Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Transcription

1 13 Biotechnology

2 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory It is possible to modify organisms with genes from other, distantly related organisms. Recombinant DNA is a DNA molecule made in the laboratory that is derived from at least two genetic sources.

3 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory Three key tools: Restriction enzymes for cutting DNA into fragments Gel electrophoresis for analysis and purification of DNA fragments DNA ligase for joining DNA fragments together in new combinations

4 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory Restriction enzymes recognize a specific DNA sequence called a recognition sequence or restriction site. 5.GAATTC 3 3.CTTAAG 5 Each sequence forms a palindrome: the opposite strands have the same sequence when read from the 5 end.

5 Figure 13.1 Bacteria Fight Invading Viruses by Making Restriction Enzymes

6 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory Some restriction enzymes cut DNA leaving a short sequence of single-stranded DNA at each end. Staggered cuts result in overhangs, or sticky ends; straight cuts result in blunt ends. Sticky ends can bind complementary sequences on other DNA molecules. Methylases add methyl groups to restriction sites and protect the bacterial cell from its own restriction enzymes.

7 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory Many restriction enzymes with unique recognition sequences have been purified. In the lab they can be used to cut DNA samples from the same source. A restriction digest combines different enzymes to cut DNA at specific places. Gel electrophoresis analysis can create a map of the intact DNA molecule from the formed fragments.

8 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory DNA fragments cut by enzymes can be separated by gel electrophoresis. A mixture of fragments is placed in a well in a semisolid gel, and an electric field is applied across the gel. Negatively charged DNA fragments move towards the positive end. Smaller fragments move faster than larger ones.

9 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory DNA fragments separate and give three types of information: The number of fragments The sizes of the fragments The relative abundance of the fragments, indicated by the intensity of the band

10 Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

11 Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

12 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory After separation on a gel, a specific DNA sequence can be found with a singlestranded probe. The gel region can be cut out and the DNA fragment removed. The purified DNA can be analyzed by sequence or used to make recombinant DNA.

13 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory DNA ligase is an enzyme that catalyzes the joining of DNA fragments, such as Okazaki fragments during replication. With restriction enzymes to cut fragments and DNA ligase to combine them, new recombinant DNA can be made.

14 Figure 13.3 Cutting, Splicing, and Joining DNA

15 Concept 13.1 Recombinant DNA Can Be Made in the Laboratory Recombinant DNA was shown to be a functional carrier of genetic information. Sequences from two E.coli plasmids, each with different antibiotic resistance genes, were recombined. The resulting plasmid, when inserted into new cells, gave resistance to both of the antibiotics.

16 Figure 13.4 Recombinant DNA (Part 1)

17 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Recombinant DNA technology can be used to clone (make identical copies) genes. Transformation: Recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal). The altered host cell is called transgenic.

18 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Usually only a few cells exposed to recombinant DNA are actually transformed. To determine which of the host cells are transgenic, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.

19 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Most research has been done using model organisms: Bacteria, especially E. coli Yeasts (Saccharomyces), commonly used as eukaryotic hosts Plant cells, able to make stem cells unspecialized, totipotent cells Cultured animal cells, used for expression of human or animal genes whole transgenic animals can be created

20 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Methods for inserting the recombinant DNA into a cell: Cells may be treated with chemicals to make plasma membranes more permeable DNA diffuses in. Electroporation a short electric shock creates temporary pores in membranes, and DNA can enter.

21 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Viruses and bacteria can be altered to carry recombinant DNA into cells. Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs. Gene guns can shoot the host cells with particles of DNA.

22 Concept 13.2 DNA Can Genetically Transform Cells and Organisms The new DNA must also replicate as the host cell divides. DNA polymerase does not bind to just any sequence. The new DNA must become part of a segment with an origin of replication a replicon or replication unit.

23 Concept 13.2 DNA Can Genetically Transform Cells and Organisms New DNA can become part of a replicon in two ways: Inserted near an origin of replication in host chromosome It can be part of a carrier sequence, or vector, that already has an origin of replication

24 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Plasmids make good vectors: Small and easy to manipulate Have one or more restriction enzyme recognition sequences that each occur only once Many have genes for antibiotic resistance which can be selectable markers

25 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Have a bacterial origin of replication (ori) and can replicate independently of the host chromosome Bacterial cells can contain hundreds of copies of a recombinant plasmid. The power of bacterial transformation to amplify a gene is extraordinary.

26 In-Text Art, Ch. 13, p. 249

27 Concept 13.2 DNA Can Genetically Transform Cells and Organisms A plasmid from the soil bacterium Agrobacterium tumefaciens is used as a vector for plant cells. A. tumefaciens contains a plasmid called Ti (for tumor-inducing). The plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants.

28 Concept 13.2 DNA Can Genetically Transform Cells and Organisms T DNA genes are removed and replaced with foreign DNA. Altered Ti plasmids transform Agrobacterium cells, then the bacterium cells infect plant cells. Whole plants can be regenerated from transgenic cells, or germ line cells can be infected.

29 In-Text Art, Ch. 13, p. 250

30 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Most eukaryotic genes are too large to be inserted into a plasmid. Viruses can be used as vectors e.g., bacteriophage. The genes that cause host cells to lyse can be cut out and replaced with other DNA. Because viruses infect cells naturally they offer an advantage over plasmids.

31 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Usually only a small proportion of host cells take up the vector (1 cell in 10,000) and they may not have the appropriate sequence. Host cells with the desired sequence must be identifiable. Selectable markers such as antibiotic resistance genes can be used.

32 Concept 13.2 DNA Can Genetically Transform Cells and Organisms If a vector carrying genes for resistance to two different antibiotics is used, one antibiotic can select cells carrying the vector. If the other antibiotic resistance gene is inactivated by the insertion of foreign DNA, then cells with the desired DNA can be identified by their sensitivity to that antibiotic.

33 Figure 13.5 Marking Recombinant DNA by Inactivating a Gene

34 Concept 13.2 DNA Can Genetically Transform Cells and Organisms Selectable markers are a type of reporter gene a gene whose expression is easily observed. Green fluorescent protein, which normally occurs in a jellyfish, emits visible light when exposed to UV light. The gene for this protein has been isolated and incorporated into vectors as a reporter gene.

35 Figure 13.6 Green Fluorescent Protein as a Reporter

36 Concept 13.3 Genes and Gene Expression Can Be Manipulated DNA fragments used for cloning come from three sources: Gene libraries Reverse transcription from mrna Products of PCR Artificial synthesis or mutation of DNA

37 Concept 13.3 Genes and Gene Expression Can Be Manipulated A genomic library is a collection of DNA fragments that comprise the genome of an organism. The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector. A vector is taken up by host cells which produce a colony of recombinant cells.

38 Concept 13.3 Genes and Gene Expression Can Be Manipulated Smaller DNA libraries can be made from complementary DNA (cdna). mrna is extracted from cells, then cdna is produced by complementary base pairing, catalyzed by reverse transcriptase. A cdna library is a snapshot of the transcription pattern of the cell. cdna libraries are used to compare gene expression in different tissues at different stages of development.

39 Figure 13.7 Constructing Libraries

40 Concept 13.3 Genes and Gene Expression Can Be Manipulated DNA can be synthesized by PCR if appropriate primers are available. The amplified DNA can then be inserted into plasmids to create recombinant DNA and cloned in host cells. Artificial synthesis of DNA is now fully automated.

41 Concept 13.3 Genes and Gene Expression Can Be Manipulated Synthetic oligonucleotides are used as primers in PCR reactions. Primers can create new sequences to create mutations in a recombinant gene. Longer synthetic sequences can be used to construct an artificial gene.

42 Concept 13.3 Genes and Gene Expression Can Be Manipulated Synthetic DNA can be manipulated to create specific mutations in order to study the consequences of the mutation. Mutagenesis techniques have revealed many cause-and-effect relationships (e.g., determining signal sequences).

43 Concept 13.3 Genes and Gene Expression Can Be Manipulated A knockout experiment inactivates a gene so that it is not transcribed and translated into a functional protein. In mice, homologous recombination targets a specific gene. The normal allele of a gene is inserted into a plasmid restriction enzymes are used to insert a reporter gene into the normal gene. The extra DNA prevents functional mrna from being made.

44 Concept 13.3 Genes and Gene Expression Can Be Manipulated The recombinant plasmid is used to transfect mouse embryonic stem cells. Stem cells unspecialized cells that divide and differentiate into specialized cells The original gene sequences line up with their homologous sequences on the mouse chromosome.

45 Concept 13.3 Genes and Gene Expression Can Be Manipulated The transfected stem cell is then transplanted into an early mouse embryo. The knockout technique has been important in determining gene functions and studying human genetic diseases. Many diseases have a knockout mouse model.

46 Figure 13.8 Making a Knockout Mouse

47 Concept 13.3 Genes and Gene Expression Can Be Manipulated Complementary RNA: Translation of mrna can be blocked by complementary micrornas antisense RNA. Antisense RNA can be synthesized and added to cells to prevent translation the effects of the missing protein can then be determined.

48 Concept 13.3 Genes and Gene Expression Can Be Manipulated RNA interference (RNAi) is a rare natural mechanism that blocks translation. RNAi occurs via the action of small interfering RNAs (sirnas). An srna is a short, double stranded RNA that is unwound to single strands by a protein complex, which also catalyzes the breakdown of the mrna. Small interfering RNA (sirna) can be synthesized in the laboratory.

49 Figure 13.9 Using Antisense RNA and sirna to Block the Translation of mrna

50 Concept 13.3 Genes and Gene Expression Can Be Manipulated DNA microarray technology provides a large array of sequences for hybridization experiments. A series of DNA sequences are attached to a glass slide in a precise order. The slide has microscopic wells, each containing thousands of copies of sequences up to 20 nucleotides long.

51 Concept 13.3 Genes and Gene Expression Can Be Manipulated DNA microarrays can be used to identify specific single nucleotide polymorphisms or other mutations. Microarrays can be used to examine gene expression patterns in different tissues in different conditions. Example: Women with a propensity for breast cancer tumors to recur have a gene expression signature.

52 Figure Using DNA Microarrays for Clinical Decision-Making

53 Concept 13.4 Biotechnology Has Wide Applications Almost any gene can be inserted into bacteria or yeasts and the resulting cells induced to make large quantities of a product. Requires specialized expression vectors with extra sequences needed for the transgene to be expressed in the host cell.

54 Figure A Transgenic Cell Can Produce Large Amounts of the Transgene s Protein Product

55 Concept 13.4 Biotechnology Has Wide Applications Expression vectors may also have: Inducible promoters that respond to a specific signal Tissue-specific promoters, expressed only in certain tissues at certain times Signal sequences e.g., a signal to secrete the product to the extracellular medium

56 Concept 13.4 Biotechnology Has Wide Applications Many medically useful products are being made using biotechnology. The two insulin polypeptides are synthesized separately along with the β- galactosidase gene. After synthesis the polypeptides are cleaved, and the two insulin peptides combined to make a functional human insulin molecule.

57 Figure Human Insulin: From Gene to Drug (Part 1)

58 Figure Human Insulin: From Gene to Drug (Part 2)

59 Concept 13.4 Biotechnology Has Wide Applications Before giving it to humans, scientists had to be sure of its effectiveness: Same size as human insulin Same amino acid sequence Same shape Binds to the insulin receptor on cells and stimulates glucose uptake

60 Concept 13.4 Biotechnology Has Wide Applications Pharming: Production of pharmaceuticals in farm animals or plants. Example: Transgenes are inserted next to the promoter for lactoglobulin a protein in milk. The transgenic animal then produces large quantities of the protein in its milk.

61 Figure Pharming

62 Concept 13.4 Biotechnology Has Wide Applications Human growth hormone (for children suffering deficiencies) can now be produced by transgenic cows. Only 15 such cows are needed to supply all the children in the world suffering from this type of dwarfism.

63 Concept 13.4 Biotechnology Has Wide Applications Through cultivation and selective breeding, humans have been altering the traits of plants and animals for thousands of years. Recombinant DNA technology has several advantages: Specific genes can be targeted Any gene can be introduced into any other organism New organisms can be generated quickly

64 Figure Genetic Modification of Plants versus Conventional Plant Breeding (Part 1)

65 Figure Genetic Modification of Plants versus Conventional Plant Breeding (Part 2)

66 Table 13.2 Potential Agricultural Applications of Biotechnology

67 Concept 13.4 Biotechnology Has Wide Applications Crop plants have been modified to produce their own insecticides: The bacterium Bacillus thuringiensis produces a protein that kills insect larvae Dried preparations of B. thuringiensis are sold as a safe alternative to synthetic insecticides. The toxin is easily biodegradable.

68 Concept 13.4 Biotechnology Has Wide Applications Genes for the toxin have been isolated, cloned, and modified, and inserted into plant cells using the Ti plasmid vector Transgenic corn, cotton, soybeans, tomatoes, and other crops are being grown. Pesticide use is reduced.

69 Concept 13.4 Biotechnology Has Wide Applications Crops with improved nutritional characteristics: Rice does not have β-carotene, but does have a precursor molecule Genes for enzymes that synthesize β- carotene from the precursor are taken from daffodils and inserted into rice by the Ti plasmid

70 Concept 13.4 Biotechnology Has Wide Applications The transgenic rice is yellow and can supply β-carotene to improve the diets of many people β-carotene is converted to vitamin A in the body

71 Figure Transgenic Rice Rich in -Carotene

72 Concept 13.4 Biotechnology Has Wide Applications Recombinant DNA is also used to adapt a crop plant to an environment. Example: Plants that are salt-tolerant. Genes from a protein that moves sodium ions into the central vacuole were isolated from Arabidopsis thaliana and inserted into tomato plants.

73 Figure Salt-tolerant Tomato Plants (Part 1)

74 Figure Salt-tolerant Tomato Plants (Part 2)

75 Concept 13.4 Biotechnology Has Wide Applications Instead of manipulating the environment to suit the plant, biotechnology may allow us to adapt the plant to the environment. Some of the negative effects of agriculture, such as water pollution, could be reduced.

76 Concept 13.4 Biotechnology Has Wide Applications Concerns over biotechnology: Genetic manipulation is an unnatural interference in nature Genetically altered foods are unsafe to eat Genetically altered crop plants are dangerous to the environment

77 Concept 13.4 Biotechnology Has Wide Applications Advocates of biotechnology point out that all crop plants have been manipulated by humans. Advocates say that since only single genes for plant function are inserted into crop plants, they are still safe for human consumption. Genes that affect human nutrition may raise more concerns.

78 Concept 13.4 Biotechnology Has Wide Applications Concern over environmental effects centers on escape of transgenes into wild populations: For example, if the gene for herbicide resistance made its way into the weed plants Beneficial insects can also be killed from eating plants with B. thuringiensis genes

79 Answer to Opening Question Bioremediation is the use, by humans, of organisms to remove contaminants from the environment. Composting and wastewater treatment use bacteria to break down large molecules, human wastes, paper, and household chemicals. Recombinant DNA technology has transformed bacteria to help clean up oil spills.

80 Figure The Spoils of War