1. Chapter 13 Notes -Genetic Engineering Prentice Hall (pg. 318-339) 13–1 Changing the Living World A.Selective Breeding 1.Hybridization 2.Inbreeding B.Increasing Variation 1.Producing New Kinds of Bacteria 2.Producing New Kinds of Plants 13–2 Manipulating DNA A.The Tools of Molecular Biology 1.DNA Extraction 2.Cutting DNA 3.Separating DNA B.Using the DNA Sequence 1.Reading the Sequence 2.Cutting and Pasting 3.Making Copies 13–3 Cell Transformation A.Transforming Bacteria B.Transforming Plant Cells C. Transforming Animal Cells 13–4 Applications of Genetic Engineering A.Transgenic Organisms 1.Transgenic Microorganisms 2.Transgenic Animals 3.Transgenic Plants B.Cloning

2. 13.1: Changing the Living World From ancient times, breeders have chosen plants and animals with the most desired traits to serve as parents of the next generation. Breeders of plants and animals want to be sure that their populations breed consistently so that each member shows the desired trait.

3. Selective Breeding Selective breeding is a method of improving a species by allowing only those individual organisms with desired characteristics to produce the next generation. The process of selective breeding requires time, patience, and several generations of offspring before the desired trait becomes common in a population. Increasing the frequency of desired alleles in a population is the essence of genetic technology.

4. Inbreeding develops pure lines To make sure that breeds consistently exhibit a trait and to eliminate any undesired traits from their breeding lines, breeders often use the method of inbreeding. Inbreeding is mating between closely related individuals. It results in offspring that are homozygous for most traits. Inbreeding can bring out harmful, recessive traits because there is a greater chance that two closely related individuals both may carry a harmful recessive allele for the trait. Horses and dogs are two examples of animals that breeders have developed as pure breeds.

5. Hybrids are usually bigger and better A hybrid is the offspring of parents that have different forms of a trait. Hybrids produced by crossing two purebred plants are often larger and stronger than their parents. Many crop plants such as wheat, corn, and rice, and garden flowers such as roses and dahlias have been developed by hybridization.

6. Increasing Variation Breeders can increase the genetic variation in a population by inducing mutations, which are the ultimate source of genetic variability. Producing new kinds of bacteria Due to their small size many bacteria can be treated with chemicals to induce mutations Example: bacteria created to clean up oil spills

7. Producing new kinds of plants Chemicals are used to prevent chromosomes from separating during mitosis, so cells have many copies of each chromosome This process is called polyploidy Polyploidy occurs when all the pairs of chromosomes do not separate during cell divisions; the resulting organism has three or more sets of chromosomes -plants are usually much hardier; artificially bred plants increase variability of the species which increases survival - up to 50% of all plants are polyploids including bananas, citrus fruits, day lilies, wheat, potatoes, and oats

8. which crosses consists of Selective Breeding for example InbreedingHybridization Similar organisms Dissimilar organisms for example Organism breed A Organism breed B Retains desired characteristics Combines desired characteristics which which crosses which Selective Breeding Conclusion

9. 13.2: Manipulating DNA Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules. The process of making changes in the DNA code of living organisms is called genetic engineering. In order to alter DNA, scientists have developed techniques to : –extract DNA from cells –to cut DNA into smaller pieces –to identify the sequence of bases in a DNA molecule –to make unlimited copies of DNA

10. DNA Extraction Scientists must first be able to open the cells in order to separate DNA from the rest of the cell parts This is accomplished by a simple chemical procedure (Strawberry DNA Lab)

11. Cutting DNA Most DNA is too large to be analyzed. Scientists use restriction enzymes to cut DNA. Restriction enzymes are bacterial proteins that have the ability to cut both strands of the DNA molecule at a specific nucleotide sequence. There are hundreds of different restriction enzymes; each can cut DNA at different sequences and in different forms.

12. Restriction enzymes cleave DNA EcoR1 example: When this DNA is cut, double-stranded fragments with single- stranded ends are formed. The same sequence of bases is found on both DNA strands. The single-stranded ends join with other single-stranded ends to become double stranded. This attraction allows scientists to cut DNA of various organisms with the same enzyme in order to combine them.

13. Separating DNA Restriction enzymes are the perfect tools for cutting DNA. Once the DNA has been cut into small pieces, the pieces will then be separated by size through a process called gel electrophoresis. DNA fragments

14. Gel Electrophoresis Procedure The process starts by making a gel that will act as a maze for the DNA to travel through. Small amounts of the fragmented DNA are placed into these holes at one end of the gel, called the wells. Since DNA has a negative charge, the wells should always be at the negative end of the chamber. The gel is placed in a buffer solution and an electric field is applied making one end of the gel positive and the other end negative. Gel Power source Negative end Positive end

15. The fragments move The negatively charged DNA fragments travel toward the positive end. The smaller the fragment, the faster it moves through the gel. The largest fragments will travel more slowly, and be closer to the wells. Completed gel Shorter fragments Longer fragments

16. DNA plus restriction enzyme Mixture of DNA fragments Gel Power source Longer fragments Shorter fragments DNA Gel Electrophoresis

17. Reading the DNA Sequence One use for gel electrophoresis is to identify the sequence of DNA. This allows researchers to study specific genes, to compare them with the genes of other organisms, and to try to discover the functions of different genes. Scientists use radioactive labels to mark each of the four DNA bases. Then, they separate the fragments on a gel. Finally, they read the sequence that appears on the gel.

18. Cutting and Pasting DNA sequences can be changed –Take gene from one organism and attach it to the DNA of another organism to form recombinant DNA Recombinant DNA is DNA combined from different sources –Ex: human insulin gene is mass produced by inserting the human insulin gene into bacterial cells Recombinant DNA Gene for human growth hormone DNA insertion Bacterial cell for containing gene for human growth hormone

19. Making Copies of DNA In order to make copies of DNA in the lab, a method called polymerase chain reaction (PCR) has been developed. This method uses heat to separate DNA strands from each other. An enzyme isolated from a bacterium is used to replicate the DNA when the appropriate nucleotides are added in a PCR machine, called a thermocycler. The machine repeatedly replicates the DNA, making millions of copies in less than a day. Having a larger sample of DNA can help scientists who are testing samples for sequencing, diseases, or even identification of individuals at a crime scene. DNA polymerase adds complementary strand DNA heated to separate strands DNA fragment to be copied PCR cycles 1 DNA copies 1 2222 3434 4848 5 etc. 16 etc.

20. 13.3: Cell Transformation Once scientists have altered DNA, they need to have a way of putting it back into an organism so it will work. Remember Griffith’s experiment of transformation? During transformation, a cell takes in DNA from outside the cell. This external DNA becomes a part of the cell’s DNA. Bacteria cells are often used as vectors. A vector is the means by which DNA from another species can be carried into the host cell. A plasmid, is a small ring of DNA found in a bacterial cell. Plasmid Bacterial chromosome Bacterial Cell

21. Insertion into a vector If a plasmid and foreign DNA have been cleaved with the same restriction enzyme, the ends of each will match and they will join together, reconnecting the plasmid ring. The foreign DNA is recombined and inserted back into the bacterium, where it can be replicated. The inserted plasmid has a genetic marker- something that allows scientists to see where the foreign DNA is. Human Cell Gene for human growth hormone Sticky ends DNA recombination Plasmid Bacterial chromosome Bacterial Cell Recombinant DNA Gene for human growth hormone DNA insertion Bacterial cell for containing gene for human growth hormone Genetic Marker

22. Human Cell Gene for human growth hormone Recombinant DNA Gene for human growth hormone Sticky ends DNA recombination DNA insertion Plasmid Bacterial chromosome Bacterial cell for containing gene for human growth hormone Bacterial Cell Making Recombinant DNA

23. Transforming Plant and Animal Cells In plants and animals, particular genes can be eliminated or transferred between species Transformed bacteria introduce plasmids into plant cells Complete plant is generated from transformed cell Recombinant plasmid Agrobacterium tumefaciens Cellular DNA Plant cell colonies Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome Gene to be transferred –Scientists can replace certain genes in a chromosome to determine their function to improve their function or to treat disorders or diseases

24. 13.4: Applications of Genetic Engineering Transgenic organisms contain recombinant DNA Plants and animals that contain DNA from an organism of a different genus (recombinant) are known as transgenic organisms because they contain foreign DNA. The first step of the process is to isolate the foreign DNA fragment that will be inserted. The second step is to attach the DNA fragment to a carrier (vector). The third step is the transfer into the host organism. This transgenic tobacco plant, which glows in the dark was grown from a tobacco cell transformed with a firefly luciferase gene. The plant shows how DNA is transferred and works from one organism to another.

25. Transgenic Organisms Bacteria are easy to modify because they reproduce rapidly and are easy to grow Makes mass quantities fast and cheap –Used to make insulin, growth hormone, clotting factor –One day hope to fight cancer, make plastic and other synthetic materials Transgenic animals are used to study genes and improve our food supply Because of the similarity in genes between humans and other organisms, scientists can easily insert human genes and study their effects in other organisms –Scientists know the locations of many genes on mouse chromosomes, which are similar to human chromosomes. –The roundworm Caenorhabditis elegans is another organism with well-understood genetics that is used for transgenic studies. –A third animal commonly used for transgenic studies is the fruit fly.

26. Transgenic animals Human genes inserted in mice to study immune responses Livestock injected with human growth hormones genes to allow for faster growth and make leaner meat Reduce food poisoning by creating chickens resistant to bacterial infections On a farm in Scotland, a transgenic sheep was produced that contained the corrected human gene for hemophilia A. (This is the same farm that cloned Dolly.) –This human gene inserted into the sheep chromosomes allows the production of the clotting protein in the sheep’s milk. –This farm also has produced transgenic sheep which produce a protein that helps lungs inflate and function properly.

27. Recombinant DNA in agriculture Recombinant DNA technology has been highly utilized in the agricultural and food industries. Crops have been developed that are better tasting, stay fresh longer, and are protected from disease and insect infestations Millions of hectares 150 100 50 0 72 36% 140 7% 34 25 16% 11% Soybeans CornCottonCanola The Most Common Genetically Modified (GM) Crops

28. Gene cloning After the foreign DNA has been inserted into the plasmid, the recombined DNA is transferred into a bacterial cell. An advantage to using bacterial cells to clone DNA is that they reproduce quickly; therefore, millions of bacteria are produced and each bacterium contains hundreds of recombinant DNA molecules. Clones are genetically identical copies. Each identical recombinant DNA molecule is called a gene clone. Plasmids also can be used to deliver genes to animal or plant cells, which incorporate the recombinant DNA. Each time the host cell divides it copies the recombinant DNA along with its own. The host cell can produce the protein encoded on the recombinant DNA. Using other vectors, recombinant DNA can be inserted into yeast, plant, and animal cells. Cleavage sites Foreign DNA (gene for human growth hormone) Recombined plasmid Recombined DNA Human growth hormone E. coli Bacterial chromosome Plasmid

29. Cloning of animals So far, you have read about cloning one gene. For decades, scientists attempted to expand the technique from a gene to an entire animal. Although their techniques are inefficient, scientists are coming closer to perfecting the process of cloning animals.

30. Cloning 1. A body cell is taken from a donor animal. 2. An egg cell is taken from a donor animal. 5. The fused cell begins dividing, becoming an embryo. 3. The nucleus is removed from the egg. 4. The body cell and egg are fused by electric shock. 6 The embryo is implanted into the uterus of a foster mother. 7. The embryo develops into a cloned animal. A donor cell is taken from a sheep’s udder. Donor Nucleus These two cells are fused using an electric shock. Fused Cell The fused cell begins dividing normally. Embryo The embryo is placed in the uterus of a foster mother. Foster Mother The embryo develops normally into a lamb—Dolly Egg Cell An egg cell is taken from an adult female sheep. The nucleus of the egg cell is removed. Cloned Lamb Cloning of the First Mammal