Tutorial 4 Mutations and Bioinformatics F2023

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Biology 1M03 - Tutorial 4 Mutations and Bioinformatics Objectives By the end of this tutorial students should be able to: Define and identify synonymous and non-synonymous substitutions. Define and identify different types of mutations. Translate the genetic code. Be familiar with amino acid nomenclature. Identify evidence for convergent evolution. Preparation 🗹 Read the introductory material in this student manual. ? Review Chapter 13.1 – 13.3 (Genotype and Phenotype, The Nature of Mutations, and Small-Scale Mutations) in Biology How Life Works, 4 th edition. ? Complete the Avenue pre-tutorial 4 quiz by 9:00am on the day of your tutorial. Introduction The Genetic Code The genetic code refers to the way in which the information contained in DNA and RNA is used by cells to make proteins. First, DNA must be transcribed into messenger RNA (mRNA) and then it can be translated into a protein, as shown in Figure 1. In a DNA molecule, the nitrogenous base adenine (A) pairs with thymine (T) on the opposite strand. Similarly, guanine (G) pairs with cytosine (C). In RNA, thymine is replaced by uracil (U) and pairs with adenine. DNA molecules have directionality; we typically write sequences in the 5' to 3' direction. The complementary strand, however, is antiparallel and would be written in the 3' to 5' direction. The directions refer to the chemical bonds in the DNA sugar-phosphate backbone. The coding strand of a double stranded DNA molecule is almost identical to the mRNA sequence, except that thymine is replaced by uracil in the mRNA. It is the non-coding (or template) strand that is transcribed into mRNA. When transcribing the mRNA, RNA polymerase reads the template strand of the DNA molecule and adds the complementary nitrogenous base to the growing mRNA. 1
Figure 1. DNA is transcribed into RNA and translated into a protein. Ribosomes in the cell translate the mRNA sequence into a protein. It does this by reading the codons in the mRNA sequence and adding the appropriate amino acid to the growing polypeptide chain (protein). The term "codon" refers to the group of 3 nucleotides that code for an amino acid in a protein. The genetic code in Figure 2 shows which codon codes for a particular amino acid. For example, a mRNA sequence usually begins with the nucleotides AUG. This codon corresponds to the start codon which is methionine. There are also 3 stop codons, which signal the end of translation of a mRNA sequence. Figure 2. The nuclear genetic code. 2
Mutations Mutations result in change to an organism’s DNA sequence. Mutations often occur randomly in individuals over time and can contribute to the process of evolution. Some nucleotide changes result in a change to the amino acid that is coded for by that particular codon, while others do not. This is possible because of the redundant nature of the nuclear genetic code. The code is redundant because the same amino acid can be coded for by multiple codons in many cases. Single nucleotide differences between copies of the same DNA sequence (e.g., in the same gene between two individuals) are called single nucleotide polymorphisms, or “SNPs”. As you can see in the table above, changing the third nucleotide in many cases does not result in an amino acid change. This third position can be referred to as the "wobble" position. Nucleotide changes that do not change the amino acid sequence in a protein are referred to as “synonymous” changes. Non-synonymous mutations change amino acids, and in some cases result in structural and/or functional changes, potentially including the protein becoming non- functional. The substitution or insertion of a single nucleotide is called a point mutation. Different types of point mutations and their outcomes can be seen in the table below. Table 1. Point mutations and their outcomes. Silent mutation Change in nucleotide that does not change the amino acid coded for by the codon. Change in genotype but not phenotype. Missense mutation Nucleotide change that results in a new amino acid coded for by the codon. Change in primary protein structure. Nonsense mutation Nucleotide change that results in a stop codon, usually resulting in early termination of translation. Frameshift mutation An insertion or deletion of a nucleotide that results in a different reading frame. Can change many amino acids at once. Mutations and Evolution Humans and chimpanzees have both lost the ability to taste PTC (depending on the individual’s alleles), but this loss of trait was derived via convergent evolution. Convergent evolution is the development of the same trait or phenotype, but through independent mechanisms. Many of the differences seen in chimpanzee and human genotypes, and the resultant phenotypes, are due to single nucleotide polymorphisms. Human and chimpanzee nucleotide sequences are 96% identical overall, and 99% identical for most protein-coding sequences. Small changes can result in big differences. The most significant difference in gene expression between humans and chimpanzees occurs in brain cells. IN-CLASS ACTIVITY: Transcribing and Translating the Genetic Code 3
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What does ATG signify in a DNA coding sequence? Start Codon (methionine) What does TGA signify in a DNA coding sequence? Stop Codon What mRNA codons code for the amino acid Arginine? CGU, CGA, CGG, CGC The following DNA sequence is found on the coding strand of a piece of double stranded DNA. What amino acid sequence would be present after translation? DNA Coding Strand: 5’ ATG ATA AGT AGC TGA 3’ mRNA: 5’ UAC UAU UCA UCG ACU 3’ Protein: Met Tyr Ser Ser Thr Imagine a mutation occurred in the DNA sequence that changed the isoleucine (Ile) amino acid to threonine in the protein sequence. What nucleotide change would have caused this missense mutation? The second base U may have switched to C IN-CLASS ACTIVITY: Identifying Sequence Differences The amino acids sequences of Homo sapiens tasters (T allele) and non-tasters (t allele) are seen below. Identify the three differences in the sequences. Taster 1 mltltrirtv syevrstflf isvlefavgf ltnafvflvn fwdvvkrq pl snsdcvllcl Non 1 mltltrirtv syevrstflf isvlefavgf ltnafvflvn fwdvvkrq al snsdcvllcl Taster 61 sisrlflhgl lflsaiqlth fqklseplnh syqaiimlwm ianqanlwla aclsllycsk Non 61 sisrlflhgl lflsaiqlth fqklseplnh syqaiimlwm ianqanlwla aclsllycsk Taster 121 lirfshtfli claswvsrki sqmllgiilc scictvlcvw cffsrphftv ttvlfmnnnt Non 121 lirfshtfli claswvsrki sqmllgiilc scictvlcvw cffsrphftv ttvlfmnnnt Taster 181 rlnwqikdln lfysflfcyl wsvppfllfl vssgmltvsl grhmrtmkvy trnsrdpsle Non 181 rlnwqikdln lfysflfcyl wsvppfllfl vssgmltvsl grhmrtmkvy trnsrdpsle Taster 241 ahikalkslv sffcffviss c aafisvpll ilwrdkigvm vcvgimaacp sghaa vlisg Non 241 ahikalkslv sffcffviss c vafisvpll ilwrdkigvm vcvgimaacp sghaa ilisg Taster 301 naklrravmt illwaqsslk vradhkadsr tlc Non 301 naklrravmt illwaqsslk vradhkadsr tlc At which amino acid positions have mutations occurred? 4
49, 262, 296 Describe the amino acid changes that presumably happened when non-tasting evolved, e.g.P49A? Proline changed to Alanine (49), Alanine changed to Valine (262), Valine changed to Isoleucine (296). Using the Homo sapiens taster and non-taster amino acid sequences, calculate the percent difference of the amino acid sequences using the following formula: (3/333) x 100 = 0.901% difference Nucleotide BLAST Instructions One of the bioinformatic tools on the NCBI website is BLAST. BLAST stands for Basic Local Alignment Search Tool. It allows you to search a query sequence against a database to find other similar sequences. Alternatively, you can use it to align two or more known sequences. You can find DNA, RNA, or protein sequences on the NCBI website as well. As part of your post-tutorial 4 assignment, you will be performing a nucleotide BLAST (blastn). The BLAST suite also includes the ability to conduct BLASTs using protein sequences and translated nucleotide sequences. Follow the instructions below to perform the nucleotide BLAST using the given sequence in question 5 of your post-tutorial assignment. We will be aligning the nucleotide sequence from humans for the PTC taster allele to the one in chimpanzees. 5
1. Access the NCBI BLAST website here: https://blast.ncbi.nlm.nih.gov/Blast.cgi 2. Select Nucleotide BLAST. 3. Copy and paste the Homo sapiens PTC taster allele nucleotide sequence in question 5 (below) into the Enter Query Sequence box. 4. Under Organism, type Pan troglodytes and select it from the list of organisms (taxid:9598). This is the scientific name for chimpanzees. 5. Don't change any other of the default settings. 6. Click BLAST at the bottom of the page to perform the search. 7. Click on the first result (with the highest total score) to view the nucleotide alignment between your Homo sapiens query sequence and the Pan troglodytes sequence. This is the best match between the two species for the PTC taster allele. 8. Take a screenshot of the nucleotide alignment between the two sequences to include in your post-tutorial assignment. 9. Calculate the percent difference between the two sequences. Include this calculation in your post-lab assignment. 6
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TUTORIAL 4: P OST -T UTORIAL A SSIGNMENT 1. a) Complete the table below. What amino acid positions have differences between tasters and non-tasters? What are the amino acids present at those positions for tasters and non- tasters? What nucleotide changes are responsible for the amino acid differences between Homo sapiens tasters and non-tasters? At which nucleotide positions in the codon and in the DNA sequence do these changes occur? (9 marks) (hints: refer to the codon table in your tutorial manual; how many nucleotides are in an amino acid codon?) Amino Acid Position # in Protein Sequence Taster Amino Acid Non-taster Amino Acid Nucleotide Change from taster to non- taster (e.g., Adenine to Thymine) Nucleotide Position # in Codon (e.g., 1, 2, or 3) Nucleotide Position # in DNA Sequence 49 p a C to G 1 145 262 a v C to U 2 785 296 v i G to A 1 886 Proline changed to Alanine (49), Alanine changed to Valine (262), Valine changed to Isoleucine (296). 1. b) Which type(s) of point mutations are occurring between tasters and non-tasters? (1 mark) They are missense mutations. 2. The amino acid sequence seen below is from a Gorilla gorilla (Western Lowland Gorilla). Make a prediction as to whether this primate is a PTC taster, or a non-taster and justify your answer. (2 marks) 1 mltltrirtv syevrstflf isvlefavgf ltnafvflvn fwdvvkrqpl snsdcvllcl 7
61 sisrlflhgl lflsaiqlth fqklseplnh syqaiimlwm ianqanlwla aclsllycsk 121 lirfshtfli claswvsrki sqmllgiilc scictvlcvw cffsrphftv ttvlfmnnnt 181 rlnwqikdln lfysflfcyl wsvppfllfl vssgmltvsl grhmrtmkvy irdsrdpsle 241 ahikalkslv sffcffviss caafisvpll ilwrdkigvm vcvgimaacp sghaavlisg 301 naklrravtt illwaqsslk vranhkadsr tpc The gorilla is likely able to taste PTC. The amino acids coded for by the gorilla’s sequence at the locations of variability regarding ability to taste PTC in Homos sapiens, is mirrored to the Homo sapiens who can taste PTC. 3. Humans and chimpanzees derived the loss of PTC tasting trait via convergent evolution. Examine the first several nucleotides of the sequences below of a non-taster human and non-taster chimpanzee. How do these sequences support the claim that the loss of trait was derived via convergent evolution? (2 marks) Homo sapiens 1 ATGTTGACTCTAACTCGCATCCGCACTGTGTCCTAT Pan troglodytes 1 AGGTTGACTCTAACTCGCATCCGCACTGTGTCCTAT The sequences above differ in the sequence for the start codon, a significant sequence change. It is likely that this change in sequence occurred further back in the evolutionary history of humans and chimpanzees than the change in alleles for PTC tasting. 4. Follow the instructions in the tutorial manual to perform a nucleotide BLAST using the Homo sapiens PTC taster allele sequence shown below. >AY258597.1 Homo sapiens PTC bitter taste receptor (PTC) gene, PTC-taster allele, complete cds ATGTTGACTCTAACTCGCATCCGCACTGTGTCCTATGAAGTCAGGAGTACATTTCTGTTCATTTCAGTCC TGGAGTTTGCAGTGGGGTTTCTGACCAATGCCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAG GCAGCCACTGAGCAACAGTGATTGTGTGCTGCTGTGTCTCAGCATCAGCCGGCTTTTCCTGCATGGACTG CTGTTCCTGAGTGCTATCCAGCTTACCCACTTCCAGAAGTTGAGTGAACCACTGAACCACAGCTACCAAG CCATCATCATGCTATGGATGATTGCAAACCAAGCCAACCTCTGGCTTGCTGCCTGCCTCAGCCTGCTTTA CTGCTCCAAGCTCATCCGTTTCTCTCACACCTTCCTGATCTGCTTGGCAAGCTGGGTCTCCAGGAAGATC TCCCAGATGCTCCTGGGTATTATTCTTTGCTCCTGCATCTGCACTGTCCTCTGTGTTTGGTGCTTTTTTA GCAGACCTCACTTCACAGTCACAACTGTGCTATTCATGAATAACAATACAAGGCTCAACTGGCAGATTAA AGATCTCAATTTATTTTATTCCTTTCTCTTCTGCTATCTGTGGTCTGTGCCTCCTTTCCTATTGTTTCTG GTTTCTTCTGGGATGCTGACTGTCTCCCTGGGAAGGCACATGAGGACAATGAAGGTCTATACCAGAAACT CTCGTGACCCCAGCCTGGAGGCCCACATTAAAGCCCTCAAGTCTCTTGTCTCCTTTTTCTGCTTCTTTGT GATATCATCCTGTGCTGCCTTCATCTCTGTGCCCCTACTGATTCTGTGGCGCGACAAAATAGGGGTGATG 8
GTTTGTGTTGGGATAATGGCAGCTTGTCCCTCTGGGCATGCAGCCGTCCTGATCTCAGGCAATGCCAAGT TGAGGAGAGCTGTGATGACCATTCTGCTCTGGGCTCAGAGCAGCCTGAAGGTAAGAGCCGACCACAAGGC AGATTCCCGGACACTGTGCTGA a) Include a screenshot of the alignment of the Homo sapiens PTC taster allele sequence with the Pan troglodytes (chimpanzee) PTC taster nucleotide sequence with the highest total score. (1 mark) b) What is the percent difference between the Homo sapiens and Pan troglodyte nucleotide sequences? Show your calculation. (2 marks) number of differences / total sequence * 100 = percent difference = (6 / 1002) x 100 = 0.599% Grade: /16 9
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