Genetic Engineering: From Gene to Protein Synthesis

1. Chapter 7 Genetically Modified OrganismsGene Expression, Mutation, and Cloning 0

2. 1 Protein Synthesis and Gene Expression In the late 1970s, genetic engineers began producing recombinant bovine growth hormone (rBGH), 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

3. 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

4. 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)

5. 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 mRNA

6. 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)

7. 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 (mRNA) See this video (start at 2:51): http://www.youtube.com/watch?v=yqESR7E4b_8 Or see this video: http://content.dnalc.org/content/c15/15510/transcription_basic.mp4

8. 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), tRNA, and some helper molecules. Ribosomes See this video (start at 4:48): http://www.youtube.com/watch?v=yqESR7E4b_8 Or see this video: https://www.dnalc.org/content/c15/15501/translation_basic.mp4

9. 1 Protein Synthesis and Gene Expression: Translation Ribosomes The ribosome is composed of ribosomal RNA (rRNA) and comprises a small and a large subunit.

10. 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

11. 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

12. 1 Protein Synthesis and Gene Expression: Translation

13. Protein Synthesis and Expression: Translation 3 A tRNA will dock if the complementary RNA codon is present on the ribosome. val ser ala 4 The amino acids join together to form a polypeptide. AGU Amino acid chain (polypeptide) arg phe ile ala UCC Stop codon CGG AAA UAU GCCUUUAUA Ribosome Figure 8.7

14. Protein Synthesis and Expression: Translation Amino acid chain (polypeptide) arg phe ala ile UCC Stop codon CGG AAA UAU GCCUUUAUA 5 The ribosome moves on to the next codon to receive the next tRNA. Ribosome 6 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. Figure 8.7

15. Protein Synthesis and Expression: Translation 7 The chain of amino acids folds, and the protein is ready to perform its job. GAG STOP AGC CUCUCGUAA Protein (such as BGH) 8 The subunits of the ribosome separate but can reassemble and begin translation of another mRNA. Figure 8.7

16. 1 Protein Synthesis and Gene Expression: Translation

17. 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 proteinsin bacteria

18. 1 Protein Synthesis and Gene Expression: Genetic Code

19. 1 Protein Synthesis and Gene Expression: Genetic Code

20. 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

21. 1 Protein Synthesis and Gene Expression: Mutation Base-substitution mutation Simple substitution of one base for another

22. 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

23. 1 Protein Synthesis and Gene Expression: Mutation Frameshift mutation Addition or deletion of a base, which changes the reading frame

24. 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

25. 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.

26. 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 copies of that gene outside of its native environment.

27. 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.

28. 2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria Step 1. Remove the gene from the cow chromosome

29. 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.

30. 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 rBGH.

31. 2 Producing Recombinant Proteins: Cloning a Gene Using Bacteria Step 3. Insert the recombinant plasmid into a bacterial cell

32. 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%. Here, rBGH 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.

33. 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).

34. 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.

35. 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.

36. 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.

37. 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.

38. 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 Somatic cell gene therapy – fix or replace the defective protein only in specific cells Disadvantage: does not propagate to children

39. 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

40. 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

41. 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

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

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

44. 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

45. 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

46. 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.

47. 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.

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

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

50. 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.