The Genetics of Human Bitter Taste Perception



This lab will explore the relationship between DNA (genotype), protein, phenotype, and inheritance by investigating…..

The Genetics of Human Bitter Taste Perception

Amy Rice Doetsch & Alex Doetsch, CSI Biology

The visible characteristics, or phenotypes, of organisms, cells, and viruses are the direct result of their genomes. Genes that are present in, and expressed by, that organism’s genome produce proteins that perform the specific functions of that cell or virus. Different organisms have different genomes that contain different sets of genes; therefore, organisms will produce different proteins which will give them different phenotypes, thereby distinguishing one organism from another. As you know from your lectures on genetics, it’s the genes on the chromosomes that are passed from one generation to the next during reproduction. If genes contain the information needed to produce proteins (and they do), and each individual has a slightly different genome from his or her neighbour, then each individual is potentially going to produce slightly different versions of the same protein (different versions of the same protein are known as isoforms). It’s the different versions, or isoforms, of proteins that produce the different phenotypes that we see. For example, a person with naturally curly hair produces a different isoform of the protein keratin than someone with naturally straight hair does.

It’s important to note that although there are small changes in the genome between one individual and the next, all of the cells within a single individual contain EXACTLY the SAME genome. For example, every one of the trillion+ cells that make up your body contains exactly the same set of genes. However, even though ALL of the cells in a multi-cellular organism, like humans, have exactly the same genome, the genome of a liver cell gets expressed in different ways from the genome of a heart cell – that’s what makes them different types of cells.

Today’s lab will focus on one particular gene, and its corresponding protein, that are involved in your ability to taste bitter compounds. The gene is called Taste Receptor type 2 member 38 or TAS2R38 for short; the protein that the gene codes for is called TAS2R38. All of us have the TAS2R38 gene and the TAS2R38 protein receptor, but we don’t necessarily share the same alleles of that gene or isoforms of the protein. Remember, different forms of a gene are called alleles, and different forms of a protein are known as isoforms. Today you will discover which alleles of the TAS2R38 gene your genome contains and which isoforms of the TAS2R38 protein are produced by your taste receptor cells.

The TAS2R38 protein, along with the taste receptors for sweet, sour, salty and umami are normally found on the surface of taste receptor cells that are located within the taste buds on the tongue (Kim et. al., 2004). While researchers have a lot to discover about human’s ability to taste bitter compounds, they do know that the bitter tasting compound phenylthiocarbamide (PTC) (See Figure 1) does interact with TAS2R38. PTC is perceived as bitter by approximately 75% of the human population, although that percentage varies slightly by ethnic groups (Kim and Drayna, 2004). For example, in the Native American population of North America, 100% of people perceive PTC as tasting bitter while about 73% of Europeans, and their descendants, perceive the bitter taste of PTC (Kim et. al., 2004). The ability to taste PTC as bitter, or not, depends on the combination of alleles an individual has for the TAS2R38 gene.

Figure 1: The chemical structure of PTC. PTC is not a naturally occurring compound, but it is structurally related to chemical compounds that occur in vegetables such as broccoli, cabbage and Brussels sprouts.

There are five known alleles of the TAS2R38 gene, and therefore five isoforms of TAS2R38. Two of these alleles (AAI and PVI) are found only in African populations, and a third allele (AAV) is found only infrequently (Kim et. al, 2004; Kim and Drayna, 2004; Wooding et. al., 2004). The major focus of our lab today will be the two most common alleles, PAV and AVI.

#1: INDIVIDUAL ACTIVITY: Are you a taster or a non-taster?

1. Obtain a single PTC taste strip from your lab instructor. To determine if you are a taster or a non-taster, place the strip on your tongue. Let the strip sit on your tongue for 2-3 seconds. Following that time period, determine if you are a strong taster, an intermediate taster or a non-taster. If you’re unsure what you are follow the guidelines below:

• A strong taster will know long before 3 seconds are up, and will desperately want to drink something to get rid of the bitter flavour. The strong tasters in the class are homozygous dominant (PAV/PAV).

• An intermediate taster will recognize that the strip has a flavour but it may take a second or two for them to realize that or they may not think it tastes that bad. The intermediate tasters are probably heterozygous (AVI/PAV).

• A non-taster will try chewing on the strip, thinking that they will uncover some sort of hidden flavour. However, no matter what a non-taster does, the strip will never taste like more than just paper. The non-tasters in the class are homozygous recessive (AVI/AVI).

2. Record the class results:

|Phenotype (taste perception) |Genotype |# students |

| | | |

| | | |

| | | |

What was the experimental control? Was it a positive or negative control?

#2: INDIVIDUAL ACTIVITY: Patterns of Inheritance

1. If there are 2 alleles but 3 possible phenotypes, what kind of inheritance pattern does that suggest to you?

2. Provide another example (it doesn’t have to be in humans) of the type of inheritance pattern you listed in Question #1.

#3: GROUP/TABLE ACTIVITY: Comparing alleles of the TAS2R38 receptor gene

So what’s the molecular or cellular basis for why some people can taste PTC and some people can’t?

1. Obtain a laptop from the computer cart, being sure to carefully unplug the computer from the cart before removing it. Make sure that your table is clean and free of any spills before setting up your laptop. If you have your own laptop, please feel free to use it instead.

2. To begin with, you will need to retrieve the two DNA sequences that correspond to the Taster & Non-taster alleles from the Biology 201 Canvas webpage. Save this sequence file (Called PTCgenes.doc) to the desktop of the laptop you are using.

3. To determine any genetic differences between the two alleles, you will need to head to the NCBI website. This is a website maintained by the National Institutes of Health and the National Library of Medicine and serves as the premiere bioinformatics website in the country. (). Access to this website is free for anyone who wants to use it, which is an example of scientific data being totally accessible to not only the public, but to other researchers as well. Contained within the NCBI site is a database known as GENBANK. This data base is the repository for many of the known DNA sequences to date, both genes and non-coding DNA sequences. GENBANK is also the location of all of the data from the Human Genome Project.

4. On the right hand side, under the heading “Popular Resources”, click on the link that says BLAST. On the new page that opens, click the box “Nucleotide BLAST”.

5. A new window will open. About ½ way down the page, make sure the box “Align two or more sequences” is checked.

6. You will see two empty boxes, one with a heading “Query”, the other “Subject”. From the file you downloaded from Canvas, select & copy the Taster DNA sequence (the nucleotide sequence only, not the heading) and then paste that copied DNA sequence into the box labeled “Query”.

7. Go back to the file you downloaded from Canvas, select & copy the Non-taster DNA sequence and then paste that copied DNA sequence into the box labeled “Subject”.

8. Double check that you’ve pasted both the Taster sequence into the “Query” box and the Non-taster sequence into the “Subject” box.

9. Once you’re sure both sequences are in place, click on the button at the bottom of the page that says “BLAST”.

10. A new window will open. This window contains the 2 DNA sequences that you pasted in, but the program has aligned them. That is, the two DNA sequences have been compared to each other, and their similarity has been determined.

11. The information at the top of this window is the parameters that you didn’t change – ignore these.

12. Scroll down a bit further until you see actual DNA sequence (A, C, T & G’s). This information is the DNA sequence you pasted in.

13. The Query sequence is your Sequence #1 (Taster) and the subject sequence is your Sequence #2 (Non-taster). The comparison of the two nucleotide sequences is shown in 60 nucleotide sections for clarity; each sequence is actually 1059 continuous nucleotides.

14. The vertical lines you see between the two sequences indicate that the nucleotide at that position is identical to the nucleotide at the same position on the other sequence. There are 1059 nucleotides in each one of the DNA sequences; the vast majority of those will be identical.

15. Scroll down the page until you see nucleotides that do NOT have a line between them. This indicates that the nucleotides at that position are different in the two sequences. It is these differences that distinguish between a person’s ability to taste PTC and a person’s inability to taste PTC.

16. How many nucleotide differences can you find between the two DNA sequences?

17. For each nucleotide difference between the two DNA sequences,

a) Determine the exact nucleotide position (this will be a number between 1-1059) of the difference between the sequences. This can be done by counting nucleotides along the row from left to right (starting counting with the nucleotide number to the left of the row) until you reach the nucleotide difference then add that number to the nucleotide position listed at the left of the row you counted. List the position of the nucleotide difference in table 2 at the end of this lab.

b) Then, list the nucleotide that occurs in the Taster DNA and the nucleotide that occurs in the Non-taster DNA.

18. You’ve determined the genetic basis for the difference between tasters, intermediate tasters & non-tasters. Individuals of each phenotype have inherited copies of the gene from their parents, one copy from their mother and one copy from their father. Depending on the combination of alleles a person inherits, they can either taste PTC (homozygous dominant), sort of taste PTC (heterozygous) or not taste PTC at all (homozygous recessive). Keep in mind that we’ve been investigating alleles, which refers to DNA sequences & nucleotide differences.

19. Now think back to what you know about macromolecules & answer the following questions:

a. What role does DNA play in the cell?

b. What type of macromolecule would typically be referred to as a receptor (lipid, DNA etc.)?

c. Is the molecule you listed in “b” a polymer?  If yes, what monomers is it composed of?

d. If the DNA nucleotide sequence is altered, what specific effect would this have on the receptor?

#4 GROUP/TABLE ACTIVITY: Comparing isoforms of the TAS2R38 receptor protein

1. To begin with, you will need to retrieve the two protein sequences that correspond to the taster & non-taster isoforms from the Biology 201 Canvas webpage. Save this sequence file (called PTCproteins.doc) to the desktop of the laptop you are using. For this activity you will only be using the 2 human proteins; you will use all 4 proteins in Activity #6.

2. To determine the differences between the two isoforms, you will need to head back to the NCBI website.

3. On the right hand side under the menu labeled “Popular Resources”, click on the link that says BLAST. On the new page that opens, click the box “Nucleotide BLAST”.

4. A new window will open. About ½ way down the page, make sure the box “Align two or more sequences” is checked.

5. A new window will open; choose blastp from the tabs at the top of the page. (BLASTP is used for protein sequences.)

6. Make sure the “Align two or more sequences” box is checked.

7. You will see two empty boxes, one with a heading “Query”, the other “Subject”. From the file you downloaded from Canvas, select & copy the human taster protein sequence (the amino acid sequence only, not the heading) and then paste that copied protein sequence into the box labeled “Query”.

8. Go back to the file you downloaded from Canvas, select & copy the human non-taster protein sequence and then paste that copied protein sequence into the box labeled “Subject”.

9. Double check that you’ve pasted both the Taster sequence into the “Query” box and the Non-taster sequence into the “Subject” box.

10. Once you’re sure both sequences are in place, click on the button at the bottom of the page that says “BLAST”.

11. A new window will open. This window contains the 2 protein sequences that you pasted in, but the program has aligned them. That is, the two proteins have been compared to each other, and their similarity has been determined.

12. The information at the top of this window is the parameters that you didn’t change – ignore these.

13. Scroll down a bit further until you see the actual amino acid sequence. These will appear as single letter abbreviations, a standard format for representing amino acids (Table 1). This amino acid information is the protein sequence you pasted in. The Query sequence is your Sequence #1 (Taster) and the subject sequence is your Sequence #2 (Non-taster). The comparison of the two protein sequences is shown in 60 amino acid sections for clarity; each protein is actually 333 continuous amino acids.

14. The letters that you see between the two sequences indicate that the amino acid at that position is identical to the amino acid at the same position on the other sequence. The vast majority of amino acids should be identical between the two proteins. This series of conserved (identical) amino acids is called the consensus sequence.

15. Scroll down the page a little further until you see amino acids that do NOT have a letter between them (i.e. the consensus sequence has a blank or another symbol like a “+” at that position). This indicates that the amino acids at that position are different in the two sequences.

16. How many amino acid differences can you find between the two proteins? Is it fewer, greater, or the same as the number of nucleotide differences?

17. For each amino acid difference between the two protein sequences,

a) Determine the exact amino acid position (this will be a number between 1-333) of the difference within the sequences. This can be done by counting amino acids along the row from left to right (start counting with the amino acid number to the left of the row) until you reach the amino acid difference then add that number to the amino acid position listed at the left of the row you counted. List the position of the amino acid difference in table 2 at the end of this lab.

b) Then, list the amino acid that occurs in the Taster protein and the amino acid that occurs in the Non-taster protein.

18. You’ve determined the protein basis for the difference between tasters, intermediate tasters & non-tasters. Individuals of each phenotype have inherited copies of the gene from their parents, one copy from their mother and one copy from their father. Depending on the combination of alleles a person inherits, they can either taste PTC (homozygous dominant), sort of taste PTC (heterozygous) or not taste PTC at all (homozygous recessive). The proteins encoded by these alleles are what are actually responsible for determining the phenotypes of different individuals.

#5: GROUP/TABLE ACTIVITY: Relating TAS2R38 allele sequences to protein isoforms

1. The amino acid sequence of a protein is determined by the nucleotide sequence of the DNA that encodes that protein. Consequently, any differences you discovered in the amino acids between the two proteins should correspond to the differences you discovered between nucleotides in the DNA sequences. This activity will investigate the specific relationship between each allele of the TAS2R38 gene and the isoform of the receptor protein it encodes.

2. To begin, you will need to determine the amino acid sequence of the protein encoded by each allele. To do this we will use a web-based translation tool called Virtual Ribosome. Go to .

3. Reopen the PTCgenes.doc file and then translate the taster allele by copying and pasting the taster nucleotide sequence into the box and clicking the “Submit query” button.

4. Open a second tab within the same browser window and again go to .

5. Go back to the PTCgenes.doc file and then translate the non-taster allele by copying and pasting the non-taster nucleotide sequence into the box and clicking the “Submit query” button.

6. You now have two tabs open in your browser window – one tab with the taster allele translated into amino acid sequence and the other tab with the non-taster allele translated into amino acid sequence. You can easily determine the differences between the two alleles and isoforms by clicking back and forth on the two tabs and look for nucleotides and amino acids that “change” as you switch back and forth between the two tabs.

7. Each amino acid in a protein is determined by three nucleotides in the DNA sequence that encodes that protein. These three nucleotide units are called codons. The first three nucleotides encodes the first amino acid, the next three nucleotides encodes the second amino acid, and so forth. The output shows the nucleotide sequence along with the amino acid sequence. Each amino acid is listed above its corresponding codon. Ignore the symbols underneath the letters.

8. Determine the codon in each allele that corresponds with each nucleotide difference between the two alleles. Find the 1st nucleotide difference in the DNA sequences by using the tab method from step 6 or by using its position number you recorded in table 2 at the end of this lab. Confirm that both the nucleotide and amino acid for each allele/isoform at their respective positions are consistent with your previous analysis, and write down the codon for each allele/isoform in the table at the end of this lab.

9. Repeat step 8 for each other nucleotide difference you found between the two alleles.

10. You have determined the protein coding differences between the two alleles. Does each nucleotide difference between the taster allele and the non-taster allele cause an amino acid substitution between the two isoforms?

11. What nucleotide change between the two alleles occurred in each codon that accounts for the differences between the isoforms of the proteins? Use Figure 2 and Table 1 as a guide.

12. Think back to what you know about proteins and macromolecules in general. Both the taster & non-taster proteins are receptor proteins. That means that they interact with other molecules to initiate some sort of chemical reaction inside a cell. Specifically, both of these proteins are receptors for the PTC molecule. That means that these proteins can interact with PTC in the paper strip and cause a series of chemical reactions that allows you to perceive (or not) a bitter taste. With that in mind, how do you think the amino acid differences you listed in the table at the end of this lab affect the interaction between PTC & the receptor? Use Figure 3 as a guide.

Figure 2. The genetic code. Because DNA sequences correspond directly to mRNA sequences the genetic code can be read as either DNA sequence (left panel) or mRNA sequence (right panel). DNA sequences contain T in place of U.

Figure 3. Schematic protein structure of the PTC receptor (TAS2R38). Horizontal lines indicate the plasma membrane; amino acids above the lines are extracellular protein domains, those between the lines are transmembrane domains and those below the lines are intracellular protein domains. The variant amino acid positions are marked with circles. (Kim et. al., 2004).

#6: GROUP/TABLE ACTIVITY: Predicting whether chimpanzees & gorillas can taste PTC

For this activity, you are going to use your new-found understanding of the relationship between amino acid sequences, protein structure and protein function to predict whether our closest relatives, chimpanzees and gorillas, are tasters or non-tasters.

1. You will be using the same file you used in Activity #4 (PTCproteins.doc), except this time you will be using all 4 protein sequences (human taster, human non-taster, chimpanzee and gorilla).

2. This time, instead of heading to NCBI, you will be using a site known as CLUSTALW ().

3. Leave all of the settings in the default mode – don’t change anything!!!!!!

4. Go back to the file you downloaded from Canvas, select & copy all 4 protein sequences as a single unit (including the protein names) and then paste them into the box under the heading “Enter your sequences…..”.

5. Double check that all 4 sequences have been pasted into the window.

6. Click on the “execute multiple alignment” button.

7. In the new window that opens, you should see a list of the proteins you copied at the top. If you scroll down the page, the alignment of those protein sequences will appear.

8. The proteins have been aligned, and will appear in 60 amino acid blocks for clarity. This does not mean that there are 20+ proteins now; this is just so that you can see the entire alignment without having to scroll over to read it. In actuality, both human isoforms, the chimpanzee and the gorilla protein are 333 continuous amino acids long.

9. An asterisk underneath a column means that all 4 amino acids in that position are identical. For example, the first amino acid is an “M” or methionine in all 4 proteins. If there is not an asterisk beneath a column that means that one or more of the amino acids at that position are different in one or more of the sequences.

10. Count the total number positions that have different amino acids (non-asterisks).

11. Are there fewer, the same number, or greater number of amino acid difference between these 4 sequences than between the 2 human isoforms? Which of these differences are likely to play a role in the ability or inability to taste bitterness?

12. Based on the amino acids present in the chimpanzee & gorilla proteins, would you predict these primates would be tasters or non-tasters? Why or why not?

___________________________________________________________________

References

Kim, U.K. and D. Drayna. 2004. Genetics of individual differences in bitter taste perception: lessons from the PTC gene. Clin. Genet. 67: 275-280.

Kim, U.K., P.A.S. Breslin, D. Reed and D. Drayna. 2004. Genetics of Human Taste Perception. J. Dent. Res. 83(6): 448-453.

Wooding, S., U.K. Kim, M.J. Bamshad, J. Larsen, L.B. Jorde et. al. 2004. Natural Selection and Molecular Evolution in PTC, a Bitter-Taste Receptor Gene. Am. J. Hum. Genet. 64: 637-646.

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QUESTIONS

1. The non-taster allele encodes a protein isoform that is not capable of detecting PTC. Do you think that this isoform is non-functional? Why or why not? If it is still functional, what function(s) do you hypothesize it is capable of performing?

2. You inherit chromosomes, and therefore genes, from your parents. Yet it’s the proteins that your cells make that determine your phenotype (eye colour, attached earlobes etc.). In your own words, explain the relationship between the genes you inherit and the proteins produced by your cells.

3. What is the probability that a non-taster mother and an intermediate taster father will have a strong taster child?

4. What is the probability that a strong taster mother and a non-taster father will have a non-taster child?

5. Johnny is a non-taster but his sister Jenny is an intermediate taster.

a. What is Johnny’s genotype?

b. What is Jenny’s genotype?

c. What are the possible genotypes for Johnny & Jenny’s parents?

• Mom’s possibilities:

• Dad’s possibilities:

6. Bitter taste is usually associated with poisonous, or toxic, compounds. Recognizing bitter taste helps prevent us from ingesting a potentially deadly substance. If recognizing bitter compounds is such a crucial mechanism of self-preservation, why are approximately 25% of people unable to recognize PTC as bitter? Formulate a hypothesis that addresses why you think the non-taster allele has been maintained throughout human evolution.

Table 2. Record the differences between the genes and proteins of bitter taste receptors in humans, chimpanzees, and gorillas.

Activity #3 - Human PTC alleles Total length of gene sequence: __________

| |1st difference |2nd difference |3rd difference |

|Position | | | |

|Taster nucleotide | | | |

|Non-taster nucleotide | | | |

Activity #4 - Human PTC receptor protein isoforms Total length of protein sequence: __________

| |1st difference |2nd difference |3rd difference |

|Position | | | |

|Taster amino acid | | | |

|Non-taster amino acid | | | |

Activity #5 - Human PTC allele mRNA codons Total number of codons: __________

| |1st difference |2nd difference |3rd difference |

|Position | | | |

|Taster codon | | | |

|Non-taster codon | | | |

Activity #6 - Primate PTC receptor protein isoforms

| |1st difference |2nd difference |3rd difference |

|Position | | | |

|Human Taster amino acid | | | |

|Human Non-taster amino acid | | | |

|Chimpanzee amino acid | | | |

|Gorilla amino acid | | | |

Prediction: Do chimpanzees taste PTC? (yes / no)

Prediction: Do gorillas taste PTC? (yes / no)

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