PhenylthiocarbamideBiochemistry, 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

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!!!

Class Data

• Rank 1: _____ people• Rank 2: _____ people • Rank 3: _____ people • Rank 4: _____ people• Total #: _____ people

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.

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.www.pediatrics.org/cgi/doi/10.1542/peds.2004-1582

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.www.pediatrics.org/cgi/doi/10.1542/peds.2004-1582

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

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.

Evolution Question #1If 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?

Reviewed in Wooding (2006). Genetics 172: 2015–2023.

Left: Fisher et al (1939), Right: Wooding et al (2006)

Evolution Question #2How 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

http://www.geojeff.org/course-materials/historical-geology-lab/lab-3-dna/atoms-molecules-dna-and-more/

Explore the DNA Sequences:Gorilla vs. Human

Explore the DNA Sequences:Chimpanzee vs. Human

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.

Your DNA Sequence

1. Find the three SNPs (Hint: there is one in each box, and the sequence numbers are irrelevant)

2. Check the sequence traces and see if any of the SNPs have heterozygous peaks

3. 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

4. Determine your genotype (e.g. PAV, AVI, or AAV)

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: 499-503.http://dx.doi.org/10.1016/j.bbrc.2014.02.140

G Protein Coupled Receptors in Sensory Systems

GAMSP 2015

A G-protein coupled receptor

abg

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

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

Fig. 3-6, p. 48

One of the olfactory receptor proteins

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A taste receptor cell illustrating the different receptor mechanisms

Fig. 15-15, p. 368

14.3 The tips of the tongues of a nontaster (a) and a supertaster (b) (pink =fungiform papillae)

Evolutionary relationships amongG- protein-coupled receptors

From Structural Biology Knowledgebase http://sbkb.org

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.

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: 499-503.http://dx.doi.org/10.1016/j.bbrc.2014.02.140

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.

Functionality of TAS2R38 Alleles

Bufe et al (2005). Current Biology, Vol. 15, 322–327.

Functionality of Chimp TAS2R38 Alleles

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

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.

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)

Bufe et al (2005). Current Biology, Vol. 15, 322–327.

PAV (-----)AVI (………..)AAI (_____)

Mean recognition for 32 subjects

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.

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.

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.

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 = 1p + q = 1

Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

Population Studies: Fareed et al Study

Hardy-Weinberg: p2 + 2pq + q2 = 1 and p = 1 - q

Example for Syed DataNon-tasters = 24 Frequency = (24/128) = 0.1875Tasters = 104 Frequency = (104/128) = 0.8125

• 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 = 0.4330p = 1 – q = 1 – 0.4330 = 0.5670 p2 = 0.3215 = TT frequency (32.15% of pop)2pq = 2(0.5670)(0.3215) = 0.4910 = Tt frequency (49.10% of pop)Frequency of Tasters = TT + Tt = 32.15 + 49.1 = 81.25% of the population

Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

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

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.

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)

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.

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.

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 childrenMennella et al (2005). Pediatrics 115: e216-e222.

www.pediatrics.org/cgi/doi/10.1542/peds.2004-1582

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.

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.

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.

Heterozygosity as an AdvantageFrom 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.

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: 499-503.http://dx.doi.org/10.1016/j.bbrc.2014.02.140

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.

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.

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.

Population Studies: Fareed et al Study

Chi-square testExample for Syed (n = 128):Expected: ¼ Non-taster (128/4 = 32) Observed: 24Expected: 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 = 64/32 + 64/96 = X2 = 2 + 0.67 =X2 = 2.67 DOESN’T MATCH PAPER

Total pop: O: 745, 235, E: 735, 245 X2 = .136+.408=.544 doesn’t match!

Fareed et al (2012). The Egyptian Journal of Medical Human Genetics 13, 161–166.

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.