1. Phenylthiocarbamide Biochemistry, Neuroscience, Evolution, and Molecular Biology
“Much of PTC’s appeal arises from the fact that it is nearly impossible to guess one’s phenotype until explicitly tested, yet, when tested, the phenotype is so striking as to be amusing.”
~ Stephen Wooding

2. PTC Tasting DON’T BIAS YOUR CLASSMATES!!! Use your neutral face
Taste the two pieces of paper
Silently rank yourself:
1: Paper is paper…it all tastes the same to me!
2: Maybe they taste a bit different?
3: Yes, I can definitely taste a difference
4: What is wrong with you people?!?!? Do you need to ask??? Are you kidding me??? Of course they are different!!! If I had an apple I would totally throw it at you!!!

3. Class Data Rank 1: _____ people Rank 2: _____ people
Total #: _____ people

4. Reviewed in Wooding (2006). Genetics 172: 2015–2023.
Importance of Taste
Taste and animal interaction with environment
Plant defense mechanism: produce noxious compounds
Bitter-taste perceptions prevent poisoning via detection of toxins in food
Less crucial in modern society
Connection between taste sensitivity and behavior connects to fitness (diet choice, smoking, etc.)
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

5. Reviewed in Mennella et al (2005). Pediatrics 115: e216-e222.
Importance of Taste
Children have different taste responses than adults
Postnatal sensory system maturation
Higher preference for sweet-tasting
Higher rejection of bitter-tasting
Biological functions of taste
Sweetness associated with readily available calories
Bitterness associated with toxins
Reviewed in Mennella et al (2005). Pediatrics 115: e216-e222.

6. Bitter vs Sweet Taste Preference
Propylthiouracil (PROP, a PTC-related compound)
Showed heritability
No age-, race/ethnicity-, or gender-related differences
Sweet preference
Less heritable
Race/ethnicity influenced sugar content of preferred cereal
Race/ethnicity did not affect lab-tested sucrose preference
Mennella et al (2005). Pediatrics 115: e216-e222.

7. Wooding (2006). Genetics 172: 2015–2023.

8. Discovering PTC Sensitivity
Accidentally discovered in 1930
Bad lab practice!!!
Two categories: tasters and non-tasters
Distinct variation regardless of age, sex, and ethnicity
Demonstrated to follow Mendelian inheritance patterns (NOTE: this is not completely true…more to come later)
Tested chimpanzees in zoos
Result: angry chimps!
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

9. Evolution Question #1
If a trait is found in both humans and chimpanzees, what does that suggest regarding the origin of the trait and the evolutionary divergence of these two species?
Left: Fisher et al (1939), Right: Wooding et al (2006)
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

10. Evolution Question #2
How could you use a cladogram (developed by using the fossil record and DNA studies) and allele DNA sequences to determine which trait is the primitive trait and which is the derived trait?
Primitive trait: inherited from distant ancestors
Derived trait: appeared by mutation in more recent ancestors

11. Explore the DNA Sequences: Gorilla vs. Human

12. Explore the DNA Sequences: Chimpanzee vs. Human

13. Chimpanzee TAS2R38 Receptor
Chimpanzee TAS2R38 non-taster allele replaces start codon, ATG, with AGG
Truncated protein is made beginning with a second ATG (M97)
Protein Gel:
Lane 1: Taster allele
Lane 2: Non-taster allele
Wooding (2006). Nature 440: 845–968.

14. Your DNA Sequence
Find the three SNPs (Hint: there is one in each box, and the sequence numbers are irrelevant)
Check the sequence traces and see if any of the SNPs have heterozygous peaks
Figure out what amino acid your sequence codes for at the SNP
SNP 49 codon is xxx
SNP 262 codon is xxx
SNP 296 codon is xxx
Determine your genotype (e.g. PAV, AVI, or AAV)

15. G Protein-Coupled Receptors (GPCRs)
7 transmembrane (TM) helices
Signal through heterotrimeric G-proteins
Respond to extracellular stimuli
Neurotransmitters
Light
Taste
Smell
Reviewed in Singh et al (2014). Biochemical and Biophysical Research Communications 446:

16. G Protein Coupled Receptors in Sensory Systems
GAMSP 2015

17. A G-protein coupled receptorb a

18. Why “G” proteins? G protein at rest is bound to GDP molecule
When GPCR binds a ligand, causes G protein to exchange GDP for GTP, which activates the G protein

19. G proteins G proteins consist of alpha, beta, gamma subunits
When activated by binding of GTP, the alpha subunit uncouples from the beta-gamma subunits
abg  a + bg

20. Figure 2. 19: (a) Rod receptor showing discs in the outer segment
Figure 2.19: (a) Rod receptor showing discs in the outer segment. (b) Close-up of one disc showing one visual pigment molecule in the membrane. (c) Close-up showing how the protein opsin in one visual pigment molecule crosses the disc membrane seven times. The light-sensitive retinal molecule is attached at the place indicated.
Fig. 3-6, p. 48

21. One of the olfactory receptor proteins
Copyright © 2002 Wadsworth Group. Wadsworth is an imprint of the Wadsworth Group, a division of Thomson Learning

22. A taste receptor cell illustrating the different receptor mechanisms
wolfe-fig jpg

23. Figure 15.15
(a) The tongue, showing the four different types of papillae. (b) A fungiform papilla on the tongue; each papilla contains a number of taste buds. (c) Cross section of a taste bud showing the taste pore where the taste stimulus enters. (d) The taste cell; the tip of the taste cell is positioned just under the pore. (e) Close-up of the membrane at the tip of the taste cell, showing the receptor sites for bitter, sour, salty, and sweet substances. Stimulation of these receptor sites, as described in the text, triggers a number of different reactions within the cell (not shown) that lead to movement of charged molecules across the membrane, which creates an electrical signal in the receptor.
Fig , p. 368

24. 14.3 The tips of the tongues of a nontaster (a) and a supertaster (b) (pink =fungiform papillae)
wolfe-fig jpg

25. Evolutionary relationships among G- protein-coupled receptors
From Structural Biology Knowledgebase

26. Reviewed in Wooding (2006). Genetics 172: 2015–2023.
TAS2R Receptors
Expressed in apical microvillae of bitter-taste receptor cells
Exposed to oral cavity through taste pore opening
Contact compounds as they enter the mouth
Ligands bind and stimulate bitter-taste perception
Bind a diversity of plant toxins
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

27. Bitter Taste Receptors (T2Rs)
25 chemosensory receptors
Members of GPCR superfamily
Mediate signal transduction
Respond to bitter agonists
Also expressed in respiratory system, brain, reproductive tissues, and airways
Mediate protective reflexes
Physiological roles (e.g. bronchodilation)
Reviewed in Singh et al (2014). Biochemical and Biophysical Research Communications 446:

28. Functionality of TAS2R38 Alleles
PAV and AVI alleles from homozygous individuals
Constructed other possible receptor types
NOTE: PAI, AVV, PVV do not correspond to known human haplotypes
Cloned into HEK293 cells
Examined elevated cytosolic [Ca2+] in response to PTC
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

29. Functionality of TAS2R38 Alleles
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

30. Functionality of Chimp TAS2R38 Alleles
Wooding (2006). Genetics 172: 2015–2023.

31. Bufe et al (2005). Current Biology, Vol. 15, 322–327.
TAS2R38 Expression
q-rt-PCR of mRNA levels relative to GAPDH
Grey bars: PAV variant
Black bars: AVI variant
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

32. Psychometric Functional Tasting
Single subjects distinguish between water and [PTC] uM
Graphed % percent correct (50/50 by chance)
Threshold values are defined as 75% performance (inflection point)
PAV (-----)
AVI (………..)
AAI (_____)
Mean recognition for 32 subjects
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

33. Population Studies
Understanding human origin and historical migration patterns
Linguistics
Cultural habits
Socioreligious affiliations
DNA
Genetic studies:
Biological affinity
Extent of diversity in populations
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

34. Population Studies
Mechanisms of evolution produce different gene frequencies in distinct populations
Mutations
Natural selection
Inbreeding
Genetic drift
Determining gene frequency distribution among human populations
Bimodal distribution of phenotypes (e.g. tasters and non-tasters)
Hardy-Weinberg analysis
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

35. Population Studies: Fareed et al Study
Sensitivity to PTC (tasters and non-tasters)
14 serial dilutions starting with 0.13% PTC
Sampled from weak to strong to determine threshold level
Non-taster: could not taste most concentrated solution
Survey of individuals in July – August 2011
State of Jammu and Kashmir
980 individuals
10-30 years old
Six populations: Gujjar and Bakarwal (n = 241), Mughal (n = 142), Khan (n = 173), Malik (n = 145), Mir (n = 151), Syed (n = 128)
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

36. Population Studies: Fareed et al Study
Population Data in Table 1
Calculations of Chi Square indicate statistical significance
Data used to determine allele frequencies
Hardy-Weinberg for determination of allele frequency
p2 + 2pq + q2 = 1
p + q = 1
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

37. Population Studies: Fareed et al Study
Hardy-Weinberg: p2 + 2pq + q2 = 1 and p = 1 - q
Example for Syed Data
Non-tasters = 24 Frequency = (24/128) =
Tasters = 104 Frequency = (104/128) =
Tasters (81.25% of the population) are combined Homozygous (TT) and Heterozygous (Tt)
Non-tasters (18.75% of the population) are Homozygous (tt), defined by H-W as q2
q = the square root of the non-taster frequency =
p = 1 – q = 1 – = p2 = = TT frequency (32.15% of pop)
2pq = 2(0.5670)(0.3215) = = Tt frequency (49.10% of pop)
Frequency of Tasters = TT + Tt = = 81.25% of the population
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

38. Freq. of Homozygous Dominant Individuals: p2
Freq. of tasters:
p2 + 2pq
Freq. of non-tasters: q2
Freq. of T allele: p
Freq. of t allele: q
Freq. of Homozygous Dominant Individuals: p2
Freq. of Heterozygotes:
2pq
Freq. of Homozygous Recessive Individuals: q2
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

39. Population Studies: Fareed et al Study
Little variation in non-taster allele among populations studied
Heterozygosity (% Tt) and Homozygosity (% TT and tt) calculated
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

40. Now, Calculate the Following for Your Class
Frequency of Tasters [(p2 + 2pq) * 100]
Frequency of Non-Tasters or Homozygous Recessive Individuals [(q2) * 100]
Frequency of Recessive Allele [(q) * 100]
Frequency of Dominant Allele [(p) * 100]
Frequency of Homozygous Dominant Individuals [(p2) * 100]
Frequency of Heterozygotes [(2pq) * 100]
Heterozygosity (%Tt)
Homozygosity (%TT + %tt)

42. Importance of Taste and PTC
PTC not found in nature, but detection correlates with ability to taste other naturally occurring bitter substances
Many are toxic (non-tasters are susceptible)
Correlates with dietary preferences that have health effects
Avoidance of bitter-tasting fruits and vegetables may contribute to unhealthy eating patterns (e.g. avoidance of naringin, the bitter ingredient in grapefruit juice) (non-tasters benefit)
Structurally similar to isothiocyanates and goitrin (in cruciferous vegetables)
Anti-cancer effects (non-tasters benefit)
Overconsumption blocks iodine metabolism and leads to thyroid enlargement and goiter-like symptoms (non-tasters more susceptible); countered by iodized salt
Reviewed in Wooding (2004). Am. J. Hum. Genet. 74:637–646. and Tepper (1998). Am. J. Hum. Genet. 63:1271–1276.

43. Importance of Taste and PTC
PROP tasters more sensitive to wide range of oral stimuli
Solutions with caffeine, quinine, and isohumulones (in beer) more bitter to PROP tasters
Some food additives (e.g. sodium benzoate, a preservative, and potassium chloride, a salt substitute) are more noticeable to PROP tasters
Sucrose is more sweet to PROP supertasters
Capsaicin (chili peppers) more hot to PROP tasters
PROP tasters have more overall food dislikes, especially strong-tasting foods (anchovies, sauerkraut, dark beer, black coffee, strong cheeses)
Reviewed in Tepper (1998). Am. J. Hum. Genet. 63:1271–1276.

44. Bitter vs Sweet Taste Preference
Children
Tasters prefer higher concentrations of sucrose solutions than non-tasters
Strong tasters liked cereals and beverages with higher sugar contents than weak tasters and non-tasters
Weak tasters: children were more sensitive than adults to low concentrations of PROP
Adults
No link between genotype and sweet preference
Race/ethnicity strongest determinants of sweet preference
Non-taster mothers perceived their taster children as more emotional than non-taster children
Mennella et al (2005). Pediatrics 115: e216-e222.

45. Discovering PTC Sensitivity
Accidentally discovered in 1930
Bad lab practice!!!
Two categories: tasters and non-tasters
Distinct variation regardless of age, sex, and ethnicity
Timing: scientists exploring human variation
T.H. Morgan Drosophila work (1933 Nobel Prize) showed Mendelian markers inform genomic organization (“linkage groups”)
L.H. Snyder (1931) exploring human Mendelian markers; published six including hair whorl direction, hairy finger joints
Blakeslee (1918) noted variation in human senses (smell of some verbena strains)
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

46. Discovering PTC Sensitivity
Snyder confirmed Fox findings and tested families: concluded non-taster allele is single locus and recessive
Blakeslee confirmed Fox findings: concluded PTC blindness is Mendelian recessive
Fox published description of PTC sensitivity (PNAS, 1932)
Polymorphism
Correlated to variety of related compounds with N=S moiety
Bitterness eliminated with sulfur to oxygen substitution
Blakeslee (1932) published sensitivity can vary by almost five orders of magnitude and suggested other genes are involved (not simple Mendelian trait)
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

47. Genetics and Evolution
R.A. Fisher, E.B. Ford, Julian Huxley: wanted to demonstrate natural selection is an important driving force in evolution
PTC sensitivity to test that natural selection has acted on a specific human gene
2% sugar solutions with 0, 6.25, 50, or 400 ppm PTC
Presented to 8 chimpanzees at Edinburgh Zoo
Angry chimps! 6 of 8 chimpanzees were tasters
Implied allele frequency of 50:50, similar to humans
Broader study: 20 of 27 were tasters; 49 and 51% taster and non-taster allele frequencies (nearly identical to humans)
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

48. Heterozygosity as an Advantage
From Fisher et al 1939 paper: “Without the conditions of stable equilibrium it is scarcely conceivable that the gene ratio should have remained over the million or more generations which have elapsed since the separation of the anthropoid and hominid stocks. The remarkable inference follows that over this period the heterozygotes for this apparently valueless character have enjoyed a selective advantage over both the homozygotes, and this, both in the lineage of the evolving chimpanzees and in that of evolving man. Wherein the selective advantages lie, it would at present be useless to conjecture, but of the existence of a stably balanced and enduring polymorphism determined by this gene there can be no room for doubt.”
Reviewed in Wooding (2006). Genetics 172: 2015–2023.

49. GPCRs and Cancer Many GPCRs are upregulated in breast tumor cells
Chemokine receptors CXCR4, CCR7
Protease-activated receptors
Lysophosphatidic acid receptors
Targets for cancer treatment
Bitter agonists (quinidine and chloroquine) trigger apoptosis through p53 dependent pathway
Bitter melon extract inhibits cell proliferation and promotes apoptosis in breast cancer cells
T2Rs are downregulated in breast cancer cells (Singh et al)
Reviewed in Singh et al (2014). Biochemical and Biophysical Research Communications 446:

50. Bufe et al (2005). Current Biology, Vol. 15, 322–327.
TAS2R38 Expression
rt-PCR of
hTAS2R38 plasmid (P)
No template control (-DNA)
Circumvallate papillae (VP)
No reverse transcriptase (-RT)
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

51. Bufe et al (2005). Current Biology, Vol. 15, 322–327.
TAS2R38 Expression
In situ hybridization of human circumvallate papilla
hTAS2R38 sense probe (left panel)
hTAS2R38 antisense probe (center and right panels)
Bufe et al (2005). Current Biology, Vol. 15, 322–327.

52. Population Studies: Fareed et al Study
Chi-Square Test for statistical analysis
Hardy-Weinberg for determination of allele frequency
P2 + 2pq + q2 = 1
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

53. Population Studies: Fareed et al Study
Chi-square test Example for Syed (n = 128): Expected: ¼ Non-taster (128/4 = 32) Observed: 24 Expected: ¾ Taster (3*138/4 = 96) Observed: 104 X2 = (24-32)2/32 + (104-96) 2/96 = X2 = (-8)2/32 + (8)2/96 = X2 = 64/ /96 = X2 = = X2 = 2.67 DOESN’T MATCH PAPER Total pop: O: 745, 235, E: 735, 245 X2 = =.544 doesn’t match!
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

54. Population Studies: Fareed et al Study
PTC taste thresholds vary
Different for the six populations
Females taste PTC at lower thresholds
Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.