26

RECOMBINANT DNA AND B IOTECHNOLOGY

CHAPTER REVIEW
To clone a gene, a vector is first prepared. To genetically engineer a plasmid or virus, restriction enzymes are used to cleave plasmid DNA and to cleave foreign DNA. The sticky ends produced facilitate the insertion of foreign DNA into vector DNA. The foreign gene is sealed into the vector DNA by DNA ligase. When the plasmid replicates or the virus reproduces, the foreign gene is cloned. A genomic library can be used as a source of genes to be cloned. A radioactive or fluorescent probe is used to identify the location of a single gene among the cloned fragment of an organisms DNA. Restriction enzymes are used to fragment DNA. If the entire genome is used for fingerprinting, the use of probes is necessary in order to see a pattern following gel electrophoresis. If just a portion of the genome is of interest or available, polymerase chain reaction (PCR) uses the enzyme DNA polymerase to make multiple copies of target DNA. Analysis of DNA segments following PCR can involve gel electrophoresis to identify the DNA as belonging to a particular organism or can involve determining the base sequence of the DNA segment. Transgenic organisms have also been made. Genetically engineered bacteria, agricultural plants, and farm animals now produce commercial products of interest to humans such as hormones and vaccines. Transgenic bacteria also perform bioremediation, extract minerals, and produce chemicals. Transgenic agricultural plants have been engineered to resist herbicides and pests. Transgenic animals have been given bovine growth hormone. Pigs have been genetically altered to serve as a source of organs for transplant patients. Cloning of animals is now possible. The Human Genome Project has two goals: constructing a genetic map of the chromosomes and sequencing the DNA bases. This information will probably be of assistance to carrying out gene therapy. Gene therapy is used to correct the genotype of humans and to cure various human ills. Ex vivo therapy involves withdrawing cells from the patient, inserting a functioning gene, usually via a retrovirus, and then returning the treated cells to the patient. Many investigators are trying to develop in vivo therapy, in which viruses, laboratory-grown cells, or synthetic chemicals will be used to carry healthy genes into the patient.

S T U DY E X E R C I S E S
Study the text section by section as you answer the questions that follow.

26.1 CLONING

OF A

GENE (P . 530)

Using recombinant DNA technology, bacteria and viruses can be genetically altered to clone a gene. A genomic library contains bacteria or viruses that carry fragments of all the DNA of a particular organism. The polymerase chain reaction (PCR) makes multiple copies of a particular piece of DNA segments. Analysis of the DNA usually follows.

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1. In the diagram, write the numbers of the following descriptions in the appropriate blanks: 1 Cloning occurs when host cell reproduces. 2 Host cell takes up recombined plasmid. 3 DNA ligase seals human gene and plasmid. 4 Restriction enzyme cleaves DNA.
plasmid human DNA bacterium human cell restriction enzyme cleaves DNA a.

insulin gene

DNA ligase seals human gene and plasmid

b.

recombinant DNA

host cell takes up recombined plasmid

c.

cloning

d.

cloned insulin gene

insulin

2. What is meant by the expression that restriction enzymes produce sticky ends? _________________________ _________________________________________________________________________________________________ 3. Change the following false statements to true statements: a. Only plasmids are used as vectors during genetic engineering experiments. Rewrite: ____________________ _______________________________________________________________________________________________ b. Recombinant DNA contains two types of bacterial DNA recombined together. Rewrite: _________________ _______________________________________________________________________________________________ c. Genetic engineering usually means that an organism receives genes from a member of its own species. Rewrite: _______________________________________________________________________________________ _______________________________________________________________________________________________ d. Gene cloning occurs when a gene produces many copies of various genes. Rewrite: _____________________ _______________________________________________________________________________________________

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4. Use the following diagram to help you complete this paragraph.

transfer clones onto membrane and add probe radioactive probe

perform radiography

The clones on the petri dish are from a genomic library. What is a genomic library? a. ______________________ If these clones are from a mammalian cell, the genes are accompanied by b. ______________ regions that are necessary for the expression of mammalian genes. If these clones have been made using reverse transcriptase, then the cDNA contains the c. ______________ but not the introns. In the diagram, a radioactive probe is being added to the petri dish. What is a probe? d. __________________________________ 5. Explain the polymerase chain reaction by telling what polymerase refers to: a. ______________ ; and what chain reaction means: b. ______________. At the beginning of the reaction, very little DNA may be available, but at the end of the reaction c. ______________ copies of a segment of DNA are available. 6. In DNA fingerprinting, a. ______________ enzymes digest the two samples to be compared. b. ______________ separates the fragments, and their different lengths are compared. If the pattern is similar, the samples are from c. ______________.

26.2 BIOTECHNOLOGY PRODUCTS (P . 534)

Bacteria, agricultural plants, and farm animals have been genetically engineered to produce commercially available products. Agricultural plants and farm animals have been genetically engineered to improve their yield. Farm animals have been genetically engineered to serve as a source of organs for human transplant patients. It is now possible to clone animals, and cloning is used to produce multiple copies of farm animals that have been genetically engineered. 7. Complete the following table on transgenic organisms:
Type of Organism Engineered for What Purpose

8. The advantage of using bacteria to produce a product is that a. __________________________________________. The advantage of using plants to produce a product is that b. ____________________________________________. The advantage of using farm animals to produce a product is that c. ______________________________________. 9. Which of these is true of xenotrasplantation? a. Xenotransplantation uses humans as a source of organs for transplants. b. Xenotransplantation uses other species such as the pig as a source of organs for transplants. c. Pigs can be genetically altered to prevent rejection of their organs by humans. d. Other species can possibly pass new and different viruses to humans.

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10. Put these statements in the proper sequence to describe the making of a transgenic female goat which will produce a medicine needed by humans in its milk. a. Development within a host animal b. Remove egg from donor animal. c. Isolate a human gene. d. Microinject the human gene into the egg of the donor animal. e. Trangenic goat is born. 11. Put these statements in the proper sequence to describe the cloning of the transgenic goat produced in question 10. a. Birth of cloned transgenic goats b. Remove nuclei from adult cells of transgenic goat. c. Collect the milk which contains the medicine of interest. d. Development within host goats e. Microinject 2n nuclei into the donor eggs. f. Remove eggs form donor animal.

26.3 THE HUMAN GENOME PROJECT (P . 538)

The Human Genome project has two goals: to genetically map each chromosome and to sequence the DNA bases of each chromosome. 12. The Human Genome project has two goals, which are (1) to make a a. _________________________ map of the human chromosomes and (2) to make a b. _________________________ map of the human chromosome. 13. Which one of these is not a step in sequencing the base pairs of DNA? a. Cut the DNA into fragments. b. Use a computer program to put the fragments in the correct order. c. Use a DNA sequencer to order the base pairs. d. Put the DNA into bacteria for cloning the fragments. 14. After eliminating the incorrect step in the preceding question, put the others in the correct order. ________________________ 15. State two benefits for human beings if we knew the sequence of the bases in the human genome. a. _______________________________________________________________________________________ b. _______________________________________________________________________________________

26.4 GENE THERAPY (P . 539)

Gene therapy is now being used to replace defective genes with healthy genes and to help cure various human ills. 16. Change these false statements to true statements. a. Ex vivo methods of gene therapy require that the therapeutic gene be placed in the body either directly or by using a viral vector. Rewrite: ____________________________________________________________ ______________________________________________________________________________________ b. A common ex vivo method is to microinject normal genes into bone marrow stem cells removed from the patient. Then the stem cells are returned to the patient. Rewrite: __________________________________ _______________________________________________________________________________________________ c. Gene therapy is currently restricted to curing genetic diseases and is not used to treat illnesses like cystic fibrosis or cardiovascular diseases. Rewrite: ____________________________________________________________________ _______________________________________________________________________________________________

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CHAPTER TEST
OBJECTIVE QUESTIONS
Do not refer to the text when taking this test. 1. Select the incorrect description of a plasmid. a. used as vector b. consists of chromosomal DNA c. found in some bacteria d. small, ringlike structure 2. Restriction enzymes a. cleave DNA into small fragments. b. restrict the growth of eukaryotic cells. c. seal pieces of DNA together. d. serve as introns in cells. 3. The problem with using pigs as a source of human organs is a. its not possible to genetically alter pig cells to avoid rejection. b. their organs are too small in comparison to human organs. c. pig organs might carry viruses that would be new to humans. d. transplant patients would rather die than receive organs from a pig. 4. To clone a transgenic animal, you have to have a. an egg donor. b. special food to feed them. c. a way to protect human beings from coming in contact with transgenic animals. d. a transgenic animal. e. both a and d 5. A final step in the use of a plasmid to clone a gene is a. to insert a foreign gene into a bacterium. b. to introduce a plasmid into a treated cell. c. to remove a plasmid from a bacterium. d. plasmids reproduce in a treated cell. 6. The bacteriophage lambda is used as a a. cleavage agent for DNA in a virus. b. cleavage agent for RNA in a virus. c. vector to carry rDNA into bacterial cells. d. vector to carry rDNA into eukaryotic cells. 7. A transgenic organism is a. free-living and receives a foreign gene. b. free-living and transmits a foreign gene. c. parasitic and receives a foreign gene. d. parasitic and transmits a foreign gene. 8. The Human Genome Project a. will probably take about 50 more years more to complete. b. has no usefulness to humans. c. will result in knowing the base pair sequences of all the DNA in a human. d. must be carried out by government employees because the results are top secret. 9. Gene therapy is a. on the back burner because human genes are so different from one another. b. going full speed ahead despite its being still investigative. c. the use of foreign genes to cure a human ill. d. both a and c e. both b and c 10. Genetically engineered plants have been or will be used to a. resist insects. b. resist herbicides. c. produce protein-enhanced beans, corn, and wheat. d. produce animal neuropeptides, blood factors, and growth hormones. e. All of these are correct. 11. ______ is a protein that activates an enzyme to dissolve blood clots. a. tPA b. Clotting factor VIII c. Atrial natriuretic factor d. bGH e. DNA ligase 12. Genetically engineered bacteria can be used to a. protect plants from frost. b. clean up oil spills on beaches. c. produce organic chemicals. d. extract copper and gold from low-grade sources. e. All of these are correct. 13. A DNA probe will seek out and bind to any complementary a. DNA. b. RNA. c. plasmid. d. organoid. e. restriction enzyme. 14. Which of these would you NOT expect to be a biotechnology product produced by a bacterium? a. steroid sex hormones b. sex hormones c. nucleic acids d. both b and c 15. If a cell is altered while outside the human body for gene therapy, it is considered ______________ therapy. a. ex vivo b. in vivo c. in vitro d. extraneous e. intravenous

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16. Which of the following is NOT needed to make a recombinant DNA molecule? a. foreign DNA b. vector DNA c. restriction enzymes d. DNA ligase e. DNA polymerase 17. Foreign DNA can be inserted into vector DNA because both DNA molecules a. have the same genes. b. have the same bases. c. have sticky ends. d. are not complementary to each other. 18. Which enzyme is used to seal breaks in a DNA molecule? a. DNA polymerase b. RNA polymerase

c. restriction enzymes d. DNA ligase e. RNA ligase 19. Possible uses for biotechnology include a. production of vaccines. b. production of drugs for health. c. altered bacteria to clean up oil spills. d. study how mammalian genes function. e. All of these are correct. 20. The gene for ______________ will result in increased milk production in cows. a. bGH b. tPA c. insulin d. erythropoietin e. interferon

THOUGHT QUESTIONS
Answer in complete sentences. 21. How do studies of genetic engineering prove that the genetic code is nearly universal?

22. What do you think are some objections our society may have regarding genetic engineering?

Test Results: ______ Number right 22 = ______ 100 = ______ %

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ANSWER KEY
STUDY EXERCISES
1. a. 4 b. 3 c. 2 d. 1 2. Cleavage results in unpaired bases. 3. a. Both plasmids and viruses are used . . . b. contains DNA from two different sources c. . . . from a member of a different species d. . . . many copies of the same gene 4. a. a collection of clones that carry all the genes of an organism b. regulatory c. exons d. single-stranded DNA or mRNA that hybridizes (pairs) with a gene of interest 5. a. DNA polymerase, the enzyme involved in DNA replication b. the reaction occurs over and over again c. many 6. a. restriction b. gel electrophoresis c. from the same individual 7.
Type of Organism Engineered for What Purpose to protect plants, for bioremediation, to produce chemicals, and to mine metals to resist insects, pesticides and herbicides, and to make products to have improved qualities and to make products

8. a. They will take up plasmids. b. They will grow from single cells (protoplasts). c. The product is easily obtainable in milk. 9. b, c, d 10. c, b, d, a, e 11. b, f, e, d, a, c 12. a. genetic b. base sequence 13. d 14. a, c, b 15. a. would be able to acquire normal genes for gene therapy b. would be able to determine the genetic illnesses of individuals 16. a. In vivo b. . . . use a viral vector to carry normal genes. . . c. . . . is not restricted to curing genetic disease and is used to treat illnesses. . .

CHAPTER TEST
1. b 2. a 3. c 4. e 5. d 6. c 7. a 8. c 9. b 10. e 11. a 12. e 13. a 14. a 15. a 16. e 17. c 18. d 19. e 20. a 21. Genes transmitted to new cells through vectors and other means are still transcribed and translated by the same process and with the same accuracy. 22. Some people may object to genetic engineering on religious grounds because we are now able to change the inherited characteristics of organisms, including human beings. Some scientists have concerns because a disease-causing transgenic bacterium may be produced for which humans have no immunity or a transgenic bacterium, plant, or animal may be produced which could wreak havoc in the environment.

bacteria

plants

animals

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