Can you Taste PTC ?

Using a Single Nucleotide Polymorphism to Predict Bitter-

Tasting Ability

Taste in MammalsMammals can

distinguish only five basic tastes

SweetSourBitterSalty Umami (the taste

of monosodium gluatmate)

Taste in MammalsTaste perception is a two-step process

1st…A taste molecule binds to a specific receptor on the surface of a taste cell

2nd …The taste cell generates a nervous impulse, which is interpreted by the brain

An Example: Taste in MammalsStimulation 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 MammalsTaste recognition is

mediated by specialized taste cells that communicate with several brain regions through direct connections to sensory neurons

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

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

Albert Blakeslee with Jimson WeedCarnegie 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

Punnett Square

Molecular Genetics of PTC TastingGene identified in 2003 by Dennis Drayna

TAS2R38 genePolymorphism associated with PTC tastingSNP(Single Nucleotide Polymorphism)--at

position 145

Taster = C Nontaster = G

Change in Amino acid 49 …. (proline) (alanine)

Analysis of the Trait--CAPSCleavage amplified polymorphismsAmplify a region of TAS2R38 gene by PCRPrimers used in the experiment:

CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG

AGGTTGGCTTGGTTTGCAATCATC

Then cut with restriction enzyme (HaeIII)

RFLP-Restriction Fragment Length Polymorphism

Analysis by eletrophoresis

2% Agarose Gel Electrophoresis

What is the relationship between this trait and our ancestors?

What is the normal state?

To taste or to not taste?

Multiple Sequence AlignmentChimp_bitter_tasteGorrilla_bitter_tasteHuman_PTC_Non-tasterHuman_PTC_tasterPan_paniscus

CCCCC

CCCCC

TTTTT

TTTTT

CCCCC

GGGGG

TTTTT

TTTTT

TTTTT

TTTTT

CCCCC

TTTTT

TTTTT

GGGGG

GGGGG

TTTTT

GGGGG

AAAAA

AAAAA

TTTTT

TTTTT

TTTTT

TTTTT

TTTTT

GGGGG

25

 Chimp_bitter_tasteGorrilla_bitter_tasteHuman_PTC_Non-tasterHuman_PTC_tasterPan_paniscus

GGGGG

GGGGG

AAAAA

TTTTT

GGGGG

TTTTT

AAAAA

GGGGG

TTTTT

GGGGG

AAAAA

AAAAA

GGGGG

AAAAA

GGGGG

GGGGG

CCCCC

AAAAA

GGGGG

CCGCC

CCCCC

AAAAA

CCCCC

TTTTT

GGGGG

50

 Chimp_bitter_tasteGorrilla_bitter_tasteHuman_PTC_Non-tasterHuman_PTC_tasterPan_paniscus

AAAAA

GGGGG

CCCCC

AAAAA

AAAAA

CCCCC

AAAAA

GGGGG

TTTTT

GGGGG

AAAAA

TTTTT

TTTTT

GGGGG

TTTTT

GGGGG

TTTTT

GGGGG

CCCCC

TTTTT

GGGGG

CCCCC

TTTTT

GGGGG

TTTTT

75

 Chimp_bitter_tasteGorrilla_bitter_tasteHuman_PTC_Non-tasterHuman_PTC_tasterPan_paniscus

GGGGG

TTTTT

CCCCC

TTTTT

CCCCC

AAAAA

GGGGG

CCCCC

AAAAA

TTTTT

CCCCC

AAAAA

GGGGG

CCCCC

CCCCC

GGGGG

GGGGG

CCCCC

TTTTT

TTTTT

TTTTT

TTTTT

CCCCC

CCCCC

TTTTT

100

Advantage: Taste or not to taste?

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)

How does HaeIII Cut the taster allele?

Hae III restriction site = GGCC

In the regions around the 145 SNP

Taster 141 GCAGGCAGCCACT Nontaster 141 GCAGGCAGGCACT

http://bioinformatics.dnalc.org/ptc/animation/ptc.html

After PCR HaeIII cut site

Taster TAGTGAAGAGGCGGCCACTG Nontaster TAGTGAAGAGGCGGGCACTG

How does the Hae III enzyme discriminate between the C-G polymorphism in the TAS2R38 gene.

HaeIII cuts at the sequence GGCCThis is at the 143-145 position of the geneThe nontaster has a GGGC and won’t cut

Many people are nontasters…more than what is expected if bitter taste was the ONLY trait under natural selectionSO…. 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.

How are these techniques different from that used in forensic crime lab?

We use a SNP and RFLPRestriction fragment length

polymorphism, or RFLP (commonly pronounced “rif-lip”), is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.

Forensics Labs use:Variable Number Tandem Repeat (VNTR)

-A tandem repeat is a short sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal locus. Tandem repeats are interspersed throughout the human genome. Some sequences are found at only one site -- a single locus -- in the human genome. For many tandem repeats, the number of repeated units vary between individuals. Such loci are termed VNTRs. One VNTR in humans is a 17 bp sequence of DNA repeated between 70 and 450 times in the genome. The total number of base pairs at this locus could vary from 1190 to 7650.

A short tandem repeat (STR) in DNA occurs in non-coding region when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 5 bp bp (for example (CATG)n in a genomic region) and is typically in the non-coding intron region.

A short tandem repeat polymorphism (STRP) occurs when homologous STR loci differ in the number of repeats between individuals. By identifying repeats of a specific sequence at specific locations in the genome, it is possible to create a genetic profile of an individual. There are currently over 10,000 published STR sequences in the human genome. STR analysis has become the prevalent analysis method for determining genetic profiles in forensic cases.