Coming into the Genome Age Part III: The Molecular Genetic Basis of PTC Tasting
By David Micklos
DNA Learning Center (DNALC), Cold Spring Harbor Laboratory
In this article, David Micklos continues his discussion of genetics, and the exciting possibilities for teaching this topic. Please see other issues of Carolina Tips® for Part I , Part II, and Part IV of this fascinating journey into the genome frontier.—Ed.
In "Coming into the Genome Age II," we examined a transposon insertion on human chromosome 16 and single nucleotide polymorphisms (SNPs) in the control region of the mitochondrial chromosome, which have no identifiable phenotypes (traits). In this article, I'll introduce an assay for an SNP that predicts the ability to taste the bitter compound phenylthiocarbamide (PTC). This experiment links genotype to phenotype and puts a contemporary spin on an old standby of classical genetics.
The story began in 1931, when Arthur Fox synthesized some PTC in his laboratory at du Pont Chemical Company in Wilmington, Delaware. After several researchers complained of a bitter taste upon entering his lab, Fox deduced that they were tasting suspended particles of PTC powder. However, he and some others could not taste the compound.
This variable-tasting ability came to the attention of Albert Blakeslee at the Carnegie Department of Genetics on Long Island, New York, the forerunner of my own institution. Blakeslee suspected that PTC tasting is genetically determined. In 1932 he published a population study showing that PTC tasting is inherited as a dominant Mendelian trait.
For 70 years, Blakeslee's genetic description of PTC was definitive: tasters inherit one or 2 copies of a "taster" allele, and nontasters inherit 2 copies of a "nontaster" allele. Then, in 2003, Dennis Drayna and his associates at the National Institutes of Health (NIH) cloned the gene that explains bitter tasting ability, TAS2R38-the 38th member of the 2R family of bitter taste receptors.
Like other taste and olfactory receptors, TAS2R38 is a small intronless gene of about 1,000 nucleotides. It is a member of the family of G protein-coupled, or 7 transmembrane-spanning, receptors. The binding of a ligand (bitter taste molecule) to the extracellular region of the receptor sets up an action potential that conducts an impulse to the sensory cortex of the brain, where it is interpreted as "bitter."
Drayna found that 3 SNPs in the TAS2R38 gene form a haplotype that correlates with bitter tasting. Each of the 3 changes also produces a change in the amino acid sequence of the receptor protein as shown below.
This allows for a fascinatingly imperfect experiment to assay for the SNP at position 145, which has the highest correlation to tasting of the 3 polymorphisms. Students isolate DNA from cheek cells obtained by a simple saline mouthwash and amplify a region of the TAS2R38 gene. The amplified fragment (amplicon) is incubated with the restriction enzyme HaeIII, which includes the SNP in its recognition sequence GGCC. HaeIII cuts the taster allele (with the sequence GGCC), but fails to cut the nontaster allele (GGGC). This creates a length polymorphism, and the 2 alleles are readily separated in an agarose gel.
For best effect, produce the genotypes first, and have each student predict their tasting phenotype. Then bring out the PTC paper and have students score their tasting ability as strong, weak, or nonexistant. The prediction of bitter tasting is good, but not perfect.
Virtually all homozygous nontasters (tt) cannot taste PTC, while homozygous tasters (TT) occasionally report weak or nontasting. The heterozygous genotype (Tt) has the "leakiest" phenotype, with weak or nontasting being fairly common. (This is formally called a heterozygote effect.) Students'; explanations for these discrepancies get to the heart of genetic variability and show the limits of genetic testing.
|Nucleotide Position||Nucleotide Change (Taster > Nontaster)||Codon Change (Taster > Nontaster)||Amino Acid Change (Taster > Nontaster)|
|145||C > G||CCA > GCA||Proline > Alanine|
|785||C > T||GCT > GTT||Alanine > Valine|
|886||G > A||GTC > ATC||Valine > Isoleucine|
Genetic testing and medicine
This experiment offers students a glimpse of the so-called "personalized medicine" that will likely be part of their future healthcare. Using SNPs to predict a tasting phenotype is a close analog to pharmacogenetics, where gene variations are used to predict drug responses. Currently, pharmacogenetics is limited mainly to cancer treatment, where therapies are increasingly tailored to the mutation profile of a specific tumor. However, genetic screening will broaden to include many potent drugs that have known, serious side effects.