2025-06-22 MUTATIONS Lecture Week 7: Readings Ch 9 & 10 1 BioA11 Introduction to the Biology of Humans • COURSE OBJECTIVES: BioA11 Instructor: Karen Williams, PhD Office: SW511 Office hours: : Virtual: Friday 1715h-1745h, In-person: Tuesday 1710-1745h, or by appointment • • • _______________ _______________ _______________ • See syllabus • Aim for this week: • 7. To use and interpret data for a case and integrate the information from multiple sources. Email: kd.williams@utoronto.ca 2 1 2025-06-22 BioA11 Introduction to the Biology of Humans Lecture week 7: Mutations • OUTLINE • Why identify mutations? CSI identifying family: DNA profiling • The connection between DNA, RNA and Protein • Mutagens cause heritable changes in DNA and can affect gene transcription and translation • How do mutations affect polypeptide structure and function? • How can we detect mutations? • Modifying, editing and manipulating the genome 3 Bio A11 Introduction to the Biology of Humans Where is DNA in the blood? • Red blood cells do not contain nuclei • See chapter 3 • Porphyrias: • The pathway for the synthesis of heme involves many enzymes defects in the function of these enzymes leads to a variety of defects ______ • http://www.omim.org/entry/263700?search=Porphyria&highlight=po rphyria • Which of the phenotypes described under “Clinical Features” remind you of vampires or werewolves? • ____________________________________________ • ____________________________________________ © 2013 by K.D.Williams -UTSC 4 2 2025-06-22 Bio A11 Introduction to the Biology of Humans See chapter 9 • DNA profiling: • ____________________ ____________________ ____________________ ____________________ • _____________(PCR) is: • ___________________ ____________________ ____________________ ___________________ 5 Why identify mutations? • Study mutations to • To identify familial identify familial patterns for disorders: patterns for disorders: • Mosquito gene Hdr • Human porphyria case 6 3 2025-06-22 BioA11 Introduction to the Biology of Humans Hdr gene let H= normal ; h= mutant hdr Female mosquito homozygous for h allele Male mosquito homozygous for H allele • Female x male • F1: • Extract DNA • PCR _______ • Lane 1; Lane 2; Lane 3 • Using primers for the hdr 1 2 3 gene • Run product on electrophoresis gel 7 Bio A11 Introduction to the Biology of Humans Mosquito DNA to PCR to gel Female mosquito homozygous for h allele Male mosquito homozygous for H allele DNA extracted from the female mosquito loaded into lane 1 of the electrophoresis gel DNA extracted from the male mosquito loaded into lane 3 of the electrophoresis gel 8 4 2025-06-22 Bio A11 Introduction to the Biology of Humans DNA profile of controlled mating cross: Lane 1 Lane 2 Lane 3 • Female mosquito homozygous “hdr”, lane 1: _____ • X • Male mosquito homozygous “Hdr”, lane 3: ____ • Progeny: ________ Hammond et al 2016 Nature Biotechnology 34: 78-83 9 Bio A11 Introduction to the Biology of Humans Describe each DNA profile • Lane 1 ____ Lane 3 • In Lane 1 the DNA from a mosquito of genotype __ , gave _________. • In Lane 2 _________________________ • In Lane 3 the DNA from a mosquito of genotype ___, gave __________. 10 5 2025-06-22 Hdr gene let H= normal Hdr; h= mutant hdr Female mosquito homozygous for h allele Male mosquito homozygous for H allele Produces __ gametes by :____ Produces __ gametes by :____ 11 BioA11 Introduction to the Biology of Humans Controlled mating between mosquitoes • Female mosquito homozygous “hdr=h”, lane 1: _____ • X • Male mosquito homozygous “Hdr=H”, lane 3: ____ Insectimages.org 12 6 2025-06-22 BioA11 Introduction to the Biology of Humans What is the nature of the hereditary material? • What is a gene? • _____________________________________ • What is the Central Dogma? https://flic.kr/p/pJFqJ4 • James Watson : the transfer of genetic information from DNA to RNA to Protein. Specifically DNA is transcribed to RNA and RNA is translated to Protein • Transcription: DNA to RNA • Translation: RNA to Protein • Mutations cause heritable changes in DNA • Variation in DNA sequence can change the proteins produced © 2012 by K.D.Williams -UTSC 13 BioA11 Introduction to the Biology of Humans Watson & Crick (with data from Wilkins and Franklin) http://www.nobelpr ize.org/nobel_priz es/medicine/laure ates/1962/index.ht ml http://www.flickr.com/photos/carolineross/7330412570/ Russell et al. 2012 Biology Nelson Education Ltd © 2012 by K.D.Williams -UTSC 14 7 2025-06-22 BioA11 Introduction to the Biology of Humans Cystic Fibrosis • OMIM 219700 and 602421 • Homozygous recessive • Most common severe form of CF patients have a mutation in a gene cystic fibrosis transmembrane conductance regulator (CFTR) • Delta F508 is a deletion in the DNA sequence at position 508 that results in the absence of phenylalanine (Phe) • What is the mRNA codon for phenylalanine (Phe)? ___________ © 2013 by K.D.Williams -UTSC 15 10.1 Protein Synthesis and Gene Expression (1 of 21) Regulating Gene Expression: controlling the amount of proteins produced • Early 1980s: first production of recombinant bovine growth hormone (rBGH) • Made by genetically engineered bacteria • Instructions for making BGH inserted into bacterial DNA • Cows injected with laboratory-produced BGH: • Increased body size • Increase milk production 16 8 2025-06-22 10.1 Protein Synthesis and Gene Expression (2 of 21) Protein synthesis: the process of using instructions carried by genes to build proteins • Gene: a sequence of DNA that encodes a protein • Protein: a large molecule composed of amino acids • Involves DNA and RNA with multiple steps One gene determines one recipe. One gene Genome in nucleus Copy of gene as mRNA Amino acids mRNA on ribosome tRNAs Protein 17 10.1 Protein Synthesis and Gene Expression (3 of 21) DNA: polymer of nucleotides • Double-stranded • Nucleotide subunit: (a) DNA • Deoxyribose • Phosphate group DNA nucleotide • Nitrogenous base • Four bases with Thymine (T) complementary bonding: • adenine and thymine • cytosine and guanine Deoxyribose Phosphate group 18 9 2025-06-22 10.1 Protein Synthesis and Gene Expression (4 of 21) RNA: polymer of nucleotides • Single-stranded • Nucleotide subunit (b) RNA • Ribose • Phosphate group RNA nucleotide • Nitrogenous base • Four bases with Uracil (U) complementary bonding: • adenine and uracil • cytosine and guanine Ribose Phosphate group 19 10.1 Protein Synthesis and Gene Expression (5 of 21) Protein synthesis: DNA → RNA → Protein • The flow of genetic information occurs in two steps: • Transcription (DNA → RNA) • Translation (RNA → protein) Polymer of nucleotides (two complementary strands) DNA Transcription Polymer of nucleotides (single strand) RNA Translation Protein ala ser val his Polymer of amino acids 20 10 2025-06-22 Bio A11 Introduction to the Biology of Humans See previous slide • Which DNA strand is this RNA transcribed from? • … CUG UUC 21 10.1 Protein Synthesis and Gene Expression (6 of 21) Transcription (DNA → RNA) • Occurs in the nucleus • Forms messenger RNA (mRNA) • RNA polymerase binds to the promoter • RNA polymerase unzips DNA and ties RNA nucleotides with complementary DNA nucleotides. RNA nucleotides Promoter DNA Promoters have distinct nucleotide sequences that RNA polymerase recognizes. RNA polymerase mRNA 22 11 2025-06-22 10.1 Protein Synthesis and Gene Expression (7 of 21) Translation (RNA → protein) • Occurs outside of the nucleus (at ribosomes) • Requirements: • mRNA (made during transcription) • Amino acids • Energy (ATP) • Ribosomes (rRNA) • Transfer RNA (tRNA) 23 10.1 Protein Synthesis and Gene Expression (8 of 21) Ribosomes • Composed of ribosomal RNA (rRNA) • A small subunit Large subunit • A large subunit Small subunit 24 12 2025-06-22 10.1 Protein Synthesis and Gene Expression (9 of 21) Transfer RNA (tRNA) • Carries amino acids • Matches its complementary anticodon with codons on mRNA • Codons and anticodons Amino acid phe Binding site for amino acid Region of internal complementarity tRNA have three nucleotides. Anticodon AAA mRNA UUU Codon 25 10.1 Protein Synthesis and Gene Expression (10 of 17) Protein synthesis: similar to baking a cake One gene determines one recipe. One gene Genome in nucleus Copy of gene as mRNA mRNA on ribosome Amino acids tRNAs Protein Copy of recipe book on index card Recipe card on counter Flour, sugar, and eggs Measuring spoon and cup Cake One recipe Cookbook on shelf 26 13 2025-06-22 10.1 Protein Synthesis and Gene Expression (11 of 21) Proteins: • Made by attaching one amino acid at a time. • Ribosome attaches to mRNA at the promoter region. • Ribosome facilitates the docking of tRNA anticodons to mRNA codons. • Peptide bonds form between the amino acids of two adjacent tRNAs. • Stop codon has no matching tRNA. • The full protein is released. 27 10.1 Protein Synthesis and Gene Expression (12 of 21) Amino acid Amino acids and tRNAs float freely in the cytoplasm. leu tRNA Enzymes facilitate the binding of a specific tRNA to its appropriate amino acid. Amino acid chain (polypeptide) ala phe ile The amino acids join together to form a polypeptide. A tRNA will dock if the complementary RNA codon is present on the ribosome. Stop codon The ribosome moves on to the next codon to receive the next tRNA. Ribosome When the ribosome reaches the stop codon, no tRNA can base-pair with the codon on the mRNA. RNA and the newly synthesized protein are released. The chain of amino acids folds, and the protein is ready to perform its job. STOP Protein (such as BGH) The subunits of the ribosome separate but can reassemble and begin translation of another mRNA. 28 14 2025-06-22 BioA11 Introduction to the Biology of Humans • The tRNA shown bind • AAA UAU ACG are mRNA what are the mRNA codons for these tRNAs: the tRNAs that add the amino acids (aa) to the polypeptide chain • What amino acids are added to the polypeptide chain? • AAA UAU ACG ? • • (you will need the codon • ______________ table 10.1) • __________________ 29 10.1 Protein Synthesis and Gene Expression (13 of 21) Genetic code: allows a specific codon to code for a specific amino acid • A codon is composed of three nucleotides. • 64 (4)3 possible combinations. • 61 codons code for amino acids. • 3 others are stop codons, which end protein synthesis. 30 15 2025-06-22 10.1 Protein Synthesis and Gene Expression (14 of 21) Additional properties of genetic code • The genetic code is redundant. • Multiple codons code for the same amino acid. • E.g. ACU, ACC, ACA, and ACG all code for the amino acid threonine. • The genetic code is universal. • E.g UUU codes for phenylalanine and nothing else. • All organisms decode the same gene to produce the same protein. 31 10.1 Protein Synthesis and Gene Expression (15 of 21) Table 10.1 The genetic code. To determine which amino acid is coded for by each mRNA codon, first look at the lefthand side of the chart for the firstbase nucleotide in the codon; there are four rows, one for each possible RNA nucleotide—A, C, G, or U. Then look at the intersection of the second-base columns at the top of the chart and the first-base rows to narrow your search. Finally, the third-base nucleotide in the codon on the right-hand side of the chart determines the amino acid that a given mRNA codon codes for. Note the three codons UAA, UAG, and UGA that do not code for an amino acid; these are stop codons. The codon AUG is a start codon, found at the beginning of most protein-coding sequences. 32 16 2025-06-22 10.1 Protein Synthesis and Gene Expression (16 of 21) Protein synthesis in all organisms • Eukaryotic cells – spatially separated transcription (in nucleus) and translation (outside of nucleus) • Prokaryotic cells (without a nucleus) – transcription and translation occur at the same time and place (a) Eukaryotic protein synthesis (b) Prokaryotic protein synthesis Nuclear pore DNA Amino acids Transcription mRNA mRNA Translation Amino acids Nucleus DNA RNA polymerase Transcription Ribosome and Translation Ribosome 33 10.1 Protein Synthesis and Gene Expression (17 of 21) Mutations: changes in genetic sequence • May affect the order of amino acids in a protein • Precise order of amino acids is necessary for properly functioning proteins. • Possible outcomes of mutation: 1. no change in protein 2. nonfunctional protein 3. different protein 34 17 2025-06-22 10.1 Protein Synthesis and Gene Expression (18 of 21) • Substitution mutation • Simple substitution of one base for another (b) Mutated DNA sequence (a) Normal DNA sequence Substitution mutation DNA DNA mRNA mRNA Protein met asp ala phe Protein Potentially nonfunctional protein Functional protein 35 10.1 Protein Synthesis and Gene Expression (19 of 21) DNA • Neutral mutation • Mutation does not change the function of the protein. • The same amino acid results from the codon. mRNA Amino acid sequence thr asn glu asn glu (a) Neutral mutation DNA mRNA Amino acid sequence thr The amino acid sequence is the same as the original. 36 18 2025-06-22 10.1 Protein Synthesis and Gene Expression (20 of 21) • Frameshift mutation • Addition or deletion of a base • Changes the reading frame • Usually results in a stop codon DNA mRNA Amino acid sequence thr asn • Shorter, nonfunctional protein glu (b) Insertion of one base pair, resulting in a frameshift mutation produced thr asn STOP The amino acid sequence is different from the original. In this case, a stop codon causes the formation of an incomplete protein. 37 10.1 Protein Synthesis and Gene Expression (21 of 21) Gene expression • Each body cell (except sperm and eggs) has the same DNA. • Cells express different genes based on their functions. • Genetic engineering: precisely controls gene expression (a) Muscle cells (b) Nerve cells 38 19 2025-06-22 How do mutations affect polypeptide structure and function? • DNA: • AAT GAA AAA changed to AAC GAA AAA • • RNA • Polypeptide 39 Bio A11 Introduction to the Biology of Humans What type of mutation? Why? Normal Mutant 10______ & 12 ______ DNA AAT GAA AAA TTA CTT TTT DNA AAT GAA AAC TTA CTT TTG RNA AAU GAA UUU RNA AAU GAA UUC Amino acids Asn Glu Phe Amino acids Asn Glu Phe DNA AAT GAA AAA TTA CTT TTT DNA AAC GAA AAA TTG CTT TTT 10 RNA Amino acids AAU GAA UUU RNA AAC GAA UUU Asn Glu Phe Amino acids Asn Glu Phe 12 40 20 2025-06-22 10.3 Genetically Modified Plants and Animals (12 of 14) Gene Editing: directly altering, deleting, or replacing a DNA sequence in an organism • Uses CRISPR: clustered regularly interspaced palindromic repeats • Faster, more effective, and less cumbersome process • Precise modification of crop plants and animals deletes mutations or adds desired gene sequences • Delete pest attracting genes in plants • to decrease pesticide use 41 10.3 Genetically Modified Plants and Animals (13 of 14) CRISPR: “crisper”; clustered regularly interspaced palindromic repeats • Part of a bacterium’s immune system • Bacteria removed genes from invading virus • Scientists use bacterial system in other organisms • Two components: • Guide finds DNA sequence for editing • “Molecular scissor” enzyme snips out unwanted DNA and replaces with correct sequence 42 21 2025-06-22 10.3 Genetically Modified Plants and Animals (14 of 14) Future uses of CRISPR • In plants: • increased shelf life • added nutrients or fiber • In animals • More muscle mass or leaner meat in livestock • Pig organs suitable for human transplantation • Editing reproduction genes in malariacausing mosquitoes • Prevent deaths of 1 million people 43 10.4 Genetically Modified Humans (1 of 10) Stem cells: unspecialized, undifferentiated precursor cells with an undetermined function • Early stem cells can become any type of cell. • As embryos develop, cells become more specialized. • Specialized, non-stem cells only make copies of those cell types. 44 22 2025-06-22 10.4 Genetically Modified Humans (4 of 10) Adult stem cells: help maintain tissues and replace damaged or diseased cells • Found in nonembryonic tissue • Newborn umbilical cord • Primary teeth of children • Bone marrow • Some blood vessels • Some muscles • Brain • Liver 45 10.4 Genetically Modified Humans (7 of 10) Gene therapy: replacement of defective genes with functional genes • Somatic cell gene therapy: fixes or replaces the defective protein only in affected cells Normal gene (DNA sequence) inserted into harmless virus Lung cell Virus enters lung cell nucleus. Nucleus Cells that now carry the normal version of the gene can produce the required protein. 46 23 2025-06-22 10.4 Genetically Modified Humans (10 of 10) Cloning humans • Nuclear transfer: process used for cloning where an adult cell nucleus is fused with an egg cell • Process currently used for animals • Ethical concerns for possible use in humans Body cell Remove the nucleus from a body cell of the person to be cloned. Transfer the nucleus from the cell of the person to be cloned into the egg cell. Embryo Grow the embryo in a culture. Egg cell Remove the nucleus from an egg cell. Implant the early embryo into the uterus of a woman who has been hormonally treated to stimulate pregnancy. Woman gives birth to a clone of the man. 47 Dolly the sheep https://s-media-cache-ak0.pinimg.com/564x/4b/6a/67/4b6a679844df050ecba2fda16200bb84.jpg 48 24 2025-06-22 BioA11 Introduction to the Biology of Humans What happened to Dolly? How did Dolly differ from other sheep? USDA ARS Photo Unit, USDA Agricultural Research Service, Bugwood.org 49 Genome editing using CRISPR/Cas 50 25 2025-06-22 Bio A11 Introduction to the Biology of Humans CRISPR for mosquito gene • What is the matching • Why would a scientist DNA sequence for this CRISPR sequence? want to use CRISPR for a mosquito? For which mosquito gene? • AAC GAA GCC • (see text chapter 10) 51 BioA11 Introduction to the Biology of Humans CRISPR/Cas and genetic manipulation Knott & Doudna 2018 Science 361:866 52 26 2025-06-22 BioA11 Introduction to the Biology of Humans CRISPR/Cas and genetic manipulation: targeted outcomes are ______________ Knott & Doudna 2018 Science 361:866 53 BioA11 Introduction to the Biology of Humans Lecture week 7: Mutations • OUTLINE • Why identify mutations? CSI identifying family DNA • The connection between DNA, RNA and Protein • Mutagens cause heritable changes in DNA and can affect gene transcription and translation • How do mutations affect polypeptide structure and function? _________________________________ • How can we detect mutations? ______________________ • Modifying, editing and manipulating the genome • How does CRISPR change the protein produced? _____________________________________________________ ____________________________________________________ 54 27
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