GENETIC ENGINEERING

1. GENETIC ENGINEERING • Excessive inbreeding of cheetahs has resulted in a lack of genetic diversity and a higher rate of mortality

2. Changing the Living World • Visit a dog show, and what do you see? • You can compare dogs of every breed imaginable, distinguished from one another by an enormous range of characteristics that are the result of genetic variation • Striking contrasts are everywhere—the size of a tiny Chihuahua and that of a massive great Dane, the short coat of a Labrador retriever and the curly fur of a poodle, the long muzzle of the wolfhound and the pug nose of a bulldog • The differences among breeds of dogs are so great that someone who had never seen such animals before might think that many of these breeds are different species • They're not, of course, but where did such differences come from? • What forces gave rise to the speed of a greyhound, the courage of a German shepherd, and the herding instincts of a border collie?

3. Selective Breeding • The answer, of course, is that we did it • Humans have kept and bred dogs for thousands of years, always looking to produce animals that might be better hunters, better retrievers, or better companions • By selective breeding, allowing only those animals with desired characteristics to produce the next generation, humans have produced many different breeds of dogs

4. CONTROLLED BREEDING • Humans allow only those plants or animals with particular traits to reproduce • Purpose is to produce offspring with traits that are desirable to humans • Often these traits make a plant or animal unfit to live in the wild

5. Selective Breeding • Humans use selective breeding, which takes advantage of naturally occurring genetic variation in plants, animals, and other organisms, to pass desired traits on to the next generation of organisms • Nearly all domestic animals—including horses, cats, and farm animals—and most crop plants have been produced by selective breeding • American botanist Luther Burbank (1849–1926) may have been the greatest selective plant breeder of all time • He developed the disease-resistant Burbank potato, which was later exported to Ireland to help fight potato blight and other diseases • During his lifetime, Burbank developed more than 800 varieties of plants

6. SELECTION • Only a few organisms with the desirable characteristics are allowed to reproduce • The offspring of these organisms stand a good chance of inheriting the desired characteristics • Mass Selection: selection from a large number of organisms • Has developed new varieties of apples, potatoes, plums, and various fruits • Used to develop a new variety of a plant or animal • Does not produce new characteristics • Works only within the limits of the existing genotypes

8. Hybridization • As one of his tools, Burbank used hybridization, crossing dissimilar individuals to bring together the best of both organisms • Hybrids, the individuals produced by such crosses, are often hardier than either of the parents • In many cases, Burbank's hybrid crosses combined the disease resistance of one plant with the food-producing capacity of another • The result was a new line of plants that had the characteristics farmers needed to increase food production

9. HYBRIDIZATION • Often organisms selected for one desirable trait will also carry, less desirable traits • Corn plants: • Variety that is hardy but small kernels • Variety with large kernels but not hardy • If the two breeds were crossed, some of the offspring might carry both desirable traits • When two breeds are crossed, the offspring are called hybrids • The breeder tries to combine the best qualities of different breeds • Often the hybrids produced by crossing two inbreed lines are larger and stronger than their parents • Hybrid vigor • Cause not fully understood • May be the result of combining favorable dominant alleles from one parent with unfavorable recessive alleles from another parent • Different pure lines probably do not carry the same unfavorable alleles • Hybrids are not usually used as parents • Usually heterozygous for many traits and their offspring would be extremely variable • Occasionally breeders will cross two different pure lines to produce a new breed • May take generations to produce a new breed • Example: • Rhode Island Red Hen contains genes from five different breeds • Cattle

10. HYBRIDIZATION

11. HYBRIDIZATION • As new , more desirable breeds have been developed, many old breeds have been ignored and, as a result, have become endangered • Some unusual-looking cattle are endangered breeds

12. ENDANGERED BREEDS

13. HYBRIDIZATION • Term hybrid may also refer to a cross between two totally different types, or species of organisms • Mule: cross between a female horse and a male donkey • Closely related species • Combines the large strength of a horse with the hardiness of a donkey • Sterile • Hinny: cross between a male horse and a female donkey • Hybrids between different species are usually sterile (unable to reproduce) • Often caused by different numbers of chromosomes in the two parent species • Hybrid has unmatched sets of chromosomes • During meiosis, these unmatched chromosomes cannot form homologous pairs

14. Inbreeding • To maintain the desired characteristics of a line of organisms, breeders often use a technique known as inbreeding • Inbreeding is the continued breeding of individuals with similar characteristics • The many breeds of dogs—from beagles to poodles—are maintained by inbreeding • Inbreeding helps to ensure that the characteristics that make each breed unique will be preserved

15. INBREEDING

16. INBREEDING • Selection can be used to establish a new breed of plant or animal • Inbreeding is a controlled breeding method in which there is the crossing of two closely related individuals • In animals, breeding of brother and sister • Since closely related individuals usually have a high percentage of genes in common, inbreeding makes it likely that the desired genes will be passed on to offspring • After many generations of inbreeding, most of the offspring will be homozygous for the desired traits • When this occurs, breeders are said to have established pure lines • Because pure lines are homozygous for the selected traits, all of the offspring will have those traits • Continued selection will not produce any new variation within a breed • Pure lines are said to breed true • All dogs probably arose from wild wolves

17. Inbreeding • Although inbreeding is useful in retaining a certain set of characteristics, it does have its risks • Most of the members of a breed are genetically similar • Because of this, there is always a chance that a cross between two individuals will bring together two recessive alleles for a genetic defect • Serious problems in many breeds of dogs, including blindness and joint deformities in German shepherds and golden retrievers, have resulted from excessive inbreeding

18. INBREEDING DEPRESSION • After many generations of inbreeding, a condition of inbreeding depression may result • Decrease in the health or fertility of each succeeding generation • Cause not fully understood • Probably caused by harmful recessive alleles that were masked by dominant alleles in the original members of a breed • As pure lines are inbreed, it becomes more and more likely that recombination will result in individuals that are homozygous for harmful alleles

19. INBREEDING DEPRESSION • The undesirable effects of inbreeding may be reduced by periodic outcrossing • Crossing an inbred organism with a less closely related individual • Introduces new genes into a line

20. Increasing Variation • Selective breeding would be nearly impossible without the wide variation that is found in natural populations • This is one of the reasons biologists are interested in preserving the diversity of plants and animals in the wild • However, sometimes breeders want more variation than exists in nature • Breeders can increase the genetic variation in a population by inducing mutations, which are the ultimate source of genetic variability

21. INDUCING MUTATIONS • Mutations are changes in the DNA of an organism • Introduces new alleles to the genetic makeup of an organism • Occurs at a very low rate in nature • Man can induce a much greater rate of mutation (x-rays, etc) • Select the mutants for selective breeding • Create new traits in many organisms that might be beneficial to humans • Example: bacteria ??????

22. Increasing Variation • As you may recall, mutations are inheritable changes in DNA • Mutations occur spontaneously, but breeders can increase the mutation rate by using radiation and chemicals • Many mutations are harmful to the organism • With luck and perseverance, however, breeders can produce a few mutants—individuals with mutations—with desirable characteristics that are not found in the original population

23. Producing New Kinds of Bacteria  • This technique has been particularly useful with bacteria • Their small size enables millions of organisms to be treated with radiation or chemicals at the same time • This increases the chances of producing a useful mutant • Using this technique, scientists have been able to develop hundreds of useful bacterial strains • It has even been possible to produce bacteria that can digest oil and that were once used to clean up oil spills • Today, naturally occurring strains of oil-digesting bacteria are used to clean up oil spills

24. Producing New Kinds of Plants  • Drugs that prevent chromosomal separation during meiosis have been particularly useful in plant breeding • Sometimes these drugs produce cells that have double or triple the normal number of chromosomes • Plants grown from such cells are called polyploid because they have many sets of chromosomes • Polyploidy is usually fatal in animals • However, for reasons that are not clear, plants are much better at tolerating extra sets of chromosomes • Polyploidy may instantly produce new species of plants that are often larger and stronger than their diploid relatives • Many important crop plants have been produced in this way, including bananas and many varieties of citrus fruits

25. INDUCING POLYPLOIDY • Polyploidy: condition in which cells contain multiple, complete sets of chromosomes • Rare and usually lethal in animals • Occurs naturally in plants • Often larger or hardier than their parents • Plant breeders: • Administer colchicine, a chemical that prohibits the formation of the cell plate during cell division • Results in two sets of chromosomes in the cell

26. INDUCING POLYPLOIDY

27. Manipulating DNA • Until very recently, animal and plant breeders could not modify the genetic code of living things • They were limited by the need to work with the variation that already exists in nature • Even when they tried to add to that variation by introducing mutations, the changes they produced in the DNA were random and unpredictable • Imagine, however, that one day biologists were able to go right to the genetic code and rewrite an organism's DNA • Imagine that biologists could transfer genes at will from one organism to another, designing new living things to meet specific needs • That day, as you may know from scientific stories in the news, is already here

28. Manipulating DNA • How are changes made to DNA? • Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules • Different techniques are used to extract DNA from cells, to cut DNA into smaller pieces, to identify the sequence of bases in a DNA molecule, and to make unlimited copies of DNA • Understanding how these techniques work will help you develop an appreciation for what is involved in genetic engineering

29. The Tools of Molecular Biology • Suppose you had a computer game you wanted to change • Knowing that the characteristics of that game are determined by a coded computer program, how would you set about rewriting parts of the program? • To make such changes, a software engineer would need a way to get the program out of the computer, read it, make changes in it, and then put the modified code back into the game • Genetic engineering, making changes in the DNA code of a living organism, works almost the same way

30. DNA Extraction  • How do biologists get DNA out of a cell? • DNA can be extracted from most cells by a simple chemical procedure: • The cells are opened and the DNA is separated from the other cell parts

31. Cutting DNA  • DNA molecules from most organisms are much too large to be analyzed, so biologists cut them precisely into smaller fragments using restriction enzymes • Hundreds of restriction enzymes are known, and each one cuts DNA at a specific sequence of nucleotides • Restriction enzymes are amazingly precise • Like a key that fits only one lock, a restriction enzyme will cut a DNA sequence only if it matches the sequence precisely

32. Cutting DNA

33. Restriction Enzymes  • Molecular biologists have developed different techniques that allow them to study and change DNA molecules • This drawing shows how restriction enzymes are used to edit DNA • The restriction enzyme EcoR I, for example, finds the sequence CTTAAG on DNA • Then, the enzyme cuts the molecule at each occurrence of CTTAAG • Different restriction enzymes recognize and cut different sequences of nucleotides on DNA molecules • The cut ends are called sticky ends because they may “stick” to complementary base sequences by means of hydrogen bonds

34. Separating DNA  • How can DNA fragments be separated and analyzed? • One way, a procedure known as gel electrophoresisIn gel • Electrophoresis, a mixture of DNA fragments is placed at one end of a porous gel, and an electric voltage is applied to the gel • When the power is turned on, DNA molecules, which are negatively charged, move toward the positive end of the gel • The smaller the DNA fragment, the faster and farther it moves • Gel electrophoresis can be used to compare the genomes, or gene composition, of different organisms or different individuals • It can also be used to locate and identify one particular gene out of the tens of thousands of genes in an individual's genome

35. Separating DNA 

36. Using the DNA Sequence • Once DNA is in a manageable form, its sequence can be read, studied, and even changed • Knowing the sequence of an organism's DNA 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 and gene combinations

37. Reading the Sequence • Researchers use a clever chemical trick to “read” DNA by determining the order of its bases • A single strand of DNA whose sequence of bases is not known is placed in a test tube • DNA polymerase, the enzyme that copies DNA, and the four nucleotide bases, A, T, G, and C, are added to the test tube • As the enzyme goes to work, it uses the unknown strand as a template to make one new DNA strand after another • The tricky part is that researchers also add a small number of bases that have a chemical dye attached

38. Reading the Sequence  • Each time a dye-labeled base is added to a new DNA strand, the synthesis of that strand is terminated • When DNA synthesis is completed, the new DNA strands are different lengths, depending on how far synthesis had progressed when the dye-tagged base was added • Since each base is labeled with a different color, the result is a series of dye-tagged DNA fragments of different lengths • These fragments are then separated according to length, often by gel electrophoresis • The order of colored bands on the gel tells the exact sequence of bases in the DNA

39. Reading the Sequence 

40. Reading the Sequence • In DNA sequencing, a complementary DNA strand is made using a small proportion of fluorescently labeled nucleotides • Each time a labeled nucleotide is added, it stops the process of replication, producing a short color-coded DNA fragment • When the mixture of fragments is separated on a gel, the DNA sequence can be read directly from the gel

41. Cutting and Pasting  • DNA sequences can be changed in a number of ways • Short sequences can be assembled using laboratory machines known as DNA synthesizers • “Synthetic” sequences can then be joined to “natural” ones using enzymes that splice DNA together • The same enzymes make it possible to take a gene from one organism and attach it to the DNA of another organism • Such DNA molecules are sometimes called recombinant DNAbecause they are produced by combining DNA from different sources

42. Making Copies  • In order to study genes, biologists often need to make many copies of a particular gene • Like a photocopy machine stuck on “print,” a technique known as polymerase chain reaction (PCR)allows biologists to do exactly that

43. Making Copies

44. Polymerase Chain Reaction  • Polymerase chain reaction (PCR) is used to make multiple copies of genes

45. Polymerase Chain Reaction  • The idea behind PCR is surprisingly simple • At one end of a piece of DNA a biologist wants to copy, he or she adds a short piece of DNA that is complementary to a portion of the sequence • At the other end, the biologist adds another short piece of complementary DNA • These short pieces are known as “primers” because they provide a place for the DNA polymerase to start working

46. Polymerase Chain Reaction  • The DNA is heated to separate its two strands, then cooled to allow the primers to bind to single-stranded DNA • DNA polymerase starts making copies of the region between the primers • Because the copies themselves can serve as templates to make still more copies, just a few dozen cycles of replication can produce millions of copies of the DNA between those primers

47. Polymerase Chain Reaction  • Where did Kary Mullis, the American inventor of PCR, find a DNA polymerase enzyme that could stand repeated cycles of heating and cooling? • Mullis found it in bacteria living in the hot springs of Yellowstone National Park—a perfect example of the importance of biodiversity to biotechnology

48. Cell Transformation • It would do little good to modify a DNA molecule in the test tube if it were not possible to put that DNA back into a living cell and make it work • This sounds tricky, and it is, but you have already seen an example of how this can be done • Remember Griffith's experiments on bacterial transformation? • During transformation, a cell takes in DNA from outside the cell • This external DNA becomes a component of the cell's DNA

49. Cell Transformation • Today, biologists understand that Griffith's extract of heat-killed bacteria must have contained DNA fragments • When he mixed those fragments with live bacteria, a few of them actually took up the DNA molecules • This suggests that bacteria can be transformed simply by placing them in a solution containing DNA molecules—and indeed they can

50. Transforming Bacteria • The figure to the right shows how bacteria can be transformed using recombinant DNA • The foreign DNA is first joined to a small, circular DNA molecule known as a plasmid • Plasmids are found naturally in some bacteria and have been very useful for DNA transfer • Why? • The plasmid DNA has two essential features: • First, it has a DNA sequence that helps promote plasmid replication • If the plasmid containing the foreign DNA manages to get inside a bacterial cell, this sequence ensures that it will be replicated