Chapter 7 Genetically Modified Organisms Gene Expression Mutation

Chapter 7 Genetically Modified Organisms Gene Expression, Mutation, and Cloning Fourth Edition BIOLOGY Science for Life | with Physiology Colleen Belk • Virginia Borden Maier © 2013 Pearson Education, Inc. Copyright © 2009 Pearson Education, Inc. Power. Point Lecture prepared by Jill Feinstein Richland Community College

1 Protein Synthesis and Gene Expression § In the late 1970 s, genetic engineers began producing recombinant bovine growth hormone (r. BGH), insulin, and interferon (IFN) § Made by genetically engineered bacteria § The bacteria were given DNA that carries instructions for making BGH, Insulin, and IFN § In cows, BGH increases body size and milk production § Insulin reverses Type I diabetes § IFN reduces viral spread during infections © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: From Gene to Protein § Protein synthesis – the process of using instructions carried on a gene to create proteins. § Several steps are involved and require both DNA and RNA. § Gene – a sequence of DNA that encodes a protein § Protein – a large molecule composed of amino acids; its sequence is predicted from the gene sequence © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: From Gene to Protein § Review: DNA § Double-stranded § Each nucleotide composed of deoxyribose, phosphate, and nitrogenous base § 4 bases: adenine (A), thymine (T), guanine(G), and cytosine (C) © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: From Gene to Protein § RNA § Single-stranded § Nucleotides comprised of ribose, phosphate, and nitrogenous base § 4 bases: A, C, G, and Uracil (U) § U and NOT Thymine is in m. RNA © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: From Gene to Protein § The flow of genetic information in a cell is DNA RNA protein (Central dogma) and occurs in 2 steps: § Transcription (DNA RNA) § Translation (RNA Protein) © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Transcription § Transcription (DNA → RNA) occurs in the nucleus. § RNA polymerase binds to the promoter (control) region of the gene. § RNA polymerase zips down the length of gene, matching RNA nucleotides with complementary DNA nucleotides § This forms messenger RNA (m. RNA) § See this video (start at 2: 51): http: //www. youtube. com/watch? v=yq. ESR 7 E 4 b_8 § Or see this video: § http: //content. dnalc. org/content/c 15/15510/transcription_basic. mp 4 © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Translation § Translation occurs in the cytoplasm (outside the nucleus). § Translation requires: m. RNA (made during transcription), amino acids, energy (ATP), t. RNA, and some helper molecules. § Ribosomes § See this video (start at 4: 48): § http: //www. youtube. com/watch? v=yq. ESR 7 E 4 b_8 § Or see this video: § https: //www. dnalc. org/content/c 15/15501/translation_basi c. mp 4 © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Translation § Ribosomes § The ribosome is composed of ribosomal RNA (r. RNA) and comprises a small and a large subunit. © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Translation § Transfer RNA: t. RNA carries amino acids and matches its anticodon with codons on m. RNA § Codons are 3 nucleotides long © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Translation § A protein is put together one amino acid at a time. § The ribosome attaches to the m. RNA at the promoter region. § Ribosome facilitates the docking of t. RNA anticodons to m. RNA codons. § When two t. RNAs are adjacent, a bond is formed between their amino acids. § Forms a peptide chain of amino acid © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Translation © 2013 Pearson Education, Inc.

Protein Synthesis and Expression: Translation 3 A t. RNA will dock if the complementary RNA codon is present on the ribosome. val r se ala 4 The amino acids join together to form a polypeptide. U Amino acid chain (polypeptide) AG arg ala phe ile GG C UCC Stop codon AAA UAU GCCUUUAUA Ribosome © 2013 Pearson Education, Inc. Figure 8. 7

Protein Synthesis and Expression: Translation Amino acid chain (polypeptide) arg ala phe ile UCC GG C Stop codon AAA UAU GCCUUUAUA 5 The ribosome moves on to the next codon to receive the next t. RNA. © 2013 Pearson Education, Inc. Ribosome 6 When the ribosome reaches the stop codon, no t. RNA can basepair with the codon on the m. RNA and the newly synthesized protein are released. Figure 8. 7

Protein Synthesis and Expression: Translation 7 The chain of amino acids folds, and the protein is ready to perform its job. GAG Protein (such as BGH) © 2013 Pearson Education, Inc. AGC STOP CUCUCGUAA 8 The subunits of the ribosome separate but can reassemble and begin translation of another m. RNA. Figure 8. 7

1 Protein Synthesis and Gene Expression: Translation © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Genetic Code § The genetic code allows a specific codon to code for a specific amino acid. § A codon is comprised of three nucleotides = 64 possible combinations (43 combinations) § 61 codons code for amino acids § 3 others are stop codons, which end protein synthesis § Genetic code expresses redundancy § The genetic code is nearly universal. We and bacteria use the same code, thus our genes can make our proteins in bacteria © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Genetic Code © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Genetic Code © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Mutations § Changes in genetic sequence = mutations § Changes in genetic sequence might affect the order of amino acids in a protein. § Protein function is dependent on the precise order of amino acids § Possible outcomes of mutation: 1 - no change in protein 2 - non-functional protein 3 - different protein © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Mutation § Base-substitution mutation § Simple substitution of one base for another © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Mutation § Neutral mutation (or silent mutation) § Mutation does not change the function of the protein, it codes for the same amino acid © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: Mutation § Frameshift mutation § Addition or deletion of a base, which changes the reading frame © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: An Overview of Gene Expression § Each cell in your body (except sperm and egg cells) has the same DNA. § But each cell only expresses a small percentage of genes. § Example: Nerve and muscle cells perform very different functions, thus they use different genes. § Turning a gene or a set of genes on or off = regulating gene expression © 2013 Pearson Education, Inc.

1 Protein Synthesis and Gene Expression: An Overview of Gene Expression § Nerves and cells have the same suite of genes, but they express different genes. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § r. BGH is a protein, and is coded by a specific gene. § Transfer of r. BGH gene to bacteria allows for growth under ideal conditions. § Bacteria can serve as “factories” for production of r. BGH. § Cloning of the gene is making copies of that gene outside of its native environment. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § Restriction enzymes – Used by bacteria as a form of defense. Restriction enzymes cut DNA at specific sequences. They are important in biotechnology because they allow scientists to make precise cuts in DNA. § Plasmid – Small, circular piece of bacterial DNA that exists separate from the bacterial chromosome. Plasmids are important because they can act as a ferry to carry a gene into and out of cells. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § Step 1. Remove the gene from the cow chromosome © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § Step 2. Insert the BGH gene into the bacterial plasmid. This creates a clone of the gene. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § Recombinant – Indicates material that has been genetically engineered: a gene that has been removed from its original genome and combined with another to change its genetic environment. § After step 2, the BGH is now referred to as recombinant BGH or r. BGH. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § Step 3. Insert the recombinant plasmid into a bacterial cell © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § About 1/3 of cows in the US are injected with r. BGH increases milk volume from cows by about 20%. § Here, r. BGH does not enter the milk, and thus does not modify the milk per se. § The same principles apply to other proteins. § Clotting proteins for hemophiliacs are produced using similar methods. § Insulin for diabetics is also produced in this way. © 2013 Pearson Education, Inc.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria § The use of recombinant genes to alter the properties of organisms (referred especially to foodsources) is defined as a genetically modified organism (GMO) § FDA approval is needed for any new food, including GMOs, that must be demonstrated to be safe for human or agricultural animal consumption § Natural food sources are generally recognized as safe (GRAS). © 2013 Pearson Education, Inc.

3 Genetically Modified Foods: Modifying Plants with the Ti Plasmid and Gene Gun § Transgenic organism – the result of the transfer of a gene from one organism to the genome of another, specifically to alter a phenotype. Also referred to as a genetically modified organism (GMO). § Benefits: Crops can be engineered for resistance to pests or drought, thus farmers can spray fewer chemicals, or irrigate less. § Concerns: § Pests can become resistant to chemicals or anti-pest proteins. § GM crops may actually lead to increased use of pesticides and herbicides. § GM crop plants may transfer genes to wild relatives § The flavor or nutritional value can be modified. © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Stem Cells § Stem cells – undifferentiated cells, capable of growing in to many different kinds of cells and tissues § Stems cells might be used to treat degenerative diseases (tissue repair less perfect as cells age, leading to permanent dysfunction) such as Alzheimer’s or Parkinson’s. § Using stem cells to produce healthy tissue is called therapeutic cloning. § Stem cells could also be used to grow specific tissues to treat burns, heart attack damage, or replacement cartilage in joints. § Some stems cells are totipotent, meaning they can become any other cell in the body. © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Stem Cells § Therapeutic cloning may be combined with acellularized tissues § Original cells are removed from an organ, leaving only the tissue matrix § Stem cells are grown in the tissue matrix instead of in a tissue culture dish. § The reconstituted organ can be reintroduced into the body. © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Human Genome Project § Human Genome Project – international effort to map the sequence of the entire human genome (~20, 000 – 25, 000 genes). § For comparative purposes, genomes of other model organisms (E. coli, yeast, fruit flies, mice) were also mapped. § It was sequenced using the technique of chromosome walking. © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Gene Therapy § Gene therapy – replacement of defective genes with functional genes § Germ line gene therapy § Embryonic treatment § Embryo supplied with a functional version of the defective gene. § Embryo + cells produced by cell division have a functional version of gene. § Risk: all progeny affected by any side-effects § Somatic cell gene therapy – fix or replace the defective protein only in specific cells § Disadvantage: does not propagate to children © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Gene Therapy § Somatic cell therapy used as a treatment of SCID (severe combined immunodeficiency) § All somatic cells have limited lifetimes. § Therapy is not permanent and requires several treatments per year and follow-ups in the future © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Cloning Humans § Human “cloning” occurs naturally whenever identical twins are produced. § Cloning of offspring from adults has already been done with cattle, goats, mice, cats, pigs, and sheep. § Cloning is achieved through the process of nuclear transfer. § Nuclear transfer has been done with fertilized egg nucleus into enucleated cell – three person mating § Being tested to cure mitochondrial genetic diseases © 2013 Pearson Education, Inc.

4 Genetically Modified Humans: Cloning Humans NOTE! Dolly died in 6. 5 years instead of 13 years with symptoms of old-age Adult DNA introduced into embryo cell Nuclear cloning from embryonic cell leads to long-lived clone © 2013 Pearson Education, Inc.

Which of the following types of RNA carries amino acids to the growing polypeptide chain? § m. RNA § t. RNA § r. RNA § RNA does not carry amino acids © 2013 Pearson Education, Inc.

Which of the following types of RNA carries amino acids to the growing polypeptide chain? § m. RNA § t. RNA § r. RNA § RNA does not carry amino acids © 2013 Pearson Education, Inc.

A sequence of m. RNA, called a codon, reads ACU. How will the set of nucleotides on the anticodon of the t. RNA read? § ACU § UGA § TGA § AUG © 2013 Pearson Education, Inc.

A sequence of m. RNA, called a codon, reads ACU. How will the set of nucleotides on the anticodon of the t. RNA read? § ACU § UGA § TGA § AUG © 2013 Pearson Education, Inc.

Which of the following statements concerning r. BGH-treated milk is correct? § The injected cows produce 20% more milk. § There is no evidence of the hormone being transferred to the milk. § Humans would be able to safely digest the hormone, just like any other protein in food. § All of the statements are correct. © 2013 Pearson Education, Inc.

Which of the following statements concerning r. BGH-treated milk is correct? § The injected cows produce 20% more milk. § There is no evidence of the hormone being transferred to the milk. § Humans would be able to safely digest the hormone, just like any other protein in food. § All of the statements are correct. © 2013 Pearson Education, Inc.

Which of the following was used to treat SCID patients? § therapeutic cloning § nuclear transfer § somatic gene therapy § germ line gene therapy © 2013 Pearson Education, Inc.

Which of the following was used to treat SCID patients? § therapeutic cloning § nuclear transfer § somatic gene therapy § germ line gene therapy © 2013 Pearson Education, Inc.

Which of the following statements is incorrect? § Stem cells are undifferentiated. § Stem cells are totipotent. § Specialized stem cells divide to make undifferentiated stem cells. § Stem cells can be used for therapeutic cloning. © 2013 Pearson Education, Inc.

Which of the following statements is incorrect? § Stem cells are undifferentiated. § Stem cells are totipotent. § Specialized stem cells divide to make undifferentiated stem cells. § Stem cells can be used for therapeutic cloning. © 2013 Pearson Education, Inc.

When scientists try to replace defective human genes with functional genes they are performing ____. § gene therapy § in vitro fertilization § therapeutic cloning § nuclear transfer © 2013 Pearson Education, Inc.

When scientists try to replace defective human genes with functional genes they are performing ____. § gene therapy § in vitro fertilization § therapeutic cloning § nuclear transfer © 2013 Pearson Education, Inc.

What is happening in step 1 in this figure? § The embryo is being grown in culture. § The egg cell and mammary cell are fused together. § The nucleus is removed from the egg cell. § The embryo is being implanted into the uterus of a third sheep. © 2013 Pearson Education, Inc.

What is happening in step 1 in this figure? § The embryo is being grown in culture. § The egg cell and mammary cell are fused together. § The nucleus is removed from the egg cell. § The embryo is being implanted into the uterus of a third sheep. © 2013 Pearson Education, Inc.

When undergoing recombination, _______. § the plasmid and the cow gene are cut with different restriction enzymes § the recombinant plasmid is reinserted into the cow’s cell to increase milk production § the r. BGH genes are injected into cows to increase their milk production § the recombinant plasmid is inserted in bacterium, making large quantities of r. BGH proteins © 2013 Pearson Education, Inc.

When undergoing recombination, _______. § the plasmid and the cow gene are cut with different restriction enzymes § the recombinant plasmid is reinserted into the cow’s cell to increase milk production § the r. BGH genes are injected into cows to increase their milk production § the recombinant plasmid is inserted in bacterium, making large quantities of r. BGH proteins © 2013 Pearson Education, Inc.