Tas2r38 variations
Tas2R38 variations tasted bitter

A V

V I

262 296

P

A

49

European EAsian

P A V 49 31 Taster

A A V 3

A V I 47 69 Non-taster


Phenotype is determined by cup at which tasted ptc
Phenotype is determined by cup # at which tasted PTC tasted bitter

x2

x2

x2

x2

x2

x2

x2

1 2 3 4 5 6 7

x2

x2

x2

x2

x2

x2

8 9 10 11 12 13 14

1M

Increasing concentration


Genotype vs phenotype
Genotype vs phenotype tasted bitter

Nontaster

Taster


Human origin of nontaster
Human origin of nontaster tasted bitter

Human PAV /AAV/ AVI

Chimp PAV

Gorilla PAV

Orangutan PAV

Macaque PAV


Population haplotype frequencies
Population haplotype frequencies tasted bitter

Non

Taster




Similarity of taste and olfactory receptors
Similarity of taste and olfactory receptors tasted bitter

Matsunami and Amrein 2003


Similarity of taste and olfactory receptors1
Similarity of taste and olfactory receptors tasted bitter

Matsunami and Amrein 2003


Taste and vno are related
Taste and VNO are related tasted bitter

V1R

T2R

V2R

T1R


Bitter taste transduction
Bitter taste transduction tasted bitter

Sheridan Nature Biotech 2004


Gpcr taste pathways
GPCR taste pathways tasted bitter


Sour and salty
Sour and salty tasted bitter

  • Ionotropic channels

    • Salty - Na+

    • Sour - H+


Salt detection likely a channel
Salt detection likely a channel tasted bitter

  • Membrane responds to Na+ gradient


Salt receptor
Salt receptor tasted bitter



Two salt pathways
Two salt pathways tasted bitter


Capsicum
Capsicum tasted bitter

Capsaicin detected by vanilloid receptor ??


Sour detection
Sour detection tasted bitter


Approach
Approach tasted bitter

30,000 genes

Screen mouse genome for genes containing TM

Screen EST libraries for genes which are not common

5 genes

884

PCR genes in papillae vs tongue

In situ hybridization to identify ones uniquely expressed

98




Knockout pdk2l1 cells knocks out sour detection
Knockout Pdk2L1 cells knocks out sour detection tasted bitter

But is this the sour channel?



2nd lab also prove
2nd lab also prove tasted bitter


Approach1
Approach tasted bitter

  • Assume TRP channels are important in taste

    • TRPM5 known to be channel in sweet, umami and bitter

  • Use in situ hybridization

    • Screen taste papillae for all 33 TRP channels

    • TrpM - 40%

    • PKD1L - 20% of circumvallate

    • No other Trp’s label




Taste receptors
Taste receptors tasted bitter


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