1. History of DNA and Recombinant DNA

2. 4.1 DNA Structure Determining that DNA is the genetic material was accomplished through decades of research by many scientists. In the late 1920s, Frederick Griffith’s studies identified a transforming substance that could change nonlethal bacteria to lethal bacteria. Figure 4.1 Griffith’s experiment. UNIT A Chapter 4: DNA Structure and Gene Expression Section 4.1 TO PREVIOUS SLIDE

3. In 1944, Oswald Avery and his research colleagues showed that Griffith’s transforming substance was DNA and that this was the genetic material. Their findings were: UNIT A Section 4.1 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression The Nature of Genetic Materia l

4. In the early 1950s, Hershey and Chase firmly established DNA as the genetic material. They used a virus (T phage) that infects bacteria, where it makes new copies of itself. In one experiment, they used virus with radioactive DNA and identified where the radioactivity went after infection UNIT A Section 4.1 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.2a Hershey-Chase experiments. The Nature of Genetic Material

5. In another experiment, they used a virus with radioactive protein and identified where the radioactivity went after infection. They discovered that radioactivity entered the bacterial cells when virus with radioactive DNA was added, but not virus with radioactive protein. Therefore, the hereditary material is DNA UNIT A Section 4.1 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.2b Hershey-Chase experiments.

6. 4.4 Gene Mutations and Cancer A gene mutation is a permanent change in DNA sequence. Germ-line mutations occur in sex cells and can be passed on to future generations Somatic mutations occur in body cells and are not passed on to future generations Both types of mutations may lead to the development of cancer. UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

7. Causes of Mutations Errors in replication are mistakes made while DNA is copied. These are rare (1 mistake per billion nucleotide pairs) Mutagens are environmental factors, such as radiation, X rays, and some chemicals, that cause mutations Transposons are DNA sequences that move within and between chromosomes. Transposons can “jump” into another gene, causing a change in gene expression UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.16 Transposon.

8. Effect of Mutations on Protein Activity Gene mutations can have a range of possible effects on protein activity, from no effect to complete inactivity or even lack of production at all. A point mutation is a single nucleotide change. It can cause no change in amino acid sequence a change in amino acid sequence that produces a protein that does not function properly introduction of a stop codon, which shortens the protein https://www.youtube.com/watch?v=DlhpvcgK_28 UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

9. Point Mutations Figure 4.17 Point mutations in hemoglobin. The effect of a point mutation can vary. a. Starting at the top: Normal sequence of bases in hemoglobin; next, the base change has no effect; next, due to base change, DNA now codes for valine instead of glutamic acid, and the result is that normal red blood cells (b) become sickle-shaped (c); next, base change will cause DNA to code for termination and the protein will be incomplete. UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

10. Nonfunctional Proteins Frameshift mutations involve one or more nucleotides being added or deleted. This can cause a change in codons that are translated and production of a nonfunctional protein. If the codons made a sentence, an example would be THE CAT ATE THE RAT; deleting the C, becomes THE ATA TET HER AT Just as the meaning of the sentence is scrambled, a nonfunctional protein can have a dramatic effect on a phenotype Many reactions in cells occur in a series called a pathway. If one protein (enzyme) is nonfunctional, it can affect the entire pathway of reactions. UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

11. Chromosomal Mutations: Deletion - a piece of a chromosome breaks off and is lost. Inversion - a piece of a chromosome breaks off and reattaches itself in reverse order. Translocation - a broken piece attaches to a nonhomologous chromosome. Nondisjunction - a pair of chromosomes fail to separate during cell division.

19. Mutations Can Cause Cancer The development of cancer involves a series of accumulating mutations, which depend on the type of cancer. Most cancers follow a common progression. They begin as a benign growth of abnormal cells They can become a malignant tumour and spread to other areas Figure 4.18 Progression of cancer. UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

20. Characteristics of Cancer Cells The primary characteristics of cancer cells: Cancer cells are genetically unstable. Tumour cells have multiple mutations and can have chromosomal changes. Cancer cells do not correctly regulate the cell cycle. The rate of division and number of cells increases. Cancer cells escape the signals for cell death. Normal cell signals for programmed cell death do not occur. Cancer cells can survive and proliferate elsewhere in the body. Invasion of new tissues can occur (metastasis), which includes new blood vessel formation (angiogenesis). UNIT A Section 4.4 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression https://www.youtube.com/watch?v=7tzaW OdvGMw

21. 4.5 DNA Cloning Genetic engineering involves altering the genome, or genetic material, of an organism. This often involves gene cloning, which is the production of copies of a gene. Gene cloning is done to study what biological functions a gene is associated with produce large quantities of protein produce transgenic organisms help cure human diseases UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

22. Recombinant DNA Technology Gene cloning involves introducing a gene into a vector (often a plasmid of a bacterium) to produce recombinant DNA (rDNA). A restriction enzyme cleaves the vector and the gene, which combine by base pairing between the “sticky ends” UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Many restriction enzymes leave overhangs of nucleotides when they cut DNA, which are called “sticky ends” because they can easily base pair with other overhangs.

23. Recombinant DNA Technology UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression DNA ligase enzyme seals the gene and vector DNAs. The rDNA is added to an organism such as bacteria, which makes many copies of the gene.

24. Making Recombinant DNA A "vector" is something that can get the DNA from one species into the other species' DNA. Often, this can be a "plasmid", a circular piece of DNA found in some bacteria. A human gene, such as the gene for insulin, is inserted into the plasmid and then the plasmid is taken up by bacteria. The bacteria reproduces the plasmid along with its own DNA when it reproduces, and translates the human gene, producing human protein.

26. Gene Cloning UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.19 Cloning a human gene. Human DNA and bacterial plasmid DNA are cleaved by a specific type of restriction enzyme. For example, human DNA containing the insulin gene is spliced into a plasmid by the enzyme DNA ligase. Gene cloning is achieved after a bacterium takes up the plasmid. If the gene functions normally as expected, the product (for example, insulin) may also be retrieved.

27. The Polymerase Chain Reaction The polymerase chain reaction (PCR) is a way of making billions of copies of a segment of DNA in a test tube. PCR involves three steps that are repeated many times in cycles. 1.Denaturation: The DNA is heated to 95 o C, and it becomes single-stranded. 2.Annealing: The temperature is lowered to 50−60 o C, and primers are added that base pair to the DNA to be copied. 3.Extension: At 72 o C, DNA polymerase used for PCR adds nucleotides to the ends of the primers. Eventually both DNA strands are copied and new double-stranded DNA forms. UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression

28. The Polymerase Chain Reaction PCR is a chain reaction because the DNA is repeatedly copied. The amount of DNA doubles with each cycle. UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.20 Polymerase chain reaction (PCR).

29. DNA Analysis PCR has numerous applications, which includes identification of people based on their DNA fingerprint. Short tandem repeat (STR) profiling identifies individuals according to how many repeats of a DNA sequence he or she has at a particular STR locus. UNIT A Section 4.5 TO PREVIOUS SLIDE Chapter 4: DNA Structure and Gene Expression Figure 4.21 The use of STR profiling to establish paternity.

30. PRODUCING BIOTECHNOLOGY PRODUCTS: genetically engineered prokaryotic and eukaryotic cells can be used to mass produce once rare medicinal proteins and hormones as well as vaccines to prevent disease (e.g. hepatitis B). The future may provide vaccines for such things as herpes and AIDS and many other diseases. it allows us to produce large amounts of proteins that are very difficult to get otherwise or are usually present only in small quantities from natural sources.

31. Human growth hormone - once took 50 pituitary glands from cadavers for a single dose. Can now be made in mass quantities, and much less expensively. Insulin used to come from the pancreatic glands of cows and pigs. This was expensive and much less pure than the cloned human DNA available today. tPA (tissue plasminogen activator) - a protein that activates an enzyme that dissolves blood clots, normally present in only tiny amounts, can now be made in large quantities and is routinely used to dissolve the coronary blood clots of heart attack victims.

33. MAKING TRANSGENIC ORGANISMS: We can alter the DNA of bacteria, plants, and farm animals to make them more valuable and less susceptible to disease. Bacteria is very useful in this capacity Bacteria are used to Protect and Enhance Plants: e.g. protect plants from frost, provide more nitrogen to roots, even produce insecticides to kill insects. Bacteria are used for Garbage Disposal: Bacteria can be engineered to eat toxic waste and clean up oils spills, filter the air, remove sulfur from coal etc. (bioremediation is the name for using organisms to clean up man’s messes) Bacteria are used to Produce Chemicals: e.g. genetically engineered bacteria produce phenylalanine, used in the production of ‘Nutrasweet’ artificial sweetener. Bacteria are used to Process Minerals: genetically engineered bacteria can be used to extract greater amounts of metals (e.g. U, Cu, Ag) from low-quality ores.

34. Transgenic plants: Are already widely used in agriculture. The bacterium grobacterium, which naturally infects many plants, is used, as well as artificial vectors called protoplasts. Over 50 types of genetically engineered plants today. Contain new genes that help resist insects, viruses, or herbicides (e.g. “Roundup” resistant wheat). Genetically engineered soybeans, cotton, alfalfa, and rice are already on the market. Transgenic plants can also be made that are resistant to temperature extremes, drought, and salty soils. There are transgenic plants that resist spoiling and bruising.

35. Transgenic animals: by injecting DNA that codes for the uptake of bovine growth hormone (bGH) into the eggs of fish, cows (e.g. 25% greater milk production), pigs, rabbits, and sheep, bigger animals can be produced more cheaply. “Gene Farming” refers to the use of transgenic farm animals to produce pharmaceutical drugs like human lactoferrin (absence of the gene for lactoferrin in humans causes reoccurring bacterial infections of the intestine). Transgenic cows, for example, will produce human lactoferrin in their milk. Drink the milk, and you get protection from the intestinal infections. There are plans to produce drugs to treat cystic fibrosis, blood diseases, and cancer through this method.

36. GENE THERAPY The idea behind gene therapy is to replace defective genes in a living organism (especially humans) with healthy genes, and is used to treat genetic disorders and diseases. Can be “Ex Vivo” (outside living organism) or “In Vivo” (inside living organism).

37. Ex Vivo gene therapy In Ex Vivo gene therapy, cells are removed from the patient, treated, then returned to the patient. Can use retroviruses to introduce the nucleic acid into the cells to be treated. e.g. has been used to treat SCID (severe combined immunodeficiency syndrome - sufferers lack an enzyme needed for certain white blood cells). Insert the correct gene into patient’s WBC or stem cell and reinsert. e.g. - has been used to treat liver cells in hypercholesterolemia (in this disorder, liver is unable to remove cholesterol from blood  heart attacks) e.g. - used to make cancer patients more resistant to chemotherapy drugs)

38. In Vivo techniques In Vivo techniques introduce genes right into the bodies of patients. e.g. an inhalant spray containing an adenovirus containing a gene to treat cystic fibrosis (must be reapplied regularly). e.g. treat or cure Parkinson’s Disease by grafting dopamine- producing cells right onto the brain. e.g. treat hemophilia with regular injections of cells that have the normal clotting-factor genes. e.g. work is being done to see if retroviruses can be used to carry genes for cytokines (soluble hormones of the immune system) to treat cancer. “antisense” technology may be used to turn off cancer-causing genes (oncogene) or turn off AIDS viruses.

39. Dnews Gene Therapy: https://www.youtube.com/watch?v=bLI1Gfb0ynw https://www.youtube.com/watch?v=bLI1Gfb0ynw