Can you Taste PTC ?

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Using a Single Nucleotide Polymorphism to Predict
Bitter-Tasting Ability

• Can you Taste PTC ?

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Important concepts

• Science evolves from past discoveries.
• Modern biological research merges genetics,
  biochemistry, comparative studies and
  bioinformatics.
• Receptors Genetic differences in taste and smell
• drug efficacy

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Taste in Mammals

• Mammals can distinguish only five basic tastes
• Sweet
• Sour
• Bitter
• Salty
• Umami (the taste of monosodium gluatmate)

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Taste in Mammals

• Taste perception is a two-step process
• 1stA taste molecule binds to a specific receptor
  on the surface of a taste cell
• Question..WHAT IS A RECEPTOR?????
• Question..WHAT DETERMINES THE STUCTURE OF A
  RECEPTOR
• 2nd The taste cell generates a nervous impulse,
  which is interpreted by the brain

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An Example Taste in Mammals

• Stimulation of sweet cells generates a
  perception of sweetness in the brain
• Taste sensation is ultimately determined by the
  wiring of a taste cell to the cortex in the brain

• If you have a sweet cell
• But it expresses a bitter taste receptor
• Bitter molecule will be perceived as being sweet!

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Taste in Mammals

• Taste recognition is mediated by specialized
  taste cells that communicate with several brain
  regions through direct connections to sensory
  neurons

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• While there are only 5 tastes there are thousand
  more olefactory (smell) receptors (OR)
• Smell is like tastea receptor a protein that
  binds to a molecule that we smell.
• Similar also to how many drugs work (the drug
  binds to a cell proteinor receptor)
• All are coded by specific genes

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A Serendipitous Observation

• The genetic basis of taste first observed by
  accident in 1930s
• PTC phenylthiocarbamide
• Prepared by Arthur Fox at Du Pont Company in
  late 1920s
• Lab partner C.R. Noller complained of bitter
  taste but Fox had no taste

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Albert Blakeslee with Jimson WeedCarnegie
Department of Genetics, Cold Spring Harbor, New
York, 1933

• Followed up by Albert Blakeslee at Carnegie
  Department of Genetics showed that inability to
  taste is recessive
• Published in 1932

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Albert Blakeslee, AAAS Convention, 1938
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Punnett Square
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Molecular Genetics of PTC Tasting

• Gene identified in 2003 by Dennis Drayna TAS2R38
  gene
• Polymorphism associated with PTC tasting
• SNP--Nucleotide position 145
• Taster C Nontaster G
• Change in Amino acid 49 . (proline) ? (alanine)

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Analysis of the Trait--CAPS

• Cleavage amplified polymorphisms
• Amplify a region of TAS2R38 gene by PCR
• Primers used in the experiment
• CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
• AGGTTGGCTTGGTTTGCAATCATC
• Then cut with restriction enzyme (HaeIII)
• RFLP-Restriction Fragment Length Polymorphism

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Analysis by eletrophoresis
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2 Agarose Gel Electrophoresis
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1ST PERIOD BIOTECHNOLOGY
NF CK NB AB PH BF
AW JF MR TW KK ZQ MH
600 U C U C U
600 C U C U C U U
600 U C C U C U C U
L H ZP JG CW PH BP
MN SL RBS KH KJK SB BE
600 U C U C U C U C C
C U C U C U C U 600
C U C U C U C U 600
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2ND PERIOD BIOTECHNOLOGY
AH ML TS HB JS
RH AL A K LB RB? JP RP
CG CC WM
600 U C U C U C U C U 600
U C C U C U C 600 U C
U C U C U C C
CT CV SB EB JS
HSM NVS DMR BM
JC SH
600 U C U C U C U C C 600 U
C U C U C 600 U
C U C U C
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• What is the relationship between this trait and
  our ancestors?
• What is the normal state?
• To taste or to not taste?

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Multiple Sequence Alignment
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• Advantage Taste or not to taste?

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More Complication More than 1 PTC Haplotypes
Postition Taster Nontaster
145 C (proline) G (alanine)
785 C (alanine) T (valine)
886 G (valine) A (isoleucine)
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Results 2010
1 KB C U C U RG AG
1 KB U C U C U C U C
CB RR AS JC
1 KB C U C U U C U
C BP BS RF
EC
SORRY mb/fd/kr
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Results 2010
1 kb U U U U C
C C C tb aa
go DP TB AA GO DP
1KB C U C U C U
AM SG KT
1KB U C U C U C U C
TF AS TJ NJ
1KB C U U C U C
C U DD CT
DN TP
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Results 2010
1KB C U C U U C C U
JO AH MS RH
1KB C U C U C U C
U TLL OSC ATM
SE
1kb U C U C U C U U C
CN AC ALG SC BT
1KB C U C U C U
DH KS WT
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How does HaeIII Cut the taster allele?

• Hae III restriction site GGCC
• In the regions around the 145 SNP
• Taster 141 GCAGGCAGCCACT
• Nontaster 141 GCAGGCAGGCACT

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Compare primer to sequence

• Primer
• CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
• primer anneal
• TTTTTGGGATGTAGTGAAGAGGCGG
• Taster TAGTGAAGAGGCAGCCACTG
• Nontaster TAGTGAAGAGGCAGGCACTG

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Compare primer to sequence

• Primer
• CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
• primer anneal
• TTTTTGGGATGTAGTGAAGAGGCGG
• 3 AAAAACCCTACATCACTTCTCCGTC
• Taster 5 TAGTGAAGAGGCAGCCACTG
• Nontaster TAGTGAAGAGGCAGGCACTG

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Compare primer to sequence

• Primer
• CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
• primer anneal
• TTTTTGGGATGTAGTGAAGAGGCGGCCACTG..
• 3AAAAACCCTA CATCACTTCTCCGTC
• Taster TAGTGAAGAGGCAGCCACTG
• Nontaster TAGTGAAGAGGCAGGCACTG

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• http//bioinformatics.dnalc.org/ptc/animation/ptc.
  html

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After PCR

• 
  HaeIII cut site
• Taster TAGTGAAGAGGCGGCCACTG
• Nontaster TAGTGAAGAGGCGGGCACTG

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Results of thePTC Taste
Receptor

• 1. According to the class data (YOUR DATA???),
  how well does TAS2R38 genotype predict
  PTC-tasting phenotype?

Phenotype Phenotype Phenotype Phenotype
Genotype Strong taste Weak Taste Nontaste
TT 4 ----24 12 28
Tt 635 12- 28
tt---- 7--- 41 19- 44
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• What does this tell you about classical dominant
  /recessive inheritance?
• Dominate If you have one copy of a gene
• you will express that trait
• Ability to taste is dominant
• RecessiveTrait that is masked by the
  dominate form of the trait..Need two copies of
  this gene if it will be expressed

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• How does the Hae III enzyme discriminate between
  the C-G polymorphism in the TAS2R38 gene.
• HaeIII cuts at the sequence GGCC
• This is at the 143-145 position of the gene
• The nontaster has a GGGC and wont cut

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• The fwd primer has the HaeIII recognition site
  GGCC. How is this different from the seq. of the
  gene?
• The gene sequence has an Athe primer a G.

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• What characteristic of the PCR reaction allows
  the Primer sequence to override the natural
  gene sequence.
• In PCRthe product produced always has primer.it
  starts with a primer and ends with a primer.so
  the sequence in the primer (and not the gene) is
  what appears in the final product
• This creates a Hae III restriction site in the
  taster allele, but not the non-taster.

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• Consider the terms below for mutations
• Synonymous
• Substitution of one base for another in an exon
  of a gene coding for a protein, so that the
  amino acid sequence produced is the same.
  Synonymous substitutions and mutations affecting
  noncoding DNA are collectively known as silent
  mutations
• Nonsynonymous
• Substitutions that result in amino acid
  replacements are said to be nonsynonymous
• What sort of mutation is the G-C polymorphism in
  the TAS2R38 gene?
• Taste..CCA proline
• NontasteGCA alanine

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Other mutations in the TAS2R38 gene
Positition Taster Nontaster
145 C (proline) G (alanine)
785 C (alanine) T (valine)
886 G (valine) A (isoleucine)
These three mutations influence the ability to
have this bitter taste perception. They are
inherited together as a unit.
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• Many people are nontastersmore than what is
  expected if bitter taste was the ONLY trait under
  natural selection
• SO. Is there some factor that makes this a
  positive outcome to balance out the negative
  effect of not tasting bitter? Is there an
  advantage to being a heterozygote (like sickle
  cell anemia)?
• Maybe.Maybe the NONTASTING form allow for
  individuals to taste another type of bitter
  molecule and so these people may know to avoid
  potentially toxic compounds.

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Methods in DNA typing

• How are these techniques different from that used
  in forensic crime lab.
• Here we use a SNP and RFLP
• Crime labs use VNTRs and STRs and sequencing
• Samples are checked carefully to insure they are
  not mixed up

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• ETHICAL ISSUES?
• Consent?
• Knowledge of use?
• After usesamples stored or destroyed?

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Genotype Taste Strong Phenotype Weak Non




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Olfactory Receptors (ORs)

• Largest mammalian gene family
• 1,000 genes or 4 of total genes
• Can detect 10,000 different odors
• Each OR gene expressed in 1 in 1,000 epithelial
  cells
• Multiple receptors bind different parts of an
  odorant molecule
• Odor code different odorant molecules are
  detected by different combinations of receptors

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OR Evolution

• Mice 20 of ORs are inactive
• Primates 30-40 of ORs are inactive
• Humans 60 of ORs are inactive
• Human-chimp comparisons
• OR genes are diverging quickly
• OR genes are under natural selection

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The future Pharmacogenetics