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

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

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

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

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!

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

Taste in Mammals While there are only 5 tastes there are thousand more olfactory (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

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 felt no taste

Albert Blakeslee with 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

Albert Blakeslee, AAAS Convention, 1938

Inheritance pattern

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)

Analysis of the Trait Isolate DNA from human cheek cells 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

Analysis of the Trait

Analysis of the Trait 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 won’t cut

Analysis by eletrophoresis

2% Agarose Gel Electrophoresis TT Tt tt Marker U C U C U C Marker 221 bp 177 bp 44 bp

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)

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

Multiple Sequence Alignment

Advantage: Taste or not to taste?

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

Compare primer to sequence: CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG primer anneal TTTTTGGGATGTAGTGAAGAGGCGG 3’ AAAAACCCTACATCACTTCTCCGTC Taster 5’ TAGTGAAGAGGCAGCCACTG Nontaster TAGTGAAGAGGCAGGCACTG

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

Evolution of .. 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.

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