Download Using a Single Nucleotide Polymorphism to Predict Bitter

Using a Single Nucleotide Polymorphism
to Predict Bitter-Tasting Ability
Can you Taste PTC ?
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
 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
 If you have a sweet cell
 Taste sensation is ultimately
determined by the wiring
of a taste cell to the cortex
in the brain
 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
 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 Weed
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
Punnett Square
Molecular Genetics of PTC Tasting
 Gene identified in 2003 by Dennis Drayna TAS2R38 gene
 Polymorphism associated with PTC tasting
 SNP(Single Nucleotide Polymorphism)--at position 145
Taster = C
Nontaster = G
 Change in Amino acid 49 …. (proline)  (alanine)
Analysis of the Trait--CAPS
 Cleavage amplified polymorphisms
 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 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 Alignment
Chimp_bitter_taste
Gorrilla_bitter_taste
Human_PTC_Non-taster
Human_PTC_taster
Pan_paniscus
CCTTCGTTTTCTTGGTGAATTTTTG
CCTTCGTTTTCTTGGTGAATTTTTG
CCTTCGTTTTCTTGGTGAATTTTTG
CCTTCGTTTTCTTGGTGAATTTTTG
CCTTCGTTTTCTTGGTGAATTTTTG
25
Chimp_bitter_taste
Gorrilla_bitter_taste
Human_PTC_Non-taster
Human_PTC_taster
Pan_paniscus
GGATGTAGTGAAGAGGCAGCCACTG
GGATGTAGTGAAGAGGCAGCCACTG
GGATGTAGTGAAGAGGCAGGCACTG
GGATGTAGTGAAGAGGCAGCCACTG
GGATGTAGTGAAGAGGCAGCCACTG
50
Chimp_bitter_taste
Gorrilla_bitter_taste
Human_PTC_Non-taster
Human_PTC_taster
Pan_paniscus
AGCAACAGTGATTGTGTGCTGCTGT
AGCAACAGTGATTGTGTGCTGCTGT
AGCAACAGTGATTGTGTGCTGCTGT
AGCAACAGTGATTGTGTGCTGCTGT
AGCAACAGTGATTGTGTGCTGCTGT
75
Chimp_bitter_taste
Gorrilla_bitter_taste
Human_PTC_Non-taster
Human_PTC_taster
Pan_paniscus
GTCTCAGCATCAGCCGGCTTTTCCT
GTCTCAGCATCAGCCGGCTTTTCCT
G T C T C A G C A T C A G C C G G C T T T T C C T 100
GTCTCAGCATCAGCCGGCTTTTCCT
GTCTCAGCATCAGCCGGCTTTTCCT
 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
Nontaster
141
141
GCAGGCAGCCACT
GCAGGCAGGCACT
 http://bioinformatics.dnalc.org/ptc/animation/ptc.html
After PCR


Taster
Nontaster
HaeIII cut site
TAGTGAAGAGGCGGCCACTG
TAGTGAAGAGGCGGGCACTG
 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
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.
How are these techniques different
from that used in forensic crime lab?
 We use a SNP and RFLP
 Restriction 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 headto-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.