Bitter Taste Perception of Phenylthiocarbamide - Research Paper Example

BIOMOLECULAR TECHNIQUES (BITTER TASTE PERCEPTION OF PHENYLTHIOCARBAMIDE (PTC) By Presented to ABSTRACT In the 1930’s a chemist in Dupont by the name of Arthur Fox, synthesized the chemical phenylthiocarbamide (PTC). A PTC cloud was generated while he transferred the compound to a bottle that caused his lab mate C. R. Noller to complain of its intense bitterness while Fox could not sense any taste (Fox, 1932). Since this discovery, there have been different variation in an individual’s ability to taste the compound (as well as other bitter compounds like those present in broccoli) has since become a common study for human genetic traits. In general, sensitivity to PTC seems to be inherited via Mendelian traits constituting two alleles: T for the taster and the nontaster- t; however in reality it is a complicated inheritance aspect. Today it is known that the sensitivity to PTC is mediated by a gene identified the TAS2R38 or PTC gene that encodes a receptor for bitter taste ( coupled receptor heteromeric G-protein) that is located on the tongue’s surface cells. The analysis focused on determining the presence or absence of the PTC gene amongst a group of 108 students. The results were compared to European and Sub-Saharan cohorts. It was found that he resulted closely matched to those of the European results (ABRF 96: Biomolecular Techniques. An International Symposium Sponsored by The Association of Biomolecular Resource Facilities, 1996). 1.0 Introduction There have been additional PTC gene studies in humans around the world which have shown that exist two main alleles, the taster and nontaster. However, five other uncommon alleles that have an effect on the taster phenotype have been revealed. These varying gene code forms for proteins differ in their being able to bind to PTC and other bitter compounds. The two main PTC gene alleles differ from each other by three single nucleotide polymorphisms (SNPs) as shown in the figure 1. SNPs are representative of the simplest genetic variation type found in individuals. SNP in a genome refers to an exact location where diverse groups of people have differing nucleotides. If more than 1% percent of any given population possess different base thin a specific location, then that location is termed as an SNP. If a population has less than 1% possessing a different base, then it is termed as a mutation (Arimoto, 2005). Fig 1 Polymorphic Sites in the PTC Taster Gene Nucleotide position Taster Nontaster 145 CCA pro GCA ala 785 GCT ala GTT val 886 GTC val ATC ile The common allele for nontaster (also referred to as haplotype) has a position 145 G nucleotide (G145), position 785 T nucleotide (T785) and position 886 A nucleotide (A886). The nontaster allele generates polypeptides with isoleucine, valine and alanine at these sites hence also called the AVI allele. On the other hand, the main taster allele has G886, C145, and C785 and generates polypeptides with valine, alanine, and proline at these sites, hence also called the PAV allele (Banting and Clark, 2012). The SNP at 785 position is used in determining individual PTC genotype as the taster sequence on this location forms a restriction site (Fnu4H1) that replaces in the nontaster allele C785 with T785 eliminating the restriction site. A restriction site refers to specific DNA sequence recognized and bound by specific restriction endonuclease or enzyme (Brewer et al., 2012). There are within the PTC gene, two additional Fnu4H1 restriction sites, but in the analysis primers that were specifically designed so that they could bind to regions that flanked the one Fnu4H1 polymorphic site excluding the two. These primers were used in PCR to generate a fragment of 303 base pair (bp). This PCR product restriction digestion with Fnu4H1 yielded one nontaster allele fragment of 303 bp and two other short 65 bp and 239 bp fragments for the taster allele. Fig 2 The Fnu4H1 Restriction Site 5’-GCNGC-3’ and 3’-CGNCG-5.” W here N is any of the four different bases The three SNPs are in blue while the nontaster allele nucleotide allele is indicated below the taster. The forward and reverse positions of PCR primers are indicated in green color and red, respectively. The underlined Fnu4H1 site at position 785 is what was exploited . The lab analysis was to determine individual genotype and phenotype at the PTC location through taste tests using phenylthiocarbamide (PTC). Individual DNA was extracted and purified from buccal, and PCR setup. The PCR amplified an SNP region of individual DNA contained within the PTC gene. The lab tests also determined individual PTC genotype via PCR product digestion with the Fnu4H1restriction enzyme that eliminates SNP taster variant but not in the nontaster variant followed by an analysis of the products using agarose gel electrophoresis. METHOD In the first week, Phenyl Thiocarbanate (PTC) Polymerase chain reaction (PCR) based genetic analysis of the PTC genotype using human-specific DNA primers was done. The practical was to match taste phenotype (super taster +/+; mild taster +/-; non-taster -/-) to individual genotype. Using a wooden sterile splint, I scraped the inside of my cheeks to remove the loose buccal cells. To an Eppendorf containing buccal cells & Chelex, four μl of Proteinase K (Stock solution; 10mg/ml) was added. This was incubated at 56˚C for 30 minutes. The tube was briefly vortexed for ten seconds then centrifuged at maximum (13,000 rpm) speed for 20 seconds while ensuring the centrifuge was balanced. The tube containing Chelex/cells was placed in a water bath/heating block at 98˚C for 15 min. The tube was vortexed for 10 seconds and then centrifuged at maximum speed for 3 min. The supernatant was transferred containing buccal cell DNA (Template), to a sterile 1.5 ml Eppendorf tube. An aliquot (5 µl) of the sample was taken as well as the aliquot to be measured using the Nanodrop nucleic acid measurement machine and concentration (ng/µl; A260, A280, 260/280, 260/230) was retained for reference. Residual DNA was frozen and retained for the PCR reaction that was undertaken the following week. In the second week, individuals were provided each with a micro-eppendorf tube with which you the PCR reaction was assembled. The master mix contained all of the PCR reaction components permitting detection of individual PTC gene. However, it did not contain the template DNA. The following was added to one tube: 43.5l Master mix (already prepared) and 6.5l Template DNA prep. (Buccal cell DNA)(Total volume = 50l). This was mixed to dissolve (flicking with a finger), and then pulse spinned to gather the liquid contents to the bottom of the tube. The tube was placed in template for thermal cycler, ensuring that the lid was securely on, and position of tube on matrix sheet was noted and run using the following program: 94˚C 4 minutes 55˚C 40 seconds 72˚C 40 seconds 40 cycles 94˚C 40 seconds 55˚C 5 minutes 72˚C 5 minutes In the third week, agarose gel electrophoresis was done following Biomolecular Techniques RFLP Protocol. PCR tube was accessed carefully using the template for ID. An aliquot (20 µl) was removed from my PCR tube, and a 0.5 ml Eppendorf tube already containing ten µl of Fnu4HI restriction enzyme master mix (total now 30 µl) Added. The tube was not vortexed. Tube contents were mixed by flicking with a finger centrifuged briefly and place in 37°C heating block/water bath. The tube was uniquely marked on tube/heating block/water bath template where the Fnu4HI digested PCR tube was located. The previous week’s PCR tube was returned to its original position in the container.The Fnu4HI restriction enzyme digest required a minimum of 2hrs. A setup of 2% submerged agarose gel; Geneflow tanks (PurpleLids, 12 well comb) require 70 ml, Peqlab / Hybaid tanks (10 well comb) require 50ml agarose, was done and left to set. In the morning, three µl of DNA loading buffer (x5) was added to a tube containing 12 µl PCR/R Enzyme Digest. Further, ) three µl of DNA loading buffer (x5) was added to a tube containing 12 µl residual PCR. finger and pulse mixed this spun both tubes (total 15 µl) and 8 – 10 µl added of this mixture to a 2% agarose gel submerged in TBE buffer while noting the lanes I had loaded (UD & Digested). 8-10 µl of 100 bp DNA Ladder was added to remaining well as a DNA marker. Electrophoresis at 90 V for 45min until blue marker is halfway through gel was done. Photograph of gel under UV trans-illumination was done. Ethidium Bromide &Agarose gel was disposed of off responsibly in a designated container. Results and Discussion Distance traveled (mm) Ladder fragment (Log) 11 3 12 2.954242509 13 2.903089987 14 2.84509804 16 2.77815125 18 2.698970004 20 2.602059991 23 2.477121255 27 2.301029996 32 2 Representative Graph plotted from the data obtained from the Log (MWT) against distance traveled by the ladder fragments (data are on the table). Calculation: From the graph the equation is y = -0.0458x + 3.5083 In which Y= Molecular Weight fragment, X= Traveled Distance Unknown MW of undigested DNA as measured by ruler ( 23 mm) y = -0.0458x + 3.5083 y = -0.0458(23) + 3.5083 y = -1.0534 + 3.5083 y = 2.4549 y = anti log 2.4549 y= 285.036 Unknown MW of Digested DNA as measured by ruler ( 25 mm) y = -0.0458x + 3.5083 y = -0.0458(25) + 3.5083 y = -1.145+ 3.5083 y = 2.3633 y = anti log 2.3633 y= 230.834 Determination of PTC tasting genotype : The agarose gel that was loaded with both my and other student samples of digested as well as undigested PCR products, and subsequently electrophoresed and displayed in an image using UV light as shown in the above figure 1. No distinct bands were seen in lanes 3,4, 5,6,11 and 12 at 303bp position for the undigested products inferring that these samples were from non-tasters. Hardy-Weinberg Equilibrium Calculations: The total student genotypic and phenotypic was used to calculate PTC gene allele frequency using the equation: ( p2 + 2pq +q2 = 1, where p represents frequency of allele that is dominant and q represents recessive allele frequency in order to determine probability of genotype frequencies for both the group’s tasters and non-tasters. The same was done for the entire class students. The results were compared to those of the RFLP determination. There were 72 tasters and 36 nontasters. The frequency from the calculations showed a result of dominant and recessive alleles was equal to 0.43 and 0.57 respectively with the Hardy-Weinberg equilibrium equation suggesting that a population of ten people should include 3 non-tasters and 7 tasters on reaching equilibrium within evolution. Data from 10 students showed, three heterozygous recessive, one homozygous dominant and three homozygous recessive replicating the similar numbers at equilibrium. Analysis using BLAST The summary of the BLAST search results are shown here below: Name: ? Genotype: tt (non- taster phenotype) Comparison with PTC Non-Taster Allele E-Value = 3.7 * 10 ^ -156 Score = 558 % Query = 98.6 Mutations: ‘-‘ in place of A at 247 bp ‘-‘ in place of A at 250 bp R in place of A at 253 bp Comparison with PTC Non-Taster Allele E-Value = 8.6 *10 ^ -145 Score = 521 % Query = 100 Mutations: G in place of A at 57 bp The ability of an individual to taste the phenylthiocarbamide is absent in some individual as was indicated by the group phenotypic data. This variation within a population is due to the polymorphism in the PTC TAS2R38 receptor gene. The study conducted was to find the relationship between the different phenotypes that is tasters and non-tasters, and the different genotype that is homozygous recessive or dominant, or heterozygous. Students that had at least one allele that is dominant that helped them in tasting PTC. In order for the different students to determine their genotypes, their extracts of DNA were digested using the recognition enzyme that was then analyzed using electrophoretic separation of both the undigested and digested PTC receptor genes. The electrophoresed gel showed no clear bands for the homozygous recessive nontasters and a combination of clear and faint for the heterozygous mild tasters. The faint bands that were also observed could indicate improper amplification resulting from the inadequate use of reagents in carrying out the reaction. Students that were unable to taste PTC bitterness including myself were already pre-determined as being homozygous recessive. The presence of a single band at the 303 bp representing the digested product indicated that the Fnu4H1 restriction enzyme failure to cut at PTC gene 303 bp fragment because of the failure of recognizing restriction site at 785 nucleotide position (where C is replaced by T) for both tt alleles. The genotypic and phenotypic results are, therefore, harmonious with each other. A BLAST search of the entire PTC genes sequenced was done. Analysis using Electrophoretic gel showed I am homozygous recessive with tt taster alleles. Comparison of the sequence of both non-taster and taster from two BLAST searches resulted in a 100% query result for non-taster and 98.6 % query result for taster allele was observed. The non-taster allele E-value was higher than that of the taster indicating that my alleles were more similar to non-taster than taster alleles. The phenotypic and genotypic results that were obtained from the student group were compared to those of the European and Sub-Saharan cohorts. A CHI mini-tab was created and it the results showed Likelihood Ratio Chi-Square = 0.001; DF = 1; P-Value = 0.974 for the European group and Likelihood Ratio Chi-Square = 38.405; DF = 1; P-Value = 0.000. The results would closely match with those of the European group. The frequency as a percentage of student tasters was 20%, mild tasters at 47% and nontasters at 33%. The European group indicates 20% for tasters, 50% for mild tasters and 30% for nontasters. The students’ allele frequency for C was 43% and 57% for T. This was similar in comparison with the European group at 43% for C and 57% for T. PTC is an unnatural organic compound; that contains isothiocyanate. In its natural form, it is present in vegetables such as Brussels sprouts, broccoli, and cabbage. PTC receptor is used as a body’s defense mechanism against bitter and harmful toxins (Wooding et al., 2004;). An individual’s ability for tasting PTC and their subsequent liking of certain food types influences their diet that affects their health (Floriano et al., 2006). This helps in a better understanding of human health ad diet relationship. PTC taste sensitivity indicates a wide and continuous distribution (for example it behaves similarly to a quantitative trait). On average, taste sensitivity to PTC is highest for tasters (the PAV/PAV) homozygotes, significantly but slightly lower for heterozygotes (PAV), and the least for non-taster (PAV/AVI) homozygotes. (Kim et al., 2003)There are rare heterozygotes (AVI/AAV) having an average TC score that is slightly and significantly higher compared to the homozygotes (AVI/AVI). All primates that are non-human that have been examined to date have shown to be homozygous haplotype (AVI/AVI). Thus, the nontaster AVI haplotype came about after humans evolved from their most common and recent primate ancestor. In addition, there exist chimps that are non-taster and having same genes as the humans, but with a different mutation from man. References ABRF 96: Biomolecular Techniques. An International Symposium Sponsored by The Association of Biomolecular Resource Facilities. (1996). Glycobiology, 6(1), pp.6-6. Arimoto, R. (2005). 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