Genetically Modified Organisms Gene Expression, Mutation, and Cloning Chapter 7 Genetically Modified Organisms Gene Expression, Mutation, and Cloning

1 Protein Synthesis and Gene Expression In the early 1980s, genetic engineers began producing recombinant bovine growth hormone (rBGH) Made by genetically engineered bacteria The bacteria were given DNA that carries instructions for making BGH In cows, growth hormones increase body size and milk production

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

1 Protein Synthesis and Gene Expression: From Gene to Protein DNA Double-stranded Each nucleotide composed of deoxyribose, phosphate, and nitrogenous base 4 bases: adenine, thymine, guanine, cytosine

1 Protein Synthesis and Gene Expression: From Gene to Protein RNA Single-stranded Nucleotides comprised of ribose, phosphate, and nitrogenous base 4 bases: A, T, G, and Uracil

1 Protein Synthesis and Gene Expression: From Gene to Protein The flow of genetic information in a cell is DNA  RNA  protein and occurs in 2 steps: Transcription (DNA  RNA) Translation (RNA  Protein)

1 Protein Synthesis and Gene Expression: Transcription Transcription occurs in the nucleus. RNA polymerase binds to the promoter region of the gene. RNA polymerase zips down the length of gene, matching RNA nucleotides with complementary DNA nucleotides This forms messenger RNA (mRNA)

Animation: Transcription Click “Go to Animation” / Click “Play”

1 Protein Synthesis and Gene Expression: Translation Translation occurs in the cytoplasm (outside the nucleus). Translation requires: mRNA (made during transcription), amino acids, energy (ATP), and some helper molecules. Ribosomes Transfer RNA (tRNA)

1 Protein Synthesis and Gene Expression: Translation Ribosomes The ribosome is composed of ribosomal RNA (rRNA) and comprises a small and a large subunit.

1 Protein Synthesis and Gene Expression: Translation Transfer RNA: tRNA carries amino acids and matches its anticodon with codons on mRNA Codons are 3 nucleotides long

1 Protein Synthesis and Gene Expression: Translation A protein is put together one amino acid at a time. The ribosome attaches to the mRNA at the promoter region. Ribosome facilitates the docking of tRNA anticodons to mRNA codons. When two tRNAs are adjacent, a bond is formed between their amino acids. Forms a peptide chain of amino acid

1 Protein Synthesis and Gene Expression: Translation

1 Protein Synthesis and Gene Expression: Translation

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 universal

1 Protein Synthesis and Gene Expression: Genetic Code

BioFlix: Protein Synthesis Animation: Translation Click “Go to Animation” / Click “Play” BioFlix: Protein Synthesis

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

1 Protein Synthesis and Gene Expression: Mutation Base-substitution mutation Simple substitution of one base for another

1 Protein Synthesis and Gene Expression: Mutation Neutral mutation Mutation does not change the function of the protein, it codes for the same amino acid

1 Protein Synthesis and Gene Expression: Mutation Frameshift mutation Addition or deletion of a base, which changes the reading frame

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

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.

1 Protein Synthesis and Gene Expression: Regulating Gene Expression Regulation of transcription Prokaryotic cells use repressors to regulate gene expression Repressors bind to the promoter and prevent the RNA polymerase from binding

1 Protein Synthesis and Gene Expression: Regulating Gene Expression Regulation of transcription Eukaryotic cells use activators to regulate gene expression Activators help the RNA polymerase bind to the promoter

1 Protein Synthesis and Gene Expression: Regulating Gene Expression Regulation by chromosome condensation Folding up of the chromosomes prevents transcription Regulation by mRNA degradation Nucleases cut mRNA Regulation of Translation Slowing of binding of the mRNA to the ribosome Regulation of Protein Degradation Proteases degrade proteins

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria rBGH is a protein, and is coded by a specific gene. Transfer of rBGH gene to bacteria allows for growth under ideal conditions. Bacteria can serve as “factories” for production of rBGH. Cloning of the gene is making many copies of that gene.

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 a cell.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria Step 1. Remove the gene from the cow chromosome

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria Step 2. Insert the BGH gene into the bacterial plasmid

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. After step 2, the GBH is now referred to as recombinant GBH or rGBH.

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria Step 3. Insert the recombinant plasmid into a bacterial cell

2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria About 1/3 of cows in the US are injected with rBGH. rBGH increases milk volume from cows by about 20%. 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. FDA approval is needed for any new food that is not generally recognized as safe (GRAS).

Animation: Producing Bovine Growth Hormone Click “Go to Animation” / Click “Play”

3 Genetically Modified Foods All agricultural products are the result of genetic modification through selective breeding. Artificial selection does not move genes from one organism to another, but does drastically change the characteristics of a population. Genetically modifying foods Increase shelf life, yield, or nutritional value Golden rice has been genetically engineered to produce beta-carotene, which increases the rice’s nutritional yield.

3 Genetically Modified Foods: Modifying Plants with the Ti Plasmid and Gene Gun Unlike rBGH, crop plants are directly modified. In order to do this, the target gene must be inserted into the plant cell. Two methods to do this: Ti plasmid Gene gun

3 Genetically Modified Foods: Modifying Plants with the Ti Plasmid

3 Genetically Modified Foods: Modifying Plants with the Gene Gun

3 Genetically Modified Foods: Modifying Plants with the Ti Plasmid and Gene Gun Transgenic organism – the result of the incorporation of a gene from one organism to the genome of another. Also referred to as a genetically modified organism (GMO). Benefits: Crops can be engineered for resistance to pests, thus farmers can spray fewer chemicals. Concerns: Pests can become resistant to chemicals. GM crops may actually lead to increased use of pesticides and herbicides. GM crop plants may transfer genes to wild relatives.

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 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. Stems cells are totipotent, meaning they can become any other cell in the body.

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.

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. Somatic cell gene therapy Somatic cell gene therapy – fix or replace the defective protein only in specific cells

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.

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.

4 Genetically Modified Humans: Cloning Humans

Which of the following types of RNA carries amino acids to the growing polypeptide chain? mRNA tRNA rRNA RNA does not carry amino acids Answer: B Transfer RNAs carry amino acids. Messenger RNAs are complementary to the DNA. Ribosomal RNAs make up ribosomes. 47

Which of the following types of RNA carries amino acids to the growing polypeptide chain? mRNA tRNA rRNA RNA does not carry amino acids 48

A sequence of mRNA, called a codon, reads ACU A sequence of mRNA, called a codon, reads ACU. How will the set of nucleotides on the anticodon of the tRNA read? ACU UGA TGA AUG Answer: B The anticodon of the tRNA is complimentary to the codon. UGA is the only sequence that is complimentary to ACU. 49

A sequence of mRNA, called a codon, reads ACU A sequence of mRNA, called a codon, reads ACU. How will the set of nucleotides on the anticodon of the tRNA read? ACU UGA TGA AUG 50

Which of the following regulation techniques will result in increased gene expression? condensing the chromosome speeding up proteases lengthening the adenosine nucleotide “tail” slowing the movement of the mRNA through the ribosome Answer: C The poly-A tail protects the mRNA from nucleases. Condensing the chromosome, speeding up proteases, and slowing the movement of the mRNA through the ribosome would decrease gene expression. 51

Which of the following regulation techniques will result in increased gene expression? condensing the chromosome speeding up proteases lengthening the adenosine nucleotide “tail” slowing the movement of the mRNA through the ribosome 52

Which of the following statements is accurate? The plasmid is cut with the same restriction enzyme as the removed gene. The plasmid is a circular piece of RNA. The plasmid is part of the bacterial chromosome. The plasmid replicates when the bacterial chromosome replicates. Answer: A The same restriction enzyme is used to cut open the plasmid and to remove the gene of interest. This creates complementary sticky ends. Plasmids are circular pieces of DNA and exist separately from the bacterial chromosome. They replicate independently of the bacterial chromosome. 53

Which of the following statements is accurate? The plasmid is cut with the same restriction enzyme as the removed gene. The plasmid is a circular piece of RNA. The plasmid is part of the bacterial chromosome. The plasmid replicates when the bacterial chromosome replicates. 54

Which of the following statements concerning rBGH-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. Answer: D All of these facts were taken into account when the FDA declared that rBGH-treated milk is safe to consume. 55

Which of the following statements concerning rBGH-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. 56

Which of the following was used to treat SCID patients? therapeutic cloning nuclear transfer somatic gene therapy germ line gene therapy Answer: C Somatic gene therapy is the taking of cells from a person and injecting it with the functional gene and then allow the cell to reproduce and then place the copies of the good cells back into the body. 57

Which of the following was used to treat SCID patients? therapeutic cloning nuclear transfer somatic gene therapy germ line gene therapy 58

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. Answer: C Once stem cells have specialized, they cannot backtrack to become a different type of cell. Stem cells are undifferentiated and totipotent. They can be used for therapeutic cloning. 59

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. 60

When scientists try to replace defective human genes with functional genes they are performing ________. gene therapy in vitro fertilization therapeutic cloning nuclear transfer Answer: A Gene therapy is the process of replacing defective genes with functional genes. In vitro fertilization is a fertility treatment resulting in excess embryos. Therapeutic cloning uses stem cells to produce healthy tissues. Nuclear transfer occurs when mammary gland cells are fused with an egg cell that has its nucleus removed. 61

When scientists try to replace defective human genes with functional genes they are performing ________. gene therapy in vitro fertilization therapeutic cloning nuclear transfer 62

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. Answer: C The mammary cells and egg cell are isolated from two sheep and the nucleus is then removed from the egg cell. The egg cell and mammary cell are then fused together. The embryo is grown in culture and then implanted into the uterus of a third sheep. 63

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. 64

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 rBGH genes are injected into cows to increase their milk production the recombinant plasmid is inserted in bacterium, making large quantities of rBGH proteins Answer: D In order to create recombinant BGH, you need to combine the bacteria plasmid with the gene that produces milk from the cow DNA. In order to do this the plasmid and cow BGH gene are cut with the same restriction enzyme. This allows for the two to have sticky ends which will allow the plasmid and the cow gene to combine into one. The recombinant plasmid is then reinserted into the bacterium. The rBGH genes produce large quantities of rBGH proteins insidie the bacterium, which are harvested, purified, and then injected into the cows. 65

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 rBGH genes are injected into cows to increase their milk production the recombinant plasmid is inserted in bacterium, making large quantities of rBGH proteins 66