10 Генетическая инженерия (1160079), страница 9
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(1990) When sciencetakes the witness stand. Sci. Am. 262 (May), 4 6 53.Potential and problems of DNA fingerprinting.McLachlin, J.R., Cornetta, K., Eglitis, M.A., &Anderson, W.F. (1990) Retroviral-mediated genetransfer. Prog. Nucleic Acid Res. Mol. Biol. 38, 91135.Paabo, S., Higuchi, R.G., & Wilson, A.C. (1989)Ancient DNA and the polymerase chain reaction.The emerging field of molecular archaeology.J. Biol. Chem. 264, 9709-9712.Murray, T.H. (1991) Ethical issues in human genome research. FASEB J.
5, 55-60.This issue also contains a number of other usefulpapers on the human genome project.Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gelelectrophoresis. J. Mol. Biol 98, 503-517.The paper that introduced the Southern hybridization method.Palmiter, R.D., Brinster, R.L., Hammer, R.E.,Trumbauer, M.E., Rosenfeld, M.G., Birnberg,N.C., & Evans, R.M. (1982) Dramatic growth ofmice that develop from eggs microinjected withmetallothionein-growth hormone fusion genes.Nature 300, 611-615.A description of how to make giant mice.Thornton, J.I.
(1989) DNA profiling: new tool linksevidence to suspects with high certainty. Chem.Eng. News 67 (November 20), 18-30.Products of RecombinantDNA TechnologyBailey, J.E. (1991) Toward a science of metabolicengineering. Science 252, 1668-1675.An overview of efforts to reengineer entire metabolicpathways in microorganisms for commercial purposes.Bains, W. (1989) Disease, DNA and diagnosis. NewScientist 122 (May 6), 48-51.Botstein, D.
& Shortle, D. (1985) Strategies andapplications of in vitro mutagenesis. Science 229,1193-1201.Buck, K. (1989) Brave new botany. New Scientist122 (June 3), 50-55.Prospects for genetic engineering in plants.Culver, K.W., Osborne, W.R.A., Miller, A.D.,Fleisher, T.A., Berger, M., Anderson, W.F., &Blaese, R.M.
(1991) Correction of ADA deficiencyin human T lymphocytes using retroviral-mediated gene transfer. Transplant Proc. 23, 170—171.Gene therapy of adenosine deaminase deficiency.Hooykaas, P.J.J. & Schilperoort, R.A. (1985) TheTi-plasmid of Agrobacterium tumefaciens: a natural genetic engineer. Trends Biochem. Sci. 10, 307309.Johnson, I.S. (1983) Human insulin from recombinant DNA technology. Science 219, 632-637.Koopman, P., Gubbay, J., Vivian, N., Goodfellow,P., & Lovell-Badge, R.
(1991) Male development ofSulston, J., Du, Z., Thomas, K., Wilson, R., Hillier, L., Staden, R., Halloran, N., Green, P., Thierry-Mieg, J., Qiu, L., Dear, S., Coulson, A.,Craxton, M., Durbin, R., Berks, M.,Metzstein, M., Hawkins, T., Ainscough, R., &Waterston, R. (1992) The C. elegans genome sequencing project: a beginning. Nature 356, 37-41.A description of strategies being developed to sequence large genomes.The telltale gene. Consumer Reports 55 (July1990), 483-488.A good summary of new genetic screening techniques based on recombinant DNA, with a focus onethical dilemmas posed by the technology.Thompson, J. & Donkersloot, J.A.
(1992) N(Carboxyalkyl)amino acids: occurrence, synthesis,and functions. Annu. Rev. Biochem. 61, 517-557.A good summary of the structure and biologicalfunctions of opines.Verma, I.M. (1990) Gene therapy. Sci. Am. 263(November), 68-84.Watson, J.D. (1990) The human genome project:past, present, and future. Science 248, 44-48.Weatherall, D.J. (1991) Gene therapy in perspective. Nature 349, 275-276.Westphal, H.
(1989) Transgenic mammals and biotechnology. FASEB J. 3, 117-120.Zambryski, P., Tempe, J., & Schell, J. (1989)Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56,193-201.Chapter 28 Recombinant DNA Technology1013Problems1. Cloning(a) Draw the structure of the end of a linearDNA fragment that was produced by an EcoRI restriction digest (include those sequences remaining from the EcoRl recognition sequence).(b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase Iand the four deoxynucleoside triphosphates.(c) Draw the sequence produced at the junctionif two ends with the structure derived in (b) areligated.(d) Draw the structure produced if the structurederived in (a) is treated with a nuclease that degrades only single-stranded DNA.(e) Draw the sequence of the junction producedif an end with structure (b) is ligated to an endwith structure (d).(f) Draw the structure of the end of a linearDNA fragment that was produced by a Pvull restriction digest (as in (a)).(g) Draw the sequence of the junction producedif an end with structure (b) is ligated to an endwith structure (f).(h) Suppose you can synthesize a short duplexDNA fragment with any sequence you desire.
Withsuch a synthetic fragment and the procedures described in (a) through (g), design a protocol thatwill remove an Eco RI restriction site from a DNAmolecule and incorporate a new BamHl restrictionsite at approximately the same location. (Hint: SeeFig. 28-3.)(i) Design four different short synthetic DNAfragments that would permit ligation of structure(a) with a DNA fragment produced by a Pstl restriction digest. In one of these synthetic fragments, design the sequence so that the final junction contains the recognition sequences for bothEcoRl and Pstl. In the second and third syntheticfragments, design the sequence so that the junction contains only the EcoRl or the Pstl recognitionsequence, respectively.
Design the sequence of thefourth fragment so that neither the Eco RI nor thePstl sequence appears in the junction.2. Selecting for Recombinant Plasmids Whencloning a foreign DNA fragment into a plasmid it isoften useful to insert the fragment at a site thatinterrupts a selectable marker (such as the tetracycline-resistance element of pBR322). The loss offunction of the interrupted gene can be used toidentify clones containing recombinant plasmidswith foreign DNA.
With a cosmid it is unnecessaryto do this, yet one can easily distinguish cosmidsthat incorporate large foreign DNA fragmentsfrom those that do not. How are these recombinantcosmids identified?3. DNA Cloning The plasmid cloning vectorpBR322 (see Fig. 28-5) is cleaved with the restriction endonuclease EcoRl. An isolated DNA fragment from a eukaryotic genome (also produced byEcoRl cleavage) is added to the prepared vectorand ligated. The mixture of ligated DNAs is thenused to transform bacteria, and plasmid-containing bacteria are selected by growth in the presenceof tetracycline. In addition to the desired recombinant plasmid, what other types of plasmids mightbe found among the transformed bacteria that aretetracycline resistant?4. Expressing a Cloned Gene You have isolateda plant gene that encodes a protein in which youare interested.
On the drawing below, indicatesequences or sites that you will need to get thisgene transcribed, translated, and regulated inE. coli.Stop codonAUG5. Identifying the Gene for a Protein with a KnownAmino Acid Sequence Design a DNA probethat would allow you to identify the gene for a protein with the following amino-terminal amino acidsequence. The probe should be 18 to 20 nucleotideslong, a size that provides adequate specificity ifthere is sufficient homology between the probe andthe gene.H 3 N-Ala-Pro-Met-Thr-Trp-Tyr-Cys-Met-AspTrp-Ile-Ala-Gly-Gly-Pro-Trp-Phe-Arg-LysAsn-Thr-Lys6.
Cloning in Plants The strategy outlined inFigure 28-17 employs Agrobacterium containingtwo separate plasmids. Suggest a reason why thesequences on the two plasmids are not combinedon one plasmid.7. Cloning in Mammals The retroviral vectorsdescribed in Figure 28-20 make it possible to integrate foreign DNA efficiently into a mammaliangenome.
Explain how these vectors, which lackgenes for replication and viral packaging (gag, pol,env), are assembled into infectious viral particles.Suggest why it is important that these vectors lackthe replication and packaging genes.Glossaryabsolute configuration: The configuration of four different substituent groupsaround an asymmetric carbon atom, inrelation to D- and L-glyceraldehyde.absorption: Transport of the products ofdigestion from the intestinal tract into theblood.acceptor control: The regulation of therate of respiration by the availability ofADP as phosphate group acceptor.accessory pigments: Visible lightabsorbing pigments (carotenoids, xanthophyll, and phycobilins) in plants and photosynthetic bacteria that complement chlorophylls in trapping energy from sunlight.acidosis: A metabolic condition in whichthe capacity of the body to buffer H+ isdiminished; usually accompanied by decreased blood pH.actin: A protein making up the thin filaments of muscle; also an important component of the cytoskeleton of many eukaryotic cells.activation energy (AG*): The amount ofenergy (in joules) required to convert allthe molecules in 1 mole of a reacting substance from the ground state to the transition state.activator: (1) A DNA-binding protein thatpositively regulates the expression of oneor more genes; that is, transcription ratesincrease when an activator is bound tothe DNA.
(2) A positive modulator of anallosteric enzyme.active site: The region of an enzyme surface that binds the substrate molecule andcatalytically transforms it; also known asthe catalytic site.active transport: Energy-requiringtransport of a solute across a membranein the direction of increasing concentration.activity: The true thermodynamic activityor potential of a substance, as distinctfrom its molar concentration.activity coefficient: The factor by whichthe numerical value of the concentrationof a solute must be multiplied to give itstrue thermodynamic activity.acyl phosphate: Any molecule with thegeneral chemical form R—C—OPO§~.0adenosine 3',5'-cyclic monophosphate:See cyclic AMP.adenosine diphosphate: See ADP.adenosine triphosphate: See ATP.adipocyte: An animal cell specialized forthe storage of fats (triacylglycerols).adipose tissue: Connective tissue specialized for the storage of large amounts oftriacylglycerols.ADP (adenosine diphosphate): A ribonucleoside 5'-diphosphate serving as phosphate group acceptor in the cell energycycle.aerobe: An organism that lives in air anduses oxygen as the terminal electron acceptor in respiration.aerobic: Requiring or occurring in thepresence of oxygen.alcohol fermentation: The anaerobicconversion of glucose to ethanol via glycolysis.
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