10 Генетическая инженерия (1160079), страница 8
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For example, gene therapy forfamilial hypercholesterolemia, caused by a defectin cholesterol metabolism (p. 679) that can lead toheart attacks at an early age, requires the introduction and expression of a functional LDL receptor in hepatocytes. The prospect of curing such diseases holds great potential for alleviating humansuffering.As this technology advances, however, so doesthe potential to alter other physical traits.
For example, the introduction and expression of a singlegene from the mouse Y chromosome (the Sry gene)into the genome of female (XX) mouse embryoscauses them to develop into male mice. The needfor continued societal involvement in debating theissues generated by this technology is obvious.Chapter 28 Recombinant DNA TechnologyRecombinant DNA Technology YieldsNew Products and ChoicesThe products of recombinant DNA technology range from proteins toengineered organisms. Large amounts of commercially useful proteinscan be produced by these techniques. Microorganisms can be designedfor special tasks; plants or animals can be engineered with traits thatare useful in agriculture.
Some products of this technology have beenapproved for use and many more are in development. During the 1980sgenetic engineering was transformed from a promising technology to amultibillion dollar industry. The first commercial product of recombinant DNA technology was human insulin, produced by Eli Lilly andCompany and approved for human use by the U.S. Food and DrugAdministration in 1982. Hundreds of companies have become involvedin product development worldwide. Much of this growth has come inhuman pharmaceuticals, and some of the major classes of new products are listed in Table 28-4.Table 28—4 Recombinant DNA products in medicineProductcategoryAnticoagulantsBlood factorsColonystimulatingfactorsErythropoietinGrowth factorsHuman growthhormoneHuman insulinInterferonsInterleukinsMonoclonalantibodiesSuperoxidedismutaseVaccinesExamples/UsesTissue plasminogen activator (TPA) activates plasmin,an enzyme involved in dissolving clots; effective intreating heart attack victims.Factor VIII promotes clotting and is deficient inhemophiliacs.
Use of factor VIII produced byrecombinant DNA technology eliminates the risksassociated with blood transfusions.Immune system growth factors that stimulate leukocyteproduction; used to treat immune deficiencies and tofight infections.Stimulates erythrocyte production; used to treat anemiain patients with kidney disease.Stimulate differentiation and growth of various celltypes; used to promote wound healing.Used to treat dwarflsm.Used to treat diabetes.Interfere with viral reproduction; also used to treatsome cancers.Activate and stimulate different classes of leukocytes;possible uses in wound healing, HIV infection, cancer,immune deficiencies.Extraordinary binding specificity is used in diagnostictests.
Also used to transport drugs, toxins, orradioactive compounds to tumors as a cancer therapy;many other uses.Prevents tissue damage from reactive oxygen specieswhen tissues deprived of O2 for short periods duringsurgery suddenly have blood flow restored.Proteins derived from viral coats are as effective in"priming" an immune system as the killed virus moretraditionally used for vaccines, but are safer. Firstdeveloped was the vaccine for hepatitis B.10091010Part IV Information PathwaysErythropoietin is typical of the newer products. Erythropoietinisaprotein hormone (Mr 51,000) that stimulates erythrocyte production.People with kidney disease often have a deficiency of this protein, acondition that leads to anemia.
Erythropoietin produced by recombinant DNA technology can be used to treat these patients, reducing theneed for repeated blood transfusions and their accompanying risks.Approved by the U.S. Food and Drug Administration in 1989, erythropoietin promises to be the most profitable pharmaceutical agent developed by recombinant DNA methods in the 1990s.Other industrial applications of this technology are likely to continue developing. Enzymes produced by recombinant DNA technologyare already used to produce detergents, sugars, and cheese. Engineered proteins are being used as food additives to supplement nutrition, flavor, and fragrance.
Microorganisms are being engineered toextract oil and minerals from ground deposits, to digest oil spills, andto detoxify hazardous waste dumps and sewage. Engineered plantswith improved resistance to drought, frost, pests, and disease are increasing crop yields and reducing the need for agricultural chemicals.The potential of this technology to benefit humankind and the worldenvironment seems readily apparent yet sometimes hard to define,with the future rendered opaque by our still limited understanding ofcellular metabolism and ecology.Every major new technology comes with associated risks and apotential for unanticipated societal or environmental impact.
As withthe automobile and nuclear energy, economic, environmental, and ethical considerations will necessarily play an increasingly important rolein determining how recombinant DNA technology is applied. One harbinger of this new relationship between biochemistry and society hasbeen the debate in the United States and elsewhere over bovine growthhormone, which is used to increase milk production.
In addition tosome potential for added stress on the animals and concerns amongconsumers about the safety of the milk for human use, increasing milkproduction when a surplus already exists may have the effect of lowering prices and imposing economic hardship on dairy farmers.Other issues raised by this technology promise to have a muchbroader impact. A particularly clear example can be seen in an array ofnew diagnostic procedures based on recombinant DNA technology.These are greatly increasing our ability to detect genetic diseases in anindividual, often many years before the onset of symptoms or evenbefore birth.
The same technology that makes it possible to identify acriminal (Box 28-1) may be used to test individuals for a genetic predisposition to conditions such as Alzheimer's disease, hypercholesterolemia, asthma, and alcoholism. This information will permit better andearlier treatments, but the same information could be used to restrictindividual access to health insurance (and thus health care), life insurance, and even certain jobs. The questions of who will have access tothis information and how it will be used will grow in importance asmore tests become widely available.These are only some of the more straightforward examples. Release of genetically engineered organisms into the environment carrieswith it a level of risk that is sometimes difficult to evaluate.
Humangene therapy (Box 28-2), with all of its promise, doubtless will presentsociety with ethical dilemmas not yet anticipated. Issues of this kindmust in the end foster a closer and more productive collaboration between science and the society it serves, as well as higher levels of scientific literacy in the general public, as we move toward the twenty-firstcentury.Chapter 28 Recombinant DNA Technology1011SummaryThe study of gene structure and function has beengreatly facilitated by recombinant DNA technology. The isolation of a gene from a large chromosome requires methods for cutting and joiningDNA fragments, the availability of small DNA vectors that can replicate autonomously and intowhich the gene can be inserted, methods to introduce the vector with its foreign DNA into a cell inwhich it can be propagated to form clones, andmethods to identify the cells containing the DNA ofinterest.
Advances in this technology are revolutionizing many aspects of medicine, agriculture,and other industries.The first organism used for DNA cloning wasE. coli. Bacterial restriction endonucleases andDNA ligases provide the most important instruments for cutting DNA at specific sequences andjoining DNA fragments. Bacterial cloning vectorsinclude plasmids, bacteriophages, and cosmids.These permit the cloning of DNA fragments of different size ranges. In each case the vectors providea replication origin for propagation in the bacterialhost, and a selectable genetic element such as antibiotic resistance to facilitate the identification ofcells harboring the recombinant vector.
DNA is introduced into cells in viral vectors or by artificialmethods that make the cell wall permeable.The first step in cloning a gene is often the construction of a DNA library that includes fragmentsrepresenting most of the genome of a given species.The library can be limited to expressed genes bycloning only the complementary DNA copies of isolated mRNAs to make a cDNA library. A specificsegment of DNA can be amplified and cloned usingthe polymerase chain reaction.
Clones containing aspecific gene in a large library can be detected byhybridization with a radioactive probe containingthe complementary nucleotide sequence.Expression vectors provide the DNA sequencesrequired for transcription, translation, and regulation of cloned genes. They allow the production oflarge amounts of cloned proteins for research andcommercial purposes. Cloned genes also can be altered by site-directed mutagenesis, which is usefulin studies of protein structure and function.Yeast is sometimes used for cloning eukaryoticDNA and it has many of the same advantages asE.
coli. Methods for cloning in plants and animalsare producing a variety of organisms with alteredtraits. Plants that are resistant to disease, insects,herbicides, and drought are being produced withthe aid of a natural gene transfer process promotedby the Ti plasmid of the parasitic soil bacteriumAgrobacterium tumefaciens. Engineered DNA canbe introduced into animal cells by microinjection orretroviral vectors.
Such procedures have producedmice with new inheritable genetic traits. The technology extends to humans, and human gene therapy is now directed at treating human genetic diseases.GeneralSambrook, J., Fritsch, E.F., & Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2ndedn, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY.In addition to detailed protocols for a wide range oftechniques, this three-volume set includes muchuseful background information on the biological,chemical, and physical principles underlying eachtechnique.Further ReadingHackett, P.B., Fuchs, J.A., & Messing, J.W.
(1988)An Introduction to Recombinant DNA Techniques,2nd edn, The Benjamin/Cummings PublishingCompany, Menlo Park, CA.Jackson, D.A., Symons, R.H., & Berg, P. (1972)Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circularSV40 DNA molecules containing lambda phagegenes and the galactose operon of Escherichia coli.Proc. Natl. Acad. Sci. USA 69, 2904-2909.The first recombinant DNA experiment linkingDNA from two different organisms.Lobban, P.E. & Kaiser, A.D. (1973) Enzymaticend-to-end joining of DNA molecules. J. Mol. Biol.78, 453-471.Report of the first recombinant DNA experiment.Libraries and Gene IsolationArnheim, N.
& Levenson, C.H. (1990) Polymerasechain reaction. Chem. Eng. News 68 (October 1),36-47.A broad overview of the technique and applications.Arnheim, N. & Erlich, H. (1992) Polymerase chainreaction strategy. Annu. Rev. Biochem. 61, 131156.1012Part IV Information PathwaysErlich, H.A., Gelfand, D., & Sninsky, J.J. (1991)Recent advances in the polymerase chain reaction.Science 252, 1643-1651.This issue of Science contains several other goodarticles on other aspects of biotechnology.chromosomally female mice transgenic for Sry.Nature 351, 117-121.Recombinant DNA technology is used to demonstrate that a single gene directs the development ofchromosomally female mice into males.Neufeld, P.J. & Colman, N.
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