1. Using a Single Nucleotide Polymorphism to Predict Bitter-Tasting Ability
Can you Taste PTC ?

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

3. Taste in Mammals Mammals can distinguish only five basic tastes Sweet
Sour
Bitter
Salty
Umami (the taste of monosodium gluatmate)

4. Taste in Mammals Taste perception is a two-step process
1st…A 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

6. 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!

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

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

9. A Serendipitous Observation
The genetic basis of taste first observed by accident in 1930’s
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

10. Albert Blakeslee with Jimson Weed Carnegie 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

11. Albert Blakeslee, AAAS Convention, 1938

12. Taste test: Phenylthiocarbamide (PTC)
Arthur Fox, "The Relationship Between Chemical
Constitution and Taste” (Arthur Fox and C.R. Noller)
Albert Blakeslee, "Genetics of Sensory Thresholds:
Taste for Phenyl Thio Carbamide"

13. Punnett Square

14. 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)

15. 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

17. Analysis by eletrophoresis

18. 2% Agarose Gel Electrophoresis

19. What is the relationship between this trait and our ancestors?
What is the normal state?
To taste or to not taste?

20. Multiple Sequence Alignment

21. Advantage: Taste or not to taste?

22. 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)

23. How does HaeIII Cut the taster allele?
Hae III restriction site = GGCC
In the regions around the 145 SNP
Taster GCAGGCAGCCACT
Nontaster GCAGGCAGGCACT

24. Compare primer to sequence:
CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
primer anneal
TTTTTGGGATGTAGTGAAGAGGCGG
Taster TAGTGAAGAGGCAGCCACTG
Nontaster TAGTGAAGAGGCAGGCACTG

25. Compare primer to sequence:
CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
primer anneal
TTTTTGGGATGTAGTGAAGAGGCGG
3’ AAAAACCCTACATCACTTCTCCGTC
Taster ’ TAGTGAAGAGGCAGCCACTG
Nontaster TAGTGAAGAGGCAGGCACTG

26. Compare primer to sequence:
CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
primer anneal
TTTTTGGGATGTAGTGAAGAGGCGGCCACTG………..
3’AAAAACCCTA CATCACTTCTCCGTC
Taster TAGTGAAGAGGCAGCCACTG
Nontaster TAGTGAAGAGGCAGGCACTG

28. After PCR HaeIII cut site Taster TAGTGAAGAGGCGGCCACTG
Nontaster TAGTGAAGAGGCGGGCACTG

29. Results of the 2012 PTC Taste Receptor
1. According to the class data (YOUR DATA???), how well does TAS2R38 genotype predict PTC-tasting phenotype?
Phenotype
Genotype
Strong taste
Weak Taste
Nontaste
TT %
12 – 28%
Tt —35%
12- 28%
tt %
19- 44%

30. 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
Recessive—Trait that is masked by the dominate form of the trait..Need two copies of this gene if it will be expressed

31. 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 position of the gene
The nontaster has a GGGC and won’t cut

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

33. What characteristic of the PCR reaction allows the Primer sequence to “override” the natural gene sequence.
In PCR—the 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.

34. 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
Nontaste……GCA = alanine

35. 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.

36. Many people are nontasters…more 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.

37. 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

38. ETHICAL ISSUES? Consent? Knowledge of use?
After use—samples stored or destroyed?

39. Genotype
Taste
Strong
Phenotype
Weak
Non

40. 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

42. 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

43. The future: Pharmacogenetics