18.0 Introduction

1. New Techniques Developed to Manipulate DNA
1. Techniques Can Be Applied to Alter an Organism's Genes fig 18.1
2. Have Great Impact on Future Lives

1. Restriction Endonucleases
1. First Human Gene Inserted into Bacteria
1. Interferon
1. Increases resistance to viral infection
2. Rare, purification of small quantities is very expensive

2. Bacterial cells made to produce protein at high rate
1. Gene for interferon placed in bacterial cell
2. Cells grew, divided, produced interferon

2. The Advent of Genetic Engineering
1. Cloning
1. Produce genetically identical cells from single altered cell
2. Cells in culture become factories for producing chemicals
1. Interferon
2. Human insulin

2. New process called genetic engineering
1. Ability to cut up DNA into pieces and rearrange them
2. Segments inserted via plasmids or viruses
3. Recognize and cleave specific nucleotide sequences

3. Discovery of Restriction Endonucleases
1. Bacteria are natural source of enzymes provide protection from viruses
2. Viruses infect bacteria, multiply within and release progeny
3. Bacteria have enzymes that chop up invading viruses
1. Enzymes are restriction endonucleases
2. Bacterial DNA not damaged because it is modified
3. Recognize sequence, bind to DNA and cleave strand

4. Methylase enzymes recognize bacterial DNA
1. Bind to same bacterial sites
2. Add methyl groups to nucleotides
3. Restriction enzymes do not recognize methylated sites
4. Bacterial DNA protected from fragmentation

4. How Restriction Endonucleases Cut DNA
1. Endonucleases recognize sites
1. Recognize a variety of four to six nucleotide sequences
2. Segments are usually palindromes
3. Nucleotides at one end are complementary to those at other end

2. Enzyme binds at and cleaves both strands of DNA at same time
1. Restriction enzymes effectively cut DNA in half
2. Site where DNA is cut has offset ends fig 18.2
3. Ends are complementary to each other

3. Some endonucleases cleave in center of recognition site
1. Do not produce ends that are offset
2. Fragments do not spontaneously reassociate

5. Why Restriction Endonucleases Are So Useful
1. Each enzyme always cuts at same sequence
1. Fragments always have same ends that are complementary to other ends
2. Sets of nucleotides called "sticky ends"

2. Ends can pair with each other
1. Two fragments can be glued together by DNA ligase
2. Any two fragments cleaved by same endonuclease can be joined together
3. Fragments can be from entirely different organisms

2. Using Restriction Endonucleases to Manipulate Genes
1. Mythological Chimera Composed of Parts of Several Animals
2. Constructing pSC101
1. Cohen and Boyer: First artificial bacterial plasmid
2. Cut plasmid containing resistance transfer factor with EcoRI
1. One fragment contained replication origin and tetracycline resistance gene
2. Complementary ends joined forming pSC101 plasmid fig 18.3

3. Using pSC101 to Make Recombinant DNA
1. Same restriction enzymes used to cut toad genome
1. Toad DNA pieces added to open pSC101 plasmids
2. Added to bacteria, cells became resistant to tetracycline
3. Also began to produce toad ribosomal RNA
4. Concluded toad gene inserted into pSC101 plasmid

2. Recombinant DNA: DNA created in laboratory
1. New genome that never existed in nature
2. Unable to evolve by natural means

4. Other Vectors
1. Vector is the genome that carried foreign DNA into host cell
2. Current plasmids induced to make copies of selves and their foreign genes
3. Bacterial viruses also used as vectors
4. Animal viruses used to insert bacterial genes into monkey cells
1. Animal genes inserted into plant cells

1. The Four Stages of a Genetic Engineering Experiment
1. Stage 1: Cleavage
1. Via restriction endonucleases
2. Large number of specific fragments produced
3. Different set of fragments for each specific sequence
4. Fragments compared by electrophoresis fig 18.4

2. Stage 2: Production of Recombinant DNA
1. Fragments put into plasmids or virus
2. Cleaved with same endonuclease as host DNA

3. Stage 3: Cloning
1. Plasmid or virus serves as vector to introduce DNA into (usually) bacteria fig 18.5
2. Cell reproduces making identical clones containing fragment
1. Each clone maintained separately
2. Whole set constitutes clone library of original DNA

4. Stage 4: Screening
1. Identify clone line containing fragment of interest
2. Among most difficult and critical steps
3. I: Preliminary screening of clones
1. Eliminate bacteria not containing vector or proper DNA fragment
2. Eliminate clones without vectors
1. Use vector with gene conferring antibiotic resistance fig 18.6a
2. Gene ampr confers resistance to ampicillin
3. Culture clones on medium containing antibiotic
4. Only bacteria resistant to antibiotic will grow on it

3. Eliminate bacteria with vector, but lacking chosen fragment
1. Use vector with z gene for b-galactosidase
2. Enables cell to metabolize X-gal sugar
3. Metabolism of X-gal produces blue product
4. Cells with vector and functional gene will turn blue fig 18.6b

4. Identify cells with vector and fragment
1. Test clones for presence of X-gal metabolism
2. DNA fragment within gene makes it inoperative
3. Clones with fragment lose ability to metabolize sugar
4. Desired cells remain colorless in presence of X-gal

5. Choose cells that grow on antibiotic, but don't turn blue

4. Finding the gene of interest
1. Clone library may contain thousands of DNA fragments
1. Many clones will be identical
2. May take hundreds of thousands of clones to assemble complete library

2. Most general procedure utilizes hybridization fig 18.7
1. Cloned genes form base pairs with probe DNA
2. Part of gene nucleotide sequence must be known to produce probe

3. In process bacterial colonies with inserted gene are grown on agar
1. Cells transferred to filter to produce replica plate
2. Filter treated with solution to denature bacterial DNA
3. Solution also contains radioactive probe
4. Probe hybridizes with complementary single-stranded bacterial DNA
5. Filter exposed to photographic film (autoradiography)
6. Only colonies with gene hybridize, become radioactive and show up on film
7. Identify colonies by comparing film to original bacterial plate

2. Working with Gene Clones
1. Getting Enough DNA to Work With: The Polymerase Chain Reaction
1. Produce multiple identical copies of DNA
1. Insert desired DNA into bacterium
2. Millions of copies exist after multiple cell divisions

2. Polymerase chain reaction (PCR) is more direct approach fig 18.8
1. Amplifies sequences
2. Add sequences (endonuclease recognition sequences) as primers to cleaved DNA

3. Three steps in PCR process
1. Step 1: Denaturation
1. Primer with excess synthetic nucleotides mixed with DNA fragment
2. Temperature of mixture increased to 98ø C
3. Fragment dissociates into single strands

2. Step 2: Annealing of primers
1. Solution cooled to 60ø C
2. Single strands of DNA reassociate into double strands
3. Fragment base-pairs with complementary primer nucleotide
4. Part of fragment still single stranded

3. Step 3: Primer extension
1. Heat stable DNA polymerase, Taq polymerase, added
2. Supply of all four nucleotides also added
3. Polymerase copies rest of fragment as in DNA replication
4. Primer lengthened into complementary copy of single-stranded fragment
5. Two copies of original now exist

4. Repeating the cycle
1. Steps 1 through 3 repeated
2. More polymerase not needed since it is heat stable
3. Repeat heating and cooling in short cycles
4. Each cycle doubles amount of DNA
5. After twenty cycles one fragment can become more than one million
6. In a few hours 100 billion copies can be produced

4. PCR process now completely automated
5. Has revolutionized aspects of science and medicine
1. PCR allows investigation of minute samples of DNA
2. Allows identification of genetic defects in embryos using minute sample
3. Examine DNA of dead organisms, extinct species as long as any DNA intact

2. Identifying DNA: Southern Blotting fig 18.8
1. When a gene is identified it can be used as a probe to identify same or similar gene
2. Procedure called Southern blot
1. DNA from sample cleaved into fragments with restriction endonuclease
2. Fragments spread apart by gel electrophoresis
3. Make gel basic, double-stranded DNA denatured into single strands
4. Gel blotted with nitrocellulose, DNA transfers to sheet
5. Probe of purified single-stranded DNA from desired gene poured onto sheet
6. Only fragments with proper sequence hybridize with probe
7. Probe may be radioactively labeled for clear identification
8. Shows as a band of radioactivity

3. Distinguishing Differences in DNA: RFLP Analysis fig 18.9
1. Used to identify an individual that possesses specific gene as a marker
2. Utilize restriction fragment length polymorphism (RFLP) analysis
1. Point mutations, sequence mutations, transposons alter length of DNA
2. Also alter length of fragments produced via action of restriction endonucleases
3. DNA from different individuals rarely have same array of restriction sites
4. Population is polymorphic for restriction fragment patterns

3. Process of RFLP analysis fig 18.10
1. Cut DNA sample with particular restriction endonuclease
2. Separate fragments according to length with electrophoresis
3. Use radioactive probe to identify fragments
4. Obtain unique pattern of bands in gel
5. Called "DNA fingerprints"
1. Used in criminal forensic investigations
2. Used as markers to identify carriers of certain genetic disorders

4. Making an Intron-Free Copy of a Eukaryotic Gene
1. Eukaryotic genes encoded in exons separated by nontranslated introns
1. Transcribed gene to form primary transcript
2. Introns cut out during RNA processing to produce mature mRNA transcript
3. Better to transfer processed, not raw DNA into bacteria
4. Bacteria lack enzymes to do RNA processing

2. Process fig 18.11
1. Isolate cytoplasmic mature mRNA for particular gene
2. Use reverse transcriptase enzyme to make DNA version of mature mRNA
3. Single strand DNA serves as template for synthesis of complementary strand
4. Produce double-stranded DNA lacking introns
5. Molecule called complementary DNA (cDNA)

5. Sequencing DNA: The Sanger Method
1. Most DNA sequencing done by "chain termination" technique
2. Process fig 18.12
1. Short, single-stranded primer added to end of unknown sequence single-strand DNA
2. Primer provides 3' end for DNA polymerase
3. Mix primed fragment, DNA polymerase, four deoxynucleotides (d-nucleotides)
4. Added to four synthesis tubes
1. Each tube contains a different dideoxynucleotide (dd-nucleotide)
2. Each dd-nucleotide lack 2' and 3' -OH groups, are chain-terminating

5. Example: Tube contains ddATP
1. Synthesis stopped when ddATP added to DNA instead of dATP
2. Low concentration of ddATP compared to dATP
3. Synthesis not stopped at first A site, produces short fragments
4. Tube contains series of fragments of varying lengths
5. Fragments separated by size by electrophoresis
6. Radioactive label allows visualization of fragments on film
7. Newly made sequence read from film
8. Original DNA has complementary sequenceá

1. DNA Sequence Technology
1. Biotechnology: Application of Genetic Engineering to Practical Problems
2. Genome Sequencing
1. Propose sequencing of entire human genome
1. Construct detailed map of human genome
2. Controversial as it requires significant resources

2. Probing the human genome
1. Localize cloned gene location via radioactive probe
2. Construction of clonal libraries
1. Use large-size restriction fragments
2. Localize chromosomal site of gene using radioactive probes and hybridization
3. Mapping genes at astounding rate
4. Examples: Dyslexia, obesity, cholesterol-proof blood

3. Attempt treatment or cure with gene therapy

3. Complete genome sequencing of organisms with smaller genomes tbl 18.1
1. Generally one-half of genes have known functions
2. Brewer's yeast is only eukaryote totally sequenced
3. Many of 6000 genes may be similar in structure to human genes

4. Complete genomes of other eukaryotes nearing completion

3. DNA Fingerprinting
1. First utilized in rape trial in 1987 fig 18.14
1. Consists of autoradiographs of parallel bars on X-ray film
2. Bar represents position of restriction endonuclease fragment
3. Include controls, patterns of two endonucleases on semen collected from victim

2. Comparison of samples
1. Suspect's two patterns match patterns of rapist, not those of victim
2. Semen of rapist and blood sample of suspect came from same person

3. Resulted in first conviction of suspect based on DNA evidence
4. Used as evidence in over 2000 court cases since then fig 18.15
5. Some probes are common in populations, others are not
6. Has revolutionized forensic science
1. Can use minute samples of hair, blood, semen
2. Laboratory analyses must be done carefully, eliminate contamination
3. National standards being developed

2. Medical Applications
1. Pharmaceuticals
1. Most obvious commercial application of gene technology
1. Bacteria can produce gene products in bulk
2. Include human insulin, interferon, growth hormone, erythropoietin fig 18.16

2. Produce medically important proteins
1. Atrial peptides: Regulate blood pressure, kidney function
2. Tissue plasminogen activator: Dissolves blood clots

3. Must separate desired protein from bacterial proteins
1. Time-consuming and expensive
2. Produce RNA transcripts of cloned genes
3. Make proteins directly in cell-free culture

2. Gene Therapy
1. First attempts of transfer of human genes in 1990
1. Obvious rationale if disease is caused by single defective gene
2. Add working copy of gene to individual

2. Clinical trials for several disorders including cystic fibrosis tbl 18.2
3. Success in adding gene encoding adenosine deaminase to bone marrow

3. Piggyback Vaccines
1. Produce subunit vaccines for herpes virus and hepatitis viruses fig 18.13
1. Part of gene for protein-polysaccharide coat isolated
2. Spliced to vaccinia virus DNA
3. Live vaccinia added to cell culture with fragments
4. Recombinant virus carries coat genes of other virus
5. Infected animal produces antibodies to outer surface of virus
6. Make antibodies against virus without exposure to it

2. Clinical trials of new DNA vaccine in 1995
1. Doesn't depend on antibodies
2. Associated with cellular immune response
1. Killer T cells in blood attack infected cells
2. Infected cells have foreign proteins on outer surface

3. First attempted used influenza virus gene encoding internal nucleoprotein
1. Gene spliced onto plasmid, injected into mice
2. Mice developed strong cellular immune response to influenza

3. Agricultural Applications
1. Manipulation of Genes in Key Crop Plants
1. Initial difficulty in identifying suitable plant vector
2. Plants lack plasmids of bacteria
3. Currently use Ti (tumor-inducing) plasmid of Agrobacterium
1. Infects broad leaf plants like tomato, tobacco, soybean
2. Attach other genes to this plasmid fig 18.18
3. Desire to develop resistance to disease, frost, other stress, nutritional balance, protein content, herbicide resistance
4. Does not infect cereal plants

4. Development of Flavr Savr tomato fig 18.19
1. Inhibit genes that make cells produce ethylene
2. Lack of ethylene delays ripening of fruit

2. Herbicide Resistance
1. Broadleaf plants engineered to be resistant to glyphosate
1. Active ingredient in Roundup herbicide fig 18.20
2. Inhibits enzyme EPSP synthetase, produces aromatic amino acids
3. Ti plasmid used to insert extra copies of EPSP synthetase gene
4. Plants overproduce enzyme overcome glyphosate suppression

2. New bacterial gene unaffected by glyophosate inserted into plants
3. Advantages of Roundup resistance
1. Crops would not need to be weeded
2. Wide variety of weeds killed and desired crop spared
3. Glyphosate readily degradable

3. Nitrogen Fixation
1. Insert legume nitrogen-fixing genes into non-leguminous plants
1. nif genes found in bacteria associated with root nodules fig 18.21
2. Bacteria convert atmospheric N₂ into NH₄

2. Plants lacking such bacteria must obtain nitrogen from soil
1. Farmland depleted of nitrogen unless fertilizer applied
2. Provide crops with ability to produce own fertilizer

3. Problems since genes do not function properly in eukaryotic cells

4. Insect Resistance
1. Insects presently controlled via chemical insecticides
2. Engineer plants for resistance to insects
1. Bacillus thuringiensis insecticidal protein genes fig 18.22
2. Ingested by tomato hornworm, converted to poison
3. Harmless to animals with different stomach enzymes
4. Genes introduced into tomato, tobacco via Ti plasmid
5. Transgenic plants safe from attack by insects that eat them fig 18.23

3. Other examples
1. Genetically altered potato kills Colorado potato beetle
2. Cotton resistant to bollworms
3. Corn resists European corn borer

4. Isolation insect-killing enzyme from a fungus
1. Cholesterol oxidase disrupts insect gut membranes
2. Fungal Bollgard gene inserted into a variety of crops
3. Kills variety of insects including cotton boll weevil and Colorado potato beetle

5. Introduce insecticidal protein into root bacteria
1. thuringiensis does not normally inhabit roots
2. Protect roots from various pests, including Pseudomonas

5. Farm Animals
1. Somatotropin growth hormone (BST) synthetically produced
1. Added to diary cow's diet to increase milk yield fig 18.24
2. Potential to increase weight of cattle and pigs fig 18.25
3. Human tests to increase size of hormonal dwarfs

2. Public resistance to BST in milk
1. Generalized fears of gene technology
2. BST is a proteins, digested in stomach

3. Development of transgenic animals faster than several generations of selective breeding

4. Cloning
1. Breeding Transgenic Animals Is Slow
1. Recombination reverses painstaking work of genetic engineer
2. Ideal is to "xerox" exact clones of the transgenic strain
3. First successful cloning of vertebrate in 1997
4. Stands to revolutionize agricultural science

2. Speman's "Fantastical Experiment"
1. First idea to clone animals in 1938
2. Proposal to remove nucleus from egg, replaced it with nucleus from another cell
1. Technology appropriate to make attempt in 1952, attempted by Briggs and King
2. Partial success in 1970 by Gurdon
1. Inserted nuclei from advanced toad embryos
2. Eggs developed into tadpoles, died before adulthood

3. The Path to Success
1. Continued nuclear transplant experiments proved unsuccessful
2. First successful cloning of sheep in 1984 used nucleus from cell of early embryo
3. Succeeded at replicating result with other animals
1. All required use of nucleus from early embryo
2. Later stages assumed to be "committed" to differentiated to work

4. Later knowledge proved this commitment idea wrong
1. Cell division doesn't occur until conditions are proper
2. Egg and donated nucleus needed to be at same stage

5. First attempts starved cells to synchronize them at G₂ checkpoint

4. Wilmut's Lamb
1. Wilmut transplanted egg from adult differentiated cell into egg
1. Removed cells ("Dolly") from udder of six year old sheep fig 18.26
2. Cells grown in tissue culture
3. Some frozen for future fingerprinting to prove identical genetic content

2. Reduced nutrient content of cell medium
3. Eggs removed from ewe, nuclei removed with micropipette
4. Mammary cells and egg cells surgically combined
1. Mammary cells inserted inside covering of egg cell
2. Electric shock causes plasma membranes of cells to become leaky
3. Contents of mammary cell passes into egg cell
4. Shock also causes cell to start division cycle

5. After six days 30 eggs reached hollow-ball blastula stage
1. 29 transplanted into surrogate mother sheep
2. Five months later one gave birth to a lamb in 1997
3. First successful cloning from differentiated animal cell

5. The Future of Cloning
1. First attempt is quite inefficient, established feasibility of process
2. Future research must improve efficiency
3. Can have major impact on medicine as well as agriculture
1. Animals with human genes to produce human hormones
2. Sheep genetically engineered to produce alpha-1 antitrypsin in milk
3. if cloned can provide source of drug used to treat cystic fibrosis

4. Question rationale for human cloning

5. Ethics and Regulation
1. Concerns Regarding Tampering with Genetic Material fig 18.27
1. Accidental production of a cancer-transmitting bacterium
2. Intentional development of a killer virus
3. Dangerous complications of genetically engineered products administered to plants or animals in future generations
4. Ecological impact of "improved" crops
5. Potential of creating "genetically superior" organisms, including humans

2. Most of Public's Concerns Not Well-Founded
1. Most organisms used in genetic engineering incompatible with human hosts
1. Recombinant technology like natural crossing, only faster
2. Genetic "dabbling" by humans minuscule compared to natural mutations

2. Benefits far outweigh the risks