Lecture Outline - Chapter 26

26.1. Cloning of a Gene (p. 500)

1. Recombinant DNA (rDNA)
   • a. Contains DNA (deoxyribonucleic acid) from two or more sources.
   • b. Vectors carry recombinant DNA into a host cell.
   • c. Plasmids (Fig. 26.2)
     • i. Are small accessory rings of DNA taken from bacteria; carry genes not on bacterial chromosome.
     • ii. Were discovered by researchers studying Escherichia coli bacteria.
     • iii. Plasmid is taken from bacteria, foreign gene is inserted, and plasmid is returned to host cell.
     • iv. Whenever cell reproduces, both genes are copied many times.
     • v. Gene cloning results in many exact copies of a foreign gene.
   • d. Viral DNA
     • i. Used to carry recombinant DNA into animal cells.
     • ii. Virus enters host cell and releases viral DNA.
     • iii. Host cell reproduces more viruses including foreign gene.
2. Making Recombinant DNA (Fig. 26.2)
   • a. Two types of enzymes are required for recombinant DNA.
     • i. Restriction Enzymes
       - found naturally in some bacteria; first isolated by Hamilton Smith in 1970.
       - hundreds now known.
       - restrict growth of viruses within bacteria.
       - cleave plasmid DNA at a specific location; action called "molecular scissors."
       - foreign DNA can be placed in gap caused by cleavage.
       - single-stranded complementary ends of two DNA molecules are called "sticky ends" (they adhere by complementary base pairing when cut by same restriction enzyme).

       ii. DNA Ligase

       - seals breaks in DNA molecules in nature.

       - seals foreign DNA into vector, completes DNA splicing.
   • b. Getting the Product (p. 501)
     • i. Bacterial cells take up recombined plasmid; calcium chloride makes bacteria more permeable.
     • ii. If foreign gene is replicated and expressed, we can recover cloned gene or protein product. (Fig. 26.2)
     • iii. To express mammalian gene in bacteria, gene must have proper regulatory regions and not contain introns.
       - reverse transcriptase is used to make DNA from mRNA (thus called complementary DNA or cDNA) without introns.
       - a machine called a DNA synthesizer can join correct sequences of nucleotides without introns.

1. Hormones and Similar Types of Proteins
   • a. Human growth hormone (previously required glands from 50 cadavers) treats abnormally slow growth.
   • b. Insulin (previously extracted from cattle and pigs, sometimes caused allergic reactions) treats diabetes.
   • c. Tissue plasminogen activator (tPA) dissolves blood clots to treat heart attacks.
   • d. Potential treatments include: clotting factor VIII for hemophilia, human lung surfactant for premature infants, atrial natriuretic factor controls hypertension.
   • e. Growth hormones (rather than steroids) produce leaner meat.
   • f. Bovine growth hormone (bGH) results in 25% greater milk production.
2. Safer Vaccines
   • a. Vaccines that generally build human immunity occasionally cause illness.
   • b. Genes for surface proteins in bacteria and viruses can be placed in a plasmid to make many copies of surface protein for use in making a safe vaccine.
   • c. Current recombinant vaccine available for hepatitis B.
   • d. Might see future recombinant vaccines for chlamydia, malaria, and AIDS.
   • e. Recombinant vaccines inoculate farm animals against hoof-and-mouth disease and scours.

1. Polymerase Chain Reaction (PCR)
   • a. Can make millions of copies of single gene or specific piece of DNA in test tube.
   • b. Very specific; can target one single DNA sequence in a million!
   • c. Allows one single gene to be amplified or copied many times.
   • d. DNA polymerase carries out DNA replication over and over again.
   • e. Primers, sequences of about 20 bases complementary to bases on either side of "target DNA," are added.
   • f. DNA polymerase does not start replication process but only continues it.
   • g. After primers bind by complimentary base pairing, DNA polymerase copies target DNA. (Fig. 26.3a, b)
   • h. Thermostable DNA polymerase from bacteria allow use of high temperatures to separate double- stranded DNA; permits automated machines to run PCR.
2. Analyzing DNA Segments (p. 503)
   • a. Following PCR, nucleotide sequences can be determined.
   • b. Uses of PCR
     • i. Estimate evolutionary relationships of current species by comparing nucleotide sequences of rRNA ribosome subunit.
     • ii. Determine nucleotide sequence for Human Genome Project.
     • iii. Molecular paleontology uses amplified mitochondrial DNA to determine evolution of humans.
   • c. DNA Probe
     • i. Allows DNA from PCR amplification to be analyzed.
     • ii. Single strand of radioactive nucleotides is made by using a DNA synthesizer.
     • iii. Single radioactive strand seeks out and binds to any complementary DNA strand. (Fig. 26.3c)
     • iv. Many probes are now available; can be used to:
       - detect viral infections with probe complementary to viral DNA.
       - diagnose genetic disorders with probe complementary to mutation.
       - diagnose cancer; probes for oncogenes, mutated tumor-suppressor genes, etc.

       v. DNA Fingerprinting (Fig. 26.4)

       - DNA is treated with restriction enzymes to cut it into different-sized fragments, or restriction fragment length polymorphisms (RFLPs).

       - different length RFLPs are separated by gel electrophoresis.

       - band pattern is recorded on X-ray film; differs for each organism.

       - DNA fingerprint is inherited; resembles that of parents.

       - single sperm is enough to identify suspected rapist when used with PCR amplification.

       - used by evolutionary biologists to determine skin sample from extinct quagga was a zebra rather than horse.

1. Free-living organisms with a foreign gene inserted in them are transgenic organisms.
2. Transgenic bacteria are engineered to perform various services.
   • a. Protection and Enhancement of Plants
     • i. Engineered bacteria protect plants against frost damage.
     • ii. Corn root bacteria are engineered to code for insect toxin.
     • iii. May be possible to transfer genes from Rhizobium bacteria, that fix nitrogen, to nonlegume crop plants such as corn, rice and wheat.
3. Bioremediation
   • a. Natural bacteria that eat oil can be engineered to be more efficient and used to clean up oil spills on beaches. (Fig. 26.5)
   • b. Engineered bacteria may be biofilters to clean air, remove sulfur from coal, etc.
4. Chemical Production
   • a. Bacteria carry out synthesis of many organic chemicals; engineered bacteria can improve efficiency.
   • b. One strain of bacteria produces phenylalanine, a chemical needed to make aspartame (NutraSweet.).
5. Mineral Processing
   • a. Bacteria already used to obtain some metals.
   • b. Improved bacteria may extract copper, uranium, and gold from low-grade sources using "bioleaching."
6. Transgenic Plants Are Here (p. 506)
   • a. Only current plasmid for plant cells with cell walls is from Agrobacterium bacteria; doesn't infect all plants.
   • b. Plant cells with plant cell wall removed are protoplasts.
   • c. Protoplasts can take up a plasmid through holes produced by an electric current.
   • d. Plant cells can then be grown into complete plants.
   • e. Current field trials are testing major crop plants engineered to be resistant to insects, viruses, and herbicides. (Fig. 26.6)
   • f. Future genetically engineered plants maybe developed for:
     • i. Increased plant growth under unfavorable conditions of heat, cold, drought, and salt.
     • ii. More nutrition.
     • iii. Making plants easier to store and transport.
     • iv. Producing plants that require less fertilizer.
     • v. Creating plants to produce chemicals and drugs.
7. Transgenic Animals Are Here (p. 506)
   • a. Animal cells do not take up bacterial plasmids.
   • b. Foreign genes can be microinjected into animal eggs by vortex mixing with silicon-carbide needles; animal eggs injected with bovine growth hormone produce larger fishes, cows, and pigs.
   • c. Gene pharming is use of transgenic farm animals to produce pharmaceuticals; product is often in milk of females.
   • d. Potential engineered animal products include:
     • i. Treatments for cystic fibrosis, cancer, blood, and other disorders.
     • ii. Cattle to produce lactoferrin, a drug to protect from bacterial gastrointestinal infection. (Fig. 26.7)
8. Ecological Concerns
   • a. Some are concerned with intentional release of genetically engineered microbes (GEMs) into environment.
   • b. Ecologists concerned with engineered bacteria replacing natural bacteria in ecosystem.
   • c. Past experience with GEMs indicates no problems.

1. Gene therapy replaces defective genes with healthy genes.
2. Some Methods Are Ex Vivo
   • a. Ex vivo means cells are removed, treated outside patient, and returned to patient.
   • b. Retrovirus (has RNA genes) is used as a vector to carry healthy genes into the cells of the patient.
   • c. Reverse transcription, where RNA is template to form DNA, carries normal gene into human genome. (Fig. 26.8)
   • d. Genetically engineered stem cells in bone marrow can provide long-lived "cure."
   • e. Other possible uses:
     - familial hypercholerterolemia where liver cells cannot remove cholesterol from blood.
     - providing failing heart muscle with additional receptors for adrenalin.
     - providing cancer patients with more tolerance to chemotherapy.
3. Some Methods Are In Vivo
   • a. Viruses, laboratory-grown cells, or synthetic carriers are used to introduce genes directly into the patient (in vivo).
   • b. Adenovirus that contains a gene to treat cystic fibrosis is placed in an aerosol spray; when patient inhales it, tracheal and bronchial cells receive the gene that prevents secretions that clog respiratory tract.
   • c. Retroviruses could carry genes for cytokines to help immune system.
   • d. May be possible to place cells in organoids (artificial organs implanted in abdominal cavity) and use them to treat various diseases.
   • e. Antisense Technology
     - new biotechnology tool for gene therapy.
     - antisense molecules are short DNA or RNA sequences with nucleotides complementary to gene sequences.
     - when antisense DNA binds to a gene, or RNA binds to mRNA it turns the gene or mRNA off.
     - may be useful in stopping microbes, oncogenes, AIDS, etc.