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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 - PowerPoint PPT Presentation
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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.