10 Генетическая инженерия (1160079), страница 6
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Auxins and cytokinins aregrowth hormones. The most common auxin is indoleacetate, derived from tryptophan. Cytokininsare adenine derivatives. Opines generally are derived from amino acid precursors; at least 14 different opines have been identified in different speciesof Agrobacterium.(b)AuxinsHOCH2HXCH 2 COO"CH2-NHCH 3HIndoleacetateZeatinCytokininsCH 3/CH 3Isopentenyl adenine(i6 Ade)OpinesH2NNHi-(CH2)3-C-COO-H.N 7ICH3—CH—COO"OctopineH2J\HHO—CH 2 —(CHOH) 4 - C H 2) CH— (CH2)3—C—COONHIH2N— C—(CH2)2—CH—COO"~OOC-(CH 2 ) 2 -CH— COO"NopalineOMannopinePart IV Information Pathways1004Agrobacterium cell(a)Ti plasmid withoutTDNA^j/•'(b)Recombinant plasmidwith foreign gene andkanamycin-resistancegene between T DNAbp repeatsbp repeatsKanamycin resistanceForeign geneThe integration of the T DNA into a plant chromosome providesthe vehicle necessary to introduce new genes into plants.
The transferof T DNA from Agrobacterium to the plant cell nucleus is mediated bytwo 25 base pair repeats that flank the T DNA and by the products ofseveral genes, called virulence (vir) genes, located elsewhere on the Tiplasmid (Fig. 28-15).The bacterial system that transfers T DNA into the plant genomecan be harnessed to transfer recombinant DNA instead. A commoncloning strategy employs an Agrobacterium that contains two differentrecombinant plasmids (Fig. 28-17).
The first is a Ti plasmid fromwhich the T DNA segment has been removed in the laboratory. Thesecond is an Agrobacterium-E. coli shuttle vector that contains theT-DNA 25 base pair repeats flanking the gene that a researcher wantsto introduce and a selectable marker (often a gene that can renderplant cells resistant to an antibiotic such as kanamycin).
The engineered Agrobacterium is used to infect a leaf (Fig. 28-17). Crown gallsare not formed because the genes for auxin, cytokinin, and opine biosynthetic enzymes are not present on either plasmid. Instead, the virgene products from the altered Ti plasmid direct the transformation ofthe plant cells by the gene flanked by the T-DNA 25 base pair repeatsin the second plasmid. The transformed cells can be selected by growthon kanamycin and induced with growth hormones to form new plants.The successful transfer of recombinant DNA into plants is vividlyillustrated by an experiment in which the luciferase gene from fireflieswas introduced into a tobacco plant (Fig. 28-18).
Note that althoughtobacco is not high on the list of beneficial plants needing improvement, it is often used experimentally because it is particularly easy totransform with Agrobacterium.The potential of this technology is not limited to the production ofglow-in-the-dark plants. The same approach has been used to produceBacteria invade at woundsites (where leaf is cut).Leaf segments aretransferred to agar dish.—'-jr Agar plate with growthhormones and kanamycinThese kanamycinresistant plantscontain the foreign gene.Plants are regeneratedfrom leaf segments.Figure 28-17 A two-plasmid strategy to create arecombinant plant. One plasmid (a) is the Ti plasmid, modified so that it lacks T DNA.
The otherplasmid (b) contains the gene of interest (e.g., thegene for the insect-killing protein described in Fig.28-19) and an antibiotic-resistance (here, kanamycin-resistance) element flanked by T-DNA 25base pair repeats. This plasmid also contains thereplication origin needed for propagation in Agrobacterium. The bacteria invade at the site of awound (the edge of the cut leaf). The vir genes onplasmid (a) mediate transfer into the plant genomeof the segment of plasmid (b) flanked by the 25base pair repeats.
New plants are generated whenthe leaf (with transformed cells) is placed on anagar dish with controlled levels of plant growthhormones and kanamycin. Nontransformed plantcells are killed by the kanamycin. The gene of interest and the antibiotic-resistance element are normally transferred together, and thus plants thatgrow in the presence of this antibiotic generallycontain the gene.Chapter 28 Recombinant DNA TechnologyFigure 28-18 A tobacco plant in which the genefor firefly luciferase is expressed.
Light was produced after the plant was watered with a solutioncontaining luciferin, the substrate for this lightproducing enzyme (see Box 13-3, Fig. 2). Don't expect glow-in-the-dark ornamental plants at yourlocal nursery anytime soon; the light is actuallyquite weak and this photograph represents a 24hour exposure. The real point—that this technologyallows the introduction of new traits into plants—isnevertheless elegantly made.plants that are resistant to herbicides, plant viruses, and insect pests(Fig.
28-19). Potential benefits include increases in yields and a reduction in the need for environmentally harmful agricultural chemicals.Cloning in Animal Cells Points theWay to Human Gene TherapyThe transformation of animal cells with foreign genetic material offersan important mechanism for advancing knowledge about the structureand function of animal genomes, as well as for the generation of animals with new traits. This potential has spawned intensive researchefforts that have produced increasingly sophisticated means for cloning in animals.Most work of this kind requires a source of individual cells. Intacttissues are often difficult to keep alive and manipulate.
Fortunately,many types of animal cells can be isolated and grown in a medium inthe laboratory if their growth requirements are carefully met. Manycells grown in this kind of tissue culture maintain the differentiatedproperties they had in the whole tissue for weeks or even months.There are several common methods for introducing DNA intoan animal cell.
Because no suitable plasmidlike vector is available,transformation requires the integration of the DNA into a host-cellchromosome.In spontaneous uptake, a calcium phosphate-DNA precipitateis taken up by the cells, perhaps by endocytosis. Generally only aboutone in 102 to 104 cells is transformed in this procedure. Another directmethod that is sometimes more efficient is to make the cells transiently permeable to DNA by exposing them to a brief high-voltagepulse in a technique called electroporation.Figure 28-19 Tomato plants engineered to be resistant to some insect larvae. The plant on theright expresses a gene for a protein toxin, derivedfrom the bacterium Bacillus thuringiensis. The protein, introduced by a protocol similar to that described in Fig.
28-17, is toxic to the larvae of somemoth species that destroy tomato leaves. This protein, however, is harmless to humans and otherorganisms. The plant on the left is not geneticallyaltered. Both plants were exposed to equal numbersof larvae. Insect resistance has also been genetically engineered in cotton and other plants.100.1006Part IV Information PathwaysRetroviral genome (single-stranded RNA)polLTRgagenvDNARecombinant defectiveretroviral DNAi4LTRReverse transcriptaseconverts RNA genometo double-stranded DNA.Viral genes are replacedwith a foreign gene.•iRecombinant DNA isintroduced into cellsin tissue culture.RNA copies ofrecombinant virusesare produced in cellscontaining a helpervirus andpackaged intoviral particles.Reverse transcriptaseand integraseRetroviral RNAgenome with foreign geneRetroviral genomewith foreign geneis integrated intothe target cellchromosome.Recombinant virusparticles infect atarget cell.Figure 28-20 €loning in mammalian cells withretroviral vectors.Microinjection entails direct injection of DNA into the nucleus ofa cell with the aid of a very fine needle.
For skilled practitioners thismethod has a high success rate, but because cells must be injected oneby one, the total number that can be treated is small.A number of eukaryotic viruses sometimes integrate their DNAinto a chromosome in the host cell. Some of these, in particular certainretroviruses (p. 882), have been modified to act as viral vectors tointroduce foreign DNA into mammalian cells. The viruses have theirown mechanisms for moving nucleic acid into cells, and transformationby this route can be very efficient.
A simplified map of a typicalretroviral genome is shown in Figure 28-20. When the virus enters acell, its RNA genome is converted to DNA by reverse transcriptase andis then integrated into the host genome in a reaction mediated by theviral integrase. The long terminal repeat (LTR) sequences are requiredfor integration of retroviral DNA in the host chromosome (see Fig.25-30), and the i// sequence is required to package the viral RNA inviral particles.The gag, pol, and env genes of the retroviral genome can be replaced with foreign DNA. This recombinant DNA lacks the genes required for retroviral replication and assembly of viral particles. To assemble viruses with the recombinant genetic information, the DNAmust be introduced into cells in tissue culture that are infected with ahelper virus, which has the genes to produce virus particles but lacksthe \\) sequence required for packaging.
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