The Biotechnology Revolution; its Relevance to the Biological and Toxin ... The biotechnology revolution actually began with three successive events in the.
THE BIOTECHNOLOGY REVOLUTION: THE SCIENCE AND APPLICATIONS
KATHRYN NIXDORFF and JENS BRAUBURGER Department of Microbiology and Genetics Darmstadt University of Technology Schnittspahnstr. 10 D-64287 Darmstadt Germany DORTE HAHLBOHM Department of Political Science Darmstadt University of Technology Markplatz 15 Residezschloss D-64283 Darmstadt Germany
1. The Biotechnology Revolution; its Relevance to the Biological and Toxin Weapons Convention
Biotechnology is a very old technology spanning a period of over 5000 years. In the earliest phases, it was concerned primarily with fermentation processes leading to bread-making as well as to the production of alcoholic beverages and cheese. With the recognition that microorganisms and their products were responsible for such processes and the establishment of microbiology as a science towards the end of the ninteenth century, biotechnology indeed acquired a scientific character. At this point the term biotechnology encompassed: 1' 2 "the use of living organisms or enzymes in the technically regulated production of organic substances." Biotechnology has since been revolutionized by molecular biology and genetic engineering, and any modern account has to take these developments into consideration. Molecular biology can be regarded as the molecular approach to studying biology, which arose· in the 1960's out of studies in bacterial genetics, biochemistry and physiology, leading to an advanced understanding of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis. 77 M. Dando et al. (eds), Verification ~(the Biological and Toxin Weapons Convention, 77-124. ©2000 Kluwer Academic Publishers.
78 The biotechnology revolution actually began with three successive events in the biological sciences. The first was the discovery in the mid 1940's to the early 1950's of natural genetic exchange in bacteria, which led to the recognition of DNA as the carrier of genetic information. This was followed by the establishment of the structure of DNA and the deciphering of the genetic code. The third event occurred in the early 1970's with the first successful recombinant DNA experiment, which led to the rapid development of genetic engineering, the method that concerns the artificial manipulation of genes and their products.3,J Today, biotechnology is highly dependent on molecular biology and genetic engineering. This is certainly reflected in the description of biotechnology in Article 2 of the Convention on Biological Diversity: 4 '"Biotechnology' means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." This broad description includes the production of derivatives of living organisms and places emphasis on modifications of products or processes, which of course refers to the application of genetic engineering or gene modification techniques. The broad term "products" can be interpreted to include living organisms. At the time the Convention on the Prohibition ofthe Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction (BTWC) 5 was negotiated in 1972, biological weapons were perceived then to be relatively impractical from a military viewpoint ,6·7 and the Convention was rapidly concluded without the incorporation of adequate provisions for verification. 8 The revolution in biotechnology was just getting underway when the Convention was brought into force in 1975. The first successful genetic engineering experiment, in which plasmid genes from one bacterium (Staphylococcus aureus) were transferred to and expressed in another unrelated bacterium (Escherichia coli), was carried out shortly after the conclusion of the BTWC. 9 It was quite apparent a few years later that this new development was perceived as a potential threat to biological weapons control. 10' 11 As a result of this awareness, activities in the area of defense research were increased at what seemed to some observers to be an exponential rate. 12 Concomitantly, there was concern that the BTWC did not cover the production of novel agents, although the wording in Article I of the Convention (Figure I) that encompasses "Microbial or other biological agents, or toxins whatever their origin or method of production" is very comprehensive in its formulation. The issue was addressed at the Second Review Conference of the BTWC in 1986. In the Final Declaration, 13 it was stated that the Conference: "reaffirms that the Convention unequivocally applies to all natural or artificially created microbial or other biological agents, or toxins, whatever their origin or methods of production."
79 The Final Declaration of the Third Review Conference 14 reaffirmed this position and added the word "altered" in stating that: "The Conference, conscious of apprehensions ansmg from relevant scientific and technological developments, inter alia, in the fields of microbiology, genetic engineering and biotechnology, and the possibilities of their use for purposes inconsistent with the objectives and the provisions of the Convention, reaffirms that the undertaking given by the States Parties in Article I applies to all such developments. The Conference also reaffirms that the Convention unequivocally covers all microbial or other biological agents or toxins, naturally or artificially created or altered, whatever their origin or method of production." In the Final Declaration of the Fourth Review Conference, 15 this position was essentially reaffirmed, with the addition of the words "as well as their components" to state that: "The Conference also reaffirms that the Convention unequivocally covers all microbial or other biological agents or toxins, naturally or artificially created or altered, as well as their components, whatever their origin or method of production, of types and in quantities that have no justification for prophylactic, protective or other peaceful purposes." A further extention applying to Article I from this Conference states that: "The Conference, conscious of apprehensions arising from relevant scientific and technological developments, inter alia, in the fields of microbiology, biotechnology, molecular biology, genetic engineering, and any applications resulting from genome studies, and the possibilities of their use for purposes inconsistent with the objectives and the provisions of the Convention, reaffirms that the undertaking given by the States Parties in Article I applies to all such developments." Thus, the coverage of developments in biotechnology and genetic engineering by the Convention has been extensively examined in the last three Review Conferences, and the affirmations in the Final Declarations of these Conferences represent a clear agreement by the States Parties that the wording in Article I of the BTWC (Figure 1) is explicit in encompassing organisms and toxins that may be created or altered by processes involving gene modifications, as well as those that may be produced or altered by biomolecular engineering. It is accordingly a resounding attest to the strength of Article I in standing the test of time. A more comprehensive review of the scope of Article I can be found in. 16 As in the two previous Review Conferences, background papers from the States Parties on new
80 scientific and technological developments relevant to the Convention were solicited preparatory to the Fourth Review Conference. A compilation of these papers 17 was available to the delegates for discussion. Specific points covered in the various papers will be referred to in later sections dealing with the different technologies. A general theme of the background papers is the continued concern about possible adverse effects the new technologies may have on compliance to the prohibitions and provisions set out in the BTWC, which has also been expressed in a recent account. 18 This continued concern is certainly one aspect that has precipitated concrete negotiations on a verification protocol to the BTWC. Article I Each State Party to this Convention undertakes never in any circumstances to develop, produce, stockpile or otherwise acquire or retain: I. Microbial or other biological agents, or toxins whatever their origin or method of production, of types and in quantities that have no justification for prophylactic, protective or other peaceful purposes; 2. Weapons, equipment or means of delivery designed to use such agents or toxins for hostile purposes or in armed conflict. Figure 1. Article I of the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction. Source: Ref. 5
The aim of the present paper is to discuss how the biotechnology revolution has both positive and negative effects on verification of the BTWC. For this purpose, the following biotechnology categories will be considered: Genetic modifications. This section will compare genetic modifications of microorganisms that occur in nature with those that can be accomplished by genetic engineering, in order to show limitations of natural processes and the possibilities that manipulative procedures can provide. Genetic engineering of plants will also be discussed because of the possibilities of changing the susceptibility of plants to insects and plant pathogens by gene technologies. Biomolecular engineering. This is the technology to design and produce proteins (structural proteins, enzymes) with specific, tailored properties. Metabolic engineering. This category involves the manipulation of complex biosynthetic pathways to improve metabolic activites of the cell. Bioproduction technologies. The processes that will be dealt with in this category include the cultivation of living cells as well as the use of living cells to manufacture products in practical quantities. Basic aspects of these biotechnologies will be discussed in order to set the stage for the consideration of some applications of the technologies that have
81 particular relevance to the BTWC and its verification. Accordingly, applications will be dealt with in the framework of dual-use considerations and will include the following topics: Targeted delivery systems. These systems are composed of agents that have been manipulated to direct or target them to specific sites in the body where their activities are desired. Vaccine development. The use of biotechnology in vaccine production will be discussed. Biosensors and diagnostic reagents. Biosensors encompassing the linkage of biomolecules to electronic, photonic, or mechanical systems will be covered in a chapter on Sensors in this volume, so that the discussion here will be limited to the use of antibodies and gene probes as diagnostic reagents. Modifications of microorganisms. Concerns that the new technologies may be used in a negative way to increase biological warfare capabilities will be considered in this section. These are biotechnology categories and applications that have also been designated as relevant in other discussions concerning the BTWC and its verification. 17 ' 18 ' 19
2. Biotechnology: The Science 2.1. GENETIC MODIFICATION
2.1.1. Natural Gene Flow and Modification Among Microorganisms When considering the question of genetic modification and its impact on biological weapons control, the topic of natural gene flow among microorganisms has to be addressed. Gene flow means simply the movement or transfer of genetic material in the form of DNA from one microorganism to another. In order to facilitate the discussion, a few basic facts about genes and what they determine should be recalled. Very simply put, DNA is the carrier of genetic information in the form of genes. Generally, a gene contains the information for the biosynthesis of a specific protein, which is either a structural protein or an enzyme needed to catalyze a reaction involved in the the biosynthesis of proteins themselves or also non-proteinaceous materials and products of a cell. This information is contained in the DNA in the form of the genetic code, reflected in the order (sequence) of the nucleotide bases or building blocks in the molecule, which in turn determines the order of the incorporation of the amino acid building blocks into the protein during its synthesis, giving the protein specific structure, characteristics, and function. Essential for the integrity of the DNA in its double helix form (Figure 2) 20 is the pairing of the nucleotide bases adenine with thymine and guanine with cytosine (Watson-Crick base pairing).Z 1 The general scheme of protein biosynthesis can be put simply as the synthesis of a single-stranded messenger RNA (mRNA) from the double-
82 stranded DNA, a process called transcription, followed by the translation of that mRNA into protein on ribosomes, the so-called protein factories in the cell. Figure 3 depicts the process of transcription and translation schematically. Accordingly, genes determine the properties of a cell and its products, and when genes are transferred from one organism to another, the ability to express these properties will be transferred. Whether the recipient organism can use the transferred genes to synthesize new proteins and thus acquire new properties depends, however, upon the rules governing genetic exchange, gene regulation, and protein biosynthesis, some of which will be discussed briefly in the framework of natural exchange of genetic material or genes among microorganisms, using bacteria as an example.
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c -- c
H ·• N
I 0 -.. H - N 11 ,
' c .,. , 1 c-c \ / N -cf N-
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Figure 2. Structure of deoxyribonucleic acid (DNA). The two single strands of DNA are held together through hydrogen bonding. Left is the DNA double helix. Right is the pairing of the bases adenine with thymine and guanine with cytosine (Watson-Crick base pairing); the dashed lines symbolize hydrogen bonding. Source: Hans G, Schlegal, Allgemeine Mikrobiologie, 7th Edition, Figure 2.10, p.29, © 1992, used with kind permission from Georg Thieme Verlag.
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Figure 3. Schematic representation of the synthesis of messenger RNA (mRNA) from DNA in the process called transcription, and the translation of mRNA into a peptide, which subsequently folds into the native protein molecule. Gly, Arg, He, Met, Val Pro, Thr: abbreviations for some amino acids that are the building blocks of the peptide chain. Only these amino acids are shown in the peptide chain for illustration.
84 Bacteria are single cells with a relatively simple type (prokaryotic) of internal cell structure. A major difference between these cells and those of higher organisms (eukaryotic) is the arrangement of DNA. Higher cell forms contain a nucleus enclosed by a nuclear membrane that contains several DNA molecules that form chromosomes. The DNA in prokaryotic cells is not surrounded by a membrane and consists of a single DNA molecule (chromosome or genome) in the form of a closed circle. 1 Some bacteria also contain DNA apart from the genome in the form of plasmids, which are smaller molecules of circular DNA that replicate autonomously, using the biosynthesis machinery of the bacterial cell. Genetic exchange among bacteria occurs naturally in the processes of conjugation, transformation, and transduction.
Conjugation. Conjugation is the process of transfer of genetic information from a donor to a recipient by means of a plasmid (conjugative plasmid) that has the ability to mobilize itself from one microorganism to another (Figure 4 ). The process is called conjugation because tight cell contact between donor and recipient is a requirement and it is believed that DNA is passed from one cell to another over a site coupling or bridging the two cells together. Contact is initially established by a hair-like projection called a pilus (Latin for "hai-r") on the donor. Because such a structure is involved in the exchange of genetic material from a bacterium that posseses the pilus to one that does not, the pilus has been termed a "sex" pilus. The ability to undergo conjugation resides in the genetic makeup of the conjugative plasmid, and 20-30 genes, such as the one directing the biosynthesis of the sex pilus, have been identified as essential for transfer. The conjugation model (Figure 4) suggests that when contact has been established, one strand of the plasmid DNA is split, a rolling circle mechanism of DNA replication is initiated, and the displaced strand is transferred to the recipient, where it is copied to form the double-stranded plasmid. 1 The genes transferred with the plasmid will be activated (expressed) in the recipient, so that the recipient acquires the traits encoded by those genes. Usually, this exchange occurs most readily among bacteria that belong to similar groups, similar genera or similar species, but there are numerous examples in the literature documenting natural conjugation between distantly related bacteria. 22 One salient feature of this type of genetic exchange is that integration of the plasmid DNA into the bacterial chromosome is not necessary for expression of the genes contained in the plasmid and biosynthesis of the respective proteins. Therefore, strict DNA sequence homology (identity) between the two DNA types is not required. Still, the biosynthetic machinery of the bacterial cell has to be able to react with the plasmid DNA and respond to its direction. The most prominant barriers to successful conjugation among different organisms are posed by this requirement and also the ability of some recipients to destroy the incoming DNA of the donor through the possession of restriction endonucleases (restriction enzymes) that can degrade the DNA of the donor (Figure 5). Studies have shown, however, that conjugation is a relatively non-specific process and accounts for most of the horizontal gene transfer between even phylogenetically
85 F. Donor
Nickmg of supercoiled plasm1d DNA and bmding of protein to s· terminus
replication and transfer of displaced strand
( ©-)0 )
Copying of transferred strand m recipient
Completion of transfer ot displaced strand
Fif?ure 4. Conjugation, the process of transfer of genetic information from a donor to a recipient bacterium by means of a plasmid. Pictured is a model for the transfer ofF (for fertility) plasmid DNA from an F'" donor cell to an F recipient by a looped rolling circle mechanism. The displaced single strand is transferred and converted into double-stranded DNA in the recipient cell. Source: Ref. 21 used with permission from Jones and Bartlett Publishers.
unrelated organisms. 23 Conjugation is probably the main mechanism of the acquisition of multiple antibiotic resistance among bacteria, especially in situations where selective pressures exist, such as in hospitals, or where antibiotics are prominantly in use.
(a) Cuts on line of symmetry
(b) Cuts symmetrically placed around line of symmetry
. .. GAAITTC ... ... CTT:AA~
... TCIGA ... ... AG:~
... TC ... AG
Separation of fragments
GA .. . CT .. .
... G ... CTTAA
t Separation of fragments
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Figure 5. Two types of cut~ made by restriction enzymes (restriction endonucleases) at specified sites in DNA. The arrows indicate cleavage sites. The dashed line is the center of symmetry of the sequence. Source: Ref. 21, used with permission from Jones and Bartlett Publishers.
Transformation. Natural transformation is the active uptake of free DNA by a bacterium from the surrounding environment and the integration of this DNA into the bacterial genome. In this process DNA fragments released into the environment are taken taken up by what is called "competent" bacterial cells. Only double-stranded DNA fragments can participate in this process, but apparently only one strand survives enzymatic degradation during uptake into the cell. This single-stranded fragment is integrated into the genome of that cell.' Competence is strictly regulated and determined by genes on the bacterial genome. It is developed only under certain physiological conditions, mostly in the transition between the exponential (early) and stationary (late) phases of growth. Furthermore, not all bacteria of even the same species can be transformed, and the conditions leading to competence induction are varied for
87 different species. Natural transformation is not as wide-spread among bacteria as conjugation, and although transformation can occur across species and even higher taxonomic barriers, it is mostly limited to the passage of chromosomal genes among members of the same genus and especially the same species. 24 To date transformation has been described in over 40 species of bacteria, which represents only a very limited number. There are strong barriers to this process, in addition to the action of restriction enzymes. One is the ability to acquire competence, and another is dictated by the requirement for integration of the "foreign" piece of DNA into the genome of the recipient bacterium before the genes present in the DNA fragment can be expressed. The single-stranded DNA is integrated into the genome by the process of homologous recombination, which requires almost perfect nucleotide base sequence homology (match) between the DNA to be integrated and that of the recipient. Divergencies in nucleotide base sequence of only 10 to 20 percent are apparently not tolerated. 23
Transduction. In the process of transduction, bacterial viruses (bacteriophages or phages) mediate the transfer of genetic information between bacteria. This can be accomplished in two ways. In generalized transduction some of the DNA of the host bacterium gets packaged into the newly formed phage particle by mistake and is carried over to the next bacterium that the phage infects. Generally, DNA in the infected bacterium requires recombination of that DNA with the bacterial genome. In specialized transduction, the phage is one that can integrate its DNA into the bacterial genome. When, in response to a particular signal, the phage DNA is excised from (cut out of) the bacterial genome, the process is not always perfect and some bacterial DNA on each side of the phage DNA is excised with it. This bacterial DNA is subsequently packaged into the phage particle and is carried over by infection to the next bacterium. Transducing phages have been described for a large number of species in 25 different genera of bacteria. The initial interaction between bacterium and phage is dependent upon very specific receptors found only on the cell surface of particular species and strains of bacteria. Therefore, although phages are widespread in the environment, they play only a minor role in interspecies gene transfer because of their specificity. 23 2.1.2. Genetic Engineering Genetic engineering is a technique that allows the artificial modification and transfer of genetic material to an organism. Through the processes of conjugation, transformation and transduction, transfer of genes from one organism to another and expression of those transferred genes in the recipient occurs naturally among bacteria. However, through the application of genetic engineering, the process can occur more rapidly and with much greater precision. Furthermore, it can overcome some barriers and restraints placed on natural genetic exchange among organisms. Genetic Engineering of Bacteria. In order to transfer a foreign gene to a bacterium, it is placed in a carrier or vector, which is usually a plasmid, although bacteriophages can be used as vectors in specific cases. Enzymes referred to
88 above, restriction endonucleases or restriction enzymes (Figure 5), can cut DNA in designated regions (called restriction sites), depending on the nucleotide base sequence. With such enzymes, a designated piece can be cut out of the foreign DNA, and another enzyme (DNA ligase) can join this fragment to a plasmid vector that has been opened with the same restriction enzyme (Figure 6). Usually, for safety reasons, plasmids that can not conjugate or move from one organism to another are used as vectors, to prevent horizontal spread of the transferred plasmid to other organisms. Transformation (also called transfection) of a bacterium with the vector can be effected artificially through certain chemical and physico-chemical procedures, and a clone of the plasmidcontaining cell can be isolated (Figure 6). If the new gene functions, the clone acquires the property afforded by the gene. The plasmid replicates along with the bacterial cell, and is transferred vertically to the next generation in that clone at cell division. Genetic Engineering of Viruses. The genetic material of viruses can also be manipulated, but a different strategy is necessary. Viruses are placed in the category of microorganisms. They are infectious, but strictly speaking, they are not living beings. They are simply particles of nucleic acid (the viral genome) that is usually packaged in a protein coat or lipid-containing envelope. In some cases viruses may also contain certain enzymes that aid in ·their replication. Viruses cannot reproduce themselves because they do not possess the biosynthetic machinery of living cells. The virus must infect a cell (host) and redirect the biosynthetic capacity and metabolic functions of the host that are necessary for virus replication; the host synthesizes the viral genome and the envelope or coat proteins to make new virus particles.' The genetic manipulation of vaccinia virus, the virus used for smallpox vaccination, will be given as an example (Figure 7), although it must be emphasized that it is difficult to generalize because of the different types of viruses and various schemes of viral multiplication. In the case of vaccinia, the gene that is to be transferred is built into a plasmid (recombination vector). The gene is constructed to contain short pieces of DNA homologous to the vaccinia genome flanking both of its ends, thus assuring that the new gene will be integrated into the viral genome by homologous recombination. The plasmid is artificially transfected into host cells in vitro, and the virus is allowed to infect these cells. Inside the host cell, a homologous recombination of the new gene with the vaccinia genome will occur. 25 Thereafter, the virus carries the new gene, which will be expressed in the host cell along with the expression of the vaccinia genome. At this point it might be mentioned that vaccinia virus has a large genome that will allow insertion of up to 20 kilobases of DNA. Furthermore, it induces a strong immune response in the host. Therefore, it was perceived as a likely carrier of multiple foreign antigen genes whose products, when expressed in the host, would lead to effective immune responses. It was speculated that protection against multiple pathogens could be achieved in this way. 26 However, most adults over the age of 21 today have had smallpox vaccinations, and it has been observed that such
Mix, anneal, ligate
Plasmid pSC101 Gene for antibiotic resistance
recombinant DNA molecule
transfer to bacterial cell
Sorting out of the cells that contain the recombinant DNA
by cultivation in the presence of antibiotic
Gi ,rn ~ ......a.~ Cii
Figure 6. The cloning of foreign DNA in a plasmid. A restriction enzyme is used to cut a fragment containing a desired gene out of the DNA from one organism. The same restriction enzyme is used to cut an opening at a single site in DNA of a plasmid vector. This plasmid contains a gene for resistance to a particular antibiotic, which can be used to select for bacteria containing the plasmid. The two DNA molecules are mixed, allowed to anneal with each other via the cut ends, and are joined tightly together (ligated) by the enzyme DNA ligase. The recombinant DNA (plasmid with inserted foreign DNA) is transferred to a bacterium. The bacteria are cultivated on an agar medium in the presence of antibiotic to allow growth of only the bacteria that have received the plasmid
90 containing the gene for resistance to that antibiotic. The single bacterial cells with the plasmid reproduce themselves, thus forming a clone. Source: Ref. 20 ,Recombinant DNA 3/E by Watson, Gilman, Witkowski, Zoller© 1992 by James D. Watson, Michael Gilman, Jan Witkowski, Mark Zoller. Used with kind permission ofW.H. Freeman and Company.
individuals carry enough antibodies to neutralize vaccinia virus before it can get established and direct the expression of its genes, 27 so that its use as a vector in these individuals is apparently limited. The use of other viruses as possible vectors will be discussed in a later section. Genetic Engineering of Plants. Transgenic plants are those with transferred genetic information that is stably maintained during mitotic and meiotic cell divisions. That is, the genes have been stably integrated into the plant genome, and these genes can be inherited in a normal Mendelian manner. In order to create transgenic plants, use is made of the unique ability of plants to completely regenerate from a single cell, when that cell is taken from young shoots or plant embryos. Under the influence of plant hormones, the cell will differentiate and grow into a plant. When the cell wall is removed from the plant cell by digestion with enzymes, the resulting naked cell or protoplast can be artificially transformed with foreign DNA by the use of chemical substances or application of a process called electroporation to change the porosity of the cell membrane, as well as microinjection or microprojectile (gene cannon) bombardment of the cell. Usually, however, these methods are not very efficient in producing stable transgenic plants. 28 The soil bacterium Agrobacterium tumefaciens contains a plasmid, the Ti plasmid, so called because it is involved in the induction of crown gall or tumors in plants. A portion of of this plasmid (T-DNA), is transferred to plant cells in a natural type of exchange that is believed to be very similar to conjugation. 29 The process is, however, different from conjugation in that the T-DNA becomes integrated into the plant genome rather efficiently by a mechanism that has not been thoroughly researched, but probably represents a form of illigitimate recombination. In any case, the T-DNA is a very good vehicle for transferring genes to plants for stable recombination with the plant genome. There are some types of plants, such as maize and grains (monocotyledons), that are naturally resistant to transfection with the T-DNA, and it has been difficult to accomplish gene transfers by this method in these plants. However, much progress has been made recently to increase the efficiency of transformation of monocotyledons by the T-DNA to a feasible level. 30 When it is to be used as a vector for transferring genes to plants, the foreign genes are integrated via engineering into the Ti plasmid DNA. Because tumor production in the plant is of course undesirable, the genes responsible for this property are removed from the T-DNA with restriction enzymes, and the new gene region is ligated into that position. Immature embryos are then incubated in culture with Agrobacterium tumefaciens containing the modified plasmid, and the T-DNA carrying the foreign gene is transferred to the plant cell, where it integrates into the plant genome.
Restriction Sites Foreign Gene
.. · . ·/ \U
Start ATG Insertion Vector
Plasmid / Origin of Replication Ligation Vaccinia Promoter
Antibiotic Resistance Marker
Tissue Culture Cells Homologous Recombination BudR Selection TK- Recombinant Virus Foreign Gene
Figure 7. Construction of vaccinia virus recombinants that express a foreign gene. In the first step, the foreign gene that is to be transferred is built into a plasmid vector containing a vaccinia virus promoter (DNA region regulating synthesis), restriction enzyme sites for insertion of a foreign gene, flanking vaccinia virus
92 DNA from the thymidine kinase (TK) gene that will specify the site of recombination, an origin of replication and an antibiotic resistance gene for propagation in Escherichia coli. After insertion of the foreign gene, the plasmid is replicated in Escherichia coli to obtain sufficient numbers of the plasmid, and then used to artificially transfect tissue culture cells that are also infected with vaccinia virus. Homologous recombination introduces the the foreign gene into the vaccinia genome. Source: Ref. 25. Reprinted from Immunology Today, Vol.6, Bernard Moss, Vaccinia virus expression vector: a new tool for immunologists, pp.243-245 © 1985, with kind permission from Elsevier Science.
Transgenic plants have found wide application in basic research, serving as analytical tools for studying plant gene regulation, for elucidating metabolic pathways, and for understanding the mechanisms of plant responses to environmental stresses. More controversial is the application of transgeneic plants in agricultural and industrial production. Engineered agronomic traits include tolerance to biotic and abiotic stresses. Genetically engineered plants with increased resistance to herbicides, pest damage, as well as viral, bacterial and fungal diseases have been generated. In addition, plants have been modified genetically to produce altered quantity and composition of endogenous products such as storage proteins, carbohydrates, fatty acids, or new compounds of plant and nonplant origin such as biopolymers or pharmaceutical substances. 28
2.1.3. Innovations in Technological Developments Useful for Genetic Engineering Polymerase Chain Reaction. One main problem in the area of artificial genetic modification is having enough of a specific gene in hand to work with. Cultivating organisms, extracting DNA, identification of specific gene sequences and amplification (reproduction) of the material by cloning can be time-consuming undertakings. The polymerase chain reaction (PCR) was developed in the mid 1980's and has since revolutionized molecular genetics. It is a method for the rapid amplification of DNA in vitro (in a test tube). It can multiply DNA molecules by up to a billionfold in the test tube in the span of a few hours, yielding sufficient amounts for cloning, sequencing, or for use in mutation studies. 1•31 For the replication of DNA, the enyme DNA polymerase synthesizes a new strand of DNA, using a single strand of the old DNA as a template. In addition, the polymerase requires a short piece of double-stranded DNA as a primer, to begin the synthesis. By using primers with specified nucleotide sequences, the starting point and the stopping point of the DNA synthesis can be precisely determined (Figure 8). Thus, one major advantage of the PCR is that a specified region of the DNA used as a template will be amplified, dictated by the nucleotide sequence of the primers that define the boundaries of the region. The PCR method requires that at least a portion of the nucleotide sequence of the gene to be amplified be known in order to make the specific primers. The primers with the desired nucleotide sequence can be readily synthesized, and many biotechnology firms offer production of these at affordable prices for research.
S" ... CTGACACMCTGTGTTCACTAGCM ..................MGGTGMCGTGGATGMGTTGGT G ... 3" 3"... GACTGTGTTGACACMGTGATCGTT.................. TTCCACTTGCACCTACTTCMCCAC ... s·
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(d) s· ... CTGACACMCTGTGTTCACTAGCM...................MGGTGMCGTGGATGMGTTGGT G ... 3
..-3·..... TTCCACTTGCACCTACTTCMCS" S"ACACMCTGTGTTCACTAGCM.... . 3 .... GACTGTGTTGACACMGTGATCGTT................... TTCCACTTGCACCTACTTCMCCAC ...S.
Figure 8. Primer for the DNA-polymerase. (a) The target sequence that is to be copied is a small segment of a gene used for illustration. The two sequences given are separated by 60 base pairs in the gene (... ). (b) When DNA is heated, the two strands separate. (c) The primers hybridize specifically with their complementary sequences on the 3' ends of both strands of the target sequence. (d) The enzyme DNA polymerase begins on the primers with the synthesis of the new strands, which are complementary to the target sequences and are extended in the 5'-+3" direction. Source: Ref. 20 , Recombinant DNA 3/E by Watson, Gilman, Witkowski, Zoller© 1992 by James D. Watson, Michael Gilman, Jan Witkowski, Mark Zoller. Used with kind permission ofW.H. Freeman and Company.
In the PCR reaction (Figure 9), a cycle of synthesis includes melting the DNA by raising the reaction temperature to about 94 °C to form single DNA strands, cooling the reaction mixture to about 72°C to allow annealing (binding) of the single strands with the primers to create the short pieces of double-stranded DNA needed by the polymerase, and extention of the primers by the polymerase, using the DNA single strands as a template (Figure 8). After extention, the mixture is heated again to separate the strands and a new cycle begins. The exact temperatures used for melting and annealing, the time intervals optimal for each step, and a variety of other parameters must be standardized for each system. A final PCR product is obtained, which represents many copies of the DNA region specified by the two primers, which has a characteristic nucleotide chain length.
94 Double-stranded DNA
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