17.1. Cloning of a Gene (p. 270)
A. Making Recombinant DNA
1. Recombinant DNA (rDNA) contains DNA from more than one source.
2. To make rDNA, a vector must first be selected.
3. A vector is a plasmid or a virus used to transfer foreign genetic material into a cell.
4. A plasmid is a ring of extrachromosomal DNA in the cytoplasm of bacteria.
5. Plasmids are removed from bacteria; a foreign gene is inserted into them. (Fig. 17.1) [transp. 107]
6. Introduction of foreign DNA into vector DNA to produce rDNA requires two enzymes.
a. Restriction enzyme is a bacterial enzyme that stops viral reproduction by cleaving viral DNA.
1) Restriction enzyme is used to cut DNA at specific points during production of rDNA.
2) It is called restriction enzyme because it restricts growth of viruses.
b. DNA ligase enzyme links DNA fragments; during production of rDNA it joins foreign DNA to vector DNA.
7. Restriction enzymes cleave vector (plasmid) and foreign (human) DNA.
a. Cleaving DNA makes DNA fragments ending in short single-stranded segments (sticky ends).
b. Sticky ends facilitate the insertion of foreign DNA into vector DNA.
8. The foreign gene is sealed into the vector DNA by DNA ligase.
a. Treated cells take up plasmids, and then bacteria and plasmids reproduce.
b. Eventually, there are many copies of the plasmid and many copies of the foreign gene.
c. In genetic engineering, cloning is production of many identical copies of a gene.
B. Getting the Product
1. Bacterial cells take up recombined plasmids when treated with CaCl2 to make them more permeable.
2. If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product.
3. Viruses can be a vector to carry rDNA into bacterial cells.
a. When a virus invades a cell, the viral DNA is released and enters the cytoplasm.
b. Inside cytoplasm of the host cell, viral DNA may direct reproduction of more viruses.
c. Each virus from a viral vector contains a copy of the foreign gene.
C. Using a Genomic Library (Fig. 17.2) [transp. 108]
1. A genome is the full set of genes of an individual.
2. A genomic library is a collection of bacterial or bacteriophage clones; each contains a segment of DNA from a foreign cell.
3. A genomic library has organism's DNA sliced into pieces and put into vectors taken up by hosts.
4. For mammalian gene expression to occur in a bacterium, the gene has to be accompanied by proper regulatory regions, and mammalian genes must not contain introns (bacterial cells do not have the necessary enzymes to process primary messenger RNA).
5. Using reverse transcriptase, a DNA copy of mature mRNA. This DNA copy is called complementary DNA (cDNA), which lacks introns.
7. A probe is a single-stranded nucleotide sequence that will hybridize with a certain piece of DNA.
a. A probe can be used to search a genomic library for a certain gene.
b. The probe can be located because it is either fluorescent or radioactive.
c. Bacterial clones carrying a particular fragment are plated on agar; after the probe hybridizes with the gene, the gene is isolated from the fragment and cloned further or analyzed for its DNA sequence. (Fig. 17.3) [transp. 109]
D. Replicating Small DNA Segments
1. The polymerase chain reaction (PCR) uses the enzyme DNA polymerase to carry out multiple replications (a chain reaction) of target DNA.
2. PCR can create millions of copies of a single gene or a specific piece of DNA in a test tube.
3. PCR is very specific---the targeted DNA sequence can be less than one part in a million of the total DNA sample; therefore, a single gene can be amplified using PCR.
4. The PCR does not replace gene cloning; cloning produces many more copies of a gene, and it is still used whenever a large quantity of a gene or a protein product is needed.
5. Before carrying out PCR, primers must be available.
a. Primers are sequences of 20 bases complementary to bases on either side of "target DNA."
b. Primers are needed because DNA polymerase only continues or extends the replication process.
c. After primers bind to the DNA strand, DNA polymerase copies the target DNA. (Fig. 17.4) [transp. 110]
6. Most labs have PCR machines; automation requires temperature-insensitive DNA polymerase an enzyme that can stand high temperatures, to separate double-stranded DNA.
E. Analyzing DNA
1. DNA fingerprinting is the technique of using DNA fragments lengths, resulting from restriction enzyme cleavage, to identify particular individuals. (Fig. 17.5) [transp. 111]
a. The DNA is first treated with restriction enzymes, which cut it into fragments.
b. Each organism's DNA results in different-sized restriction fragment length polymorphisms (RFLPs).
c. During gel electrophoresis, fragments separate according to length, resulting in a pattern of bands.
d. Radioactive probes allow specific sequences to be separated from all other fragments, and the resulting pattern to be recorded on X-ray film (autoradiography).
e. DNA fingerprinting can identify deceased individuals from skeletal remains, perpetrators of crimes from blood or semen samples, and genetic makeup of long-dead individuals or extinct organisms.
2. Following a PCR, DNA segments can be analyzed.
3. Therefore, PCR amplification and analysis is used to
a. detect viral infections, genetic disorders, and cancer, and in forensic laboratories;
b. estimate evolutionary relationships of present-day species by comparing nucleotide sequences;
c. determine the nucleotide sequence of human genes: the Human Genome Project; and
d. conduct molecular paleontology by determining the evolutionary history of human populations.
4. Automated DNA sequencers that make use of computers can sequence genes at a fairly rapid rate.
17.2. Biotechnology Products Are Many (p. 274)
A. Hormones and Similar Types of Proteins
1. Biotechnology allows mass production of proteins that are difficult to obtain otherwise.
2. Human growth hormone treats people who grow slower than normal.
3. Insulin treats diabetics; previously required pancreas of cattle and pigs and could cause reactions.
4. Tissue plasminogen activator (tPA) activates an enzyme to dissolve blood clots; treats heart attack victims by dissolving blood clots in coronary arteries.
5. Diseases may soon be treatable by biotechnology products: clotting factor VIII for hemophilia, a human lung surfactant for premature infants, and atrial natriuretic factor to control hypertension.
6. Biotechnology is used with animals: farm animals given growth hormone produce leaner meat, and bovine growth hormone (bGH) stimulates milk cows to produce 25% more milk.
B. Safer Vaccines
1. Vaccines that use treated bacteria or viruses to trigger immunity response may cause the disease.
2. Genes for surface proteins can be used to genetically engineer harmless bacteria to copy the protein.
3. A vaccine for hepatitis B is available; vaccine studies are underway for chlamydia and other diseases.
4. Farm animals benefit from biotechnology vaccines for hoof-and-mouth disease and dysentery.
17.3. Making Transgenic Organisms (p. 275)
A. Transgenic Organisms
1. Free-living organisms that have had a foreign gene inserted into them.
B. Transgenic Bacteria Perform Services
1. Transgenic bacteria have been produced to protect and improve the health of plants.
a. Frost-minus bacteria protect the vegetative parts of plants from frost damage.
b. Root-colonizing bacteria receive genes from bacteria with insect toxin, protecting the roots.
c. Infecting nonleguminous plants with genetically engineered nitrogen-fixing bacteria might reduce fertilizer needed on agricultural fields.
2. Transgenic bacteria perform bioremediation.
a. Bacteria selected for ability to degrade oil can be improved by genetic engineering. (Fig. 17.6)
b. Bacteria can be biofilters to prevent airborne chemical pollutants from being vented into the air.
c. Bacteria can also remove sulfur from coal before it is burned and help clean up toxic dumps.
3. Transgenic bacteria produce chemical products.
a. We can manipulate genes coding for enzymes to catalyze synthesis of valuable chemicals.
b. Phenylalanine used in aspartame sweetener can be grown by engineered bacteria.
4. Transgenic bacteria process minerals.
a. Many major mining companies already use bacteria to obtain various metals.
b. Genetically engineered "bioleaching" bacteria extract copper, uranium, gold from low-grade ore.
C. Transgenic Plants Are Here
1. The bacterium Agrobacterium has the only known plasmid for genetically engineering plant cells; therefore, other techniques are used to introduce foreign DNA into plants.
a. Plant cells that have had the cell wall removed are called protoplasts.
b. Electric current makes tiny holes in a plasma membrane through which genetic material enters.
2. About fifty types of genetically engineered plants have entered field trials. (Fig. 17.7)
3. Major crops that can be improved in this way are soybean, cotton, alfalfa, rice, potato, and corn.
4. One day genetically engineered plants will have one or more of the following attributes: they will be heat-, cold-, drought-, or salt-tolerant; they will be more nutritious; they can be stored and transported without fear of damage; they will require less fertilizer; or they will produce chemicals and drugs that are of value to humans.
5. Plants can be engineered to produce human proteins, such as hormones, in their seeds.
6. Mouse-eared cress has been engineered to produce a biodegradable plastic in cell granules.
D. Transgenic Animals Are Here
1. Animal cells will not take up bacterial plasmids; other methods insert genes into eggs of animals.
a. It is possible to microinject foreign genes into eggs by hand.
b. Vortex mixing places eggs in an agitator with DNA and silicon-carbide needles that make tiny holes through which the DNA can enter.
c. Using this technique, many types of animal eggs have been injected with bovine growth hormone (bGH) to produce larger fishes, cows, pigs, rabbits, and sheep.
2. Gene pharming is the use of transgenic farm animals to produce pharmaceuticals; the product is obtainable from the milk of females.
a. Genes for therapeutic proteins are inserted into animal's DNA; animal's milk produces proteins.
b. Drugs obtained through gene pharming are planned for the treatment of cystic fibrosis, cancer, blood diseases, and other disorders.
c. Testing is underway for antithrombin III to prevent blood clots during surgery. (Fig. 17.8)
E. Ecological Concerns
1. Some are very concerned about deliberate release of genetically modified microbes.
2. These bacteria might displace normal residents in an ecosystem and the effect could be deleterious.
3. Tools are currently available to detect, measure, and disable cell activity in the natural environment; such tools may pave way for genetically modified microbes to play role in agriculture and environmental protection.
17.4. Gene Therapy Is a Reality (p. 278)
A. Gene Therapy Supplies Healthy Genes
1. To make up for a faulty gene.
2. Includes use of genes to treat genetic disorders and various human illnesses.
B. Some Methods Are Ex Vivo
1. In ex vivo therapy (outside the patient), cells are removed from a patient, treated, and returned.
a. A retrovirus is often used as a vector to carry normal genes into the cells of the patient.
b. If recombinant RNA enters a human bone marrow stem cell, reverse transcription occurs.
c. During reverse transcription, RNA is used as a template for the formation of DNA; it is the resulting DNA that carries the normal gene into the human genome. (Fig. 17.9) [transp. 112]
2. Girls with severe combined immunodeficiency syndrome (SCD) underwent ex vivo gene therapy.
a. Lacking an enzyme involved in maturation of T and B cells, they faced life-threatening infections.
b. White blood cells are removed, infected with retrovirus that carries a normal gene, and returned.
c. Use of bone marrow stem cells genetically engineered in the same way may produce a permanent cure.
3. Over a hundred gene therapy trials are underway for other treatments.
a. Familial hypercholesterolemia develops when liver cells lack a receptor for removing cholesterol from blood; surgically removed portion of liver is infected with retrovirus with normal gene for receptor.
b. Chemotherapy in cancer cells kills off healthy as well as cancer cells; genes given to cancer patients make healthy cells more tolerant or make tumors more vulnerable.
C. Some Methods Are In Vivo
1. In vivo methods use viruses, laboratory-grown cells, or synthetic carriers to introduce genes directly into patients; no cells are removed from the patient.
a. Liposomes, microscopic vesicles that form when lipoproteins are in solution, are coated with healthy cystic fibrosis genes and sprayed into a patient's nostrils.
b. Retroviruses carry genes for cytokines, soluble hormones of the immune system, directly into tumors of patients; presence of cytokines stimulates immune system to rid body of cancer cells.
2. It may be possible to use in vivo therapy to cure hemophilia, diabetes, Parkinson disease, or AIDS.
a. Hemophilia patients could get regular doses of cells with normal clotting-factor genes.
b. Organoids (artificial organs), could be implanted in the abdominal cavity.
c. To cure Parkinson disease, dopamine-producing cells could be grafted directly into the brain.
d. Requires laboratory-grown cells to be stripped of antigens to prevent an immune response.
17.5. Mapping the Human Chromosomes (p. 279)
A. Human Genome Project Goals
1. To identify the location of approximately 100,000 human genes on all chromosomes.
2. To determine the sequence of the three billion base pairs in the human genome.
B. The Genetic Map
1. Several methods have been used to attempt mapping human chromosomes. (Fig. 17.10)
2. mRNA has been isolated; reverse transcriptase has been used to produce a cDNA copy of a gene.
a. A cDNA copy is attached to fluorescent dye; probe determines to which chromosome it belongs.
b. Base pairing of probe and chromosome occurs on the microscope slide; use of fluorescence allows us to see where the gene is located.
3. Genes can sometimes be assigned a location on a chromosome according to their relative relationship to genetic markers on specific chromosomes.
a. Genetic markers are DNA base sequences linked to a disease-causing gene.
b. Sickle-cell and Huntington disease and Duchenne muscular dystrophy tests use genetic markers.
C. Physical Map
1. Completed physical map of the human genome will indicate sequence of all DNA pairs.
2. Labs using fluorescent dyes detect fragment ends with A, T, C, or G to create physical map.
3. Some genetic markers contain unique stretches of DNA called expressed sequence-tags (ESTs).
4. The human genome project helps in understanding and curing human genetic diseases.
5. Improvements in genetic testing make the need for ethical guidelines more pressing.