PART 1: DNA Cloning and Plasmids

1. PART 1:DNA Cloning and Plasmids DNA Technology and Genomics

2. Biotechnology • DNA technology has launched a revolution in the area of biotechnology • The manipulation of organisms or their genetic components to make useful products • Example: gene cloning • Usually uses bacterial plasmids

3. Plasmid • A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.

4. Cell containing geneof interest Bacterium Gene of interest Plasmid Bacterialchromosome DNA ofchromosome RecombinantDNA (plasmid) Recombinantbacterium 3 Gene of interest Protein expressedby gene of interest Copies of gene Protein harvested Basic research on protein Basic research on gene Gene used to alterbacteria for cleaningup toxic waste Human growth hormone treatsstunted growth Gene for pestresistance inserted into plants Protein dissolvesblood clots in heartattack therapy Figure 20.2 • Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

5. How do You Make Recombinant DNA? • Use bacterial restriction enzymes • Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

6. Restriction Enzymes: Proteins that cut the DNA in a specific place Recombinant Plasmid

7. Transformation bacteria takes in plasmid from environment

8. Why make DNA recombinants? • There are 3 main reasons for creating recombinant DNA • to create a protein product • to create multiple copies of genes • to insert foreign genes into other organisms to give those organisms a new trait • Make DNA fingerprints • Recombinant DNA is used widely today to create large amounts of protein for treating illnesses • Ex: In 1982, insulin became the first recombinant DNA drug to hit the market

9. PLANTS Disease-resistant Insect-resistant crops Hardier and bigger fruit Increased vitamin content “Pharming” purposes (ex. Vaccine) 70-75% of food in supermarket is genetically modified. The DNA of plants and animals is altered for several reasons

10. TRANSGENIC ANIMALS Fish – grow faster than normal aquaculture salmon Chickens – more resistant to infections Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk Genetically modified organisms are called transgenic organisms.

11. What are Genetically Modified Foods? (“Frankenfoods”?) You have already eaten GM foods. Some GM tomatoes, for example, have had their genes altered to stop them from going soft while they are still growing. For several years they were widely sold in tomato paste. The GM foods we eat have all been tested for safety. But some people worry about the long term effects of eating genetically modified foods!

12. 'Frankenfood' Fears Experts say this science, like any other, has no guarantees. Risks include: • Introducing allergens and toxins to food • Accidental contamination between genetically modified and non-genetically modified foods • Antibiotic resistance • Adversely changing the nutrient content of a crop • Creation of "super" weeds and other environmental risks Benefits include: • Increased pest and disease resistance • Drought tolerance • Increased food supply

13. Advantages: • The rice can be considered a particular advantage to poor people in underdeveloped countries. They eat only an extremely limited diet lacking in the essential bodily vitamins. The consequences of this restricted diet causes many people to die or become blind. This is particularly true in areas of Asia, where most of the population live on rice from morning to evening. Golden rice is genetically modified rice that now contains a large amount of A-vitamins. Or more correctly, the rice contains the element beta-carotene which is converted in the body into Vitamin-A. So when you eat golden rice, you get more vitamin A. Can We End World Hunger? Malnutrition and vitamin deficiencies? Golden rice

15. Going Bananas? According to recent reports, the world may soon be out of bananas. Bananas are propagated through asexual cloning, so all bananas are genetically identical Banana plantations in Africa, Asia and Central America are uniquely susceptible to fungi, viruses and pests. Unless scientists can find a way to genetically enhance the banana’s ability to ward off parasites, we could be bananaless in ten years. http://gslc.genetics.utah.edu/features/gmfoods/

16. Examples • For example, taking a gene from a jellyfish and inserting into the zebrafish genome created the “glofish” – fish that glow in the dark which are now being sold on the market. • Another example is genes from a bacteria inserted into food crops so that the crop can create it’s own insecticide. These plants and animals are also known as “transgenic”.

17. Transgenic Goat This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk. Human DNA in a Goat Cell .

18. Part 2: The Genetic Basis of Development:Stem Cells and Cloning DNA Technology and Genomics

19. DROSOPHILA MELANOGASTER (FRUIT FLY) CAENORHABDITIS ELEGANS (NEMATODE) 0.25 mm Model Organisms • used understand broad biological principles Small, easily grown, short generation time, embryos outside body, 13,700 genes Small, easily grown, short generation time, transparent, 19,00 genes

20. ARABIDOPSIS THAMANA (COMMON WALL CRESS) MUS MUSCULUS (MOUSE) DANIO RERIO (ZEBRAFISH) Other “model organisms” Small, easily grown, body, 25,000 genes

21. Embryonic development • Embryonic development involves • cell division (a.k.a.- mitosis), • cell differentiation • In embryonic development of most organisms • A single-celled zygote gives rise to cells of many different types, each with a different structure and corresponding function

22. 3 Interrelated Processes… • Through a succession of mitotic cell divisions • zygote gives rise to large # of cells • In cell differentiation • Cells become specialized in structure and function

23. Specialized cells perform specific functions • Cell differentiation is the process by which unspecialized cells develop into their mature forms and functions. • Your body began as a single fertilized egg. • While almost every cell in your body has a full set of DNA, each type of cell uses only the specific genes it needs to carry out its function. That is, a cell differentiates among the genes and uses only certain ones • Cookbook has recipe for dessert, pasta, etc but same cookbook.

24. CELLS...Totipotency • A totipotentcell • cell capable of generating a complete new organism • Cloning • using 1+ body cells from a multicellular organism to make another genetically identical individual

25. Nuclear Transplantation in Animals • In nuclear transplantation (nuclear transfer) • nucleus of unfertilized egg cell or zygote is replaced with nucleus of a differentiated cell

26. Reproductive Cloning of Mammals • In 1997, Scottish researchers • 1st cloned ANIMAL a lamb from an adult sheep by nuclear transplantation • Named “Dolly” • Aged quickly and didn’t live long. • Possibly because she was cloned from an adult sheep that had “old DNA”. • Died at age 6 of complications that usually affect older sheep • Incomplete reprogramming of original nucleus?

27. Mammalian Cloning

28. Lamb (“Dolly”) genetically identical to mammary cell donor

29. “Copy Cat” • Was the first cat ever cloned • Clones in same species do NOT necessarily look or behave alike  epigenetics

30. Problems With Cloning • In most nuclear transplantation studies • Only a small % of cloned embryos develop normally • Most (all?) have various defects if clone lives • It takes hundreds of tries to produce one clone

31. The Stem Cells of Animals ONLY • A stem cell (topipotent cells) • A relatively unspecialized cell • Can reproduce itself indefinitely • Can differentiate into specialized cells of one or more types, given appropriate conditions • 2 types: embryonic & adult

32. Stem Cell Classification • Totipotent stem cells can grow into any other cell type. Only a fertilized egg and the cells produced by the first few divisions of an embryo are totipotent. • Pluripotent stem cells can grow into any cell type except for totipotent stem cells. • Multipotent stem cells can only grow into cells of a closely related cell family.

33. Stem Cell Classification

34. Adult stem cells • Said to be pluripotent • able to give rise to multiple but not all cell types Embryonic stem cells • Said to be topipotent • able to give rise toall cell types

35. Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) From bone marrow in this example Adult stem cells Embryonic stem cells Totipotent cells Pluripotent cells Cultured stem cells Different culture conditions Different types of Differentiated cells Liver cells Blood cells Nerve cells • Stem cells can be isolated • From early embryos at the blastocyst stage