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Exploitation of Recombinant Techniques Robin Augustine

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Exploitation of Recombinant Techniques

The Recombinant DNA technique was first proposed by Peter Lobban, a graduate student,with A. Dale Kaiser at the Stanford University Department of Biochemistry. The technique was then

realized by Lobban and Kaiser; Jackson, Symons and Berg; and Stanley Norman Cohen, Chang,Herbert Boyer and Helling., in 197274. They published their findings in papers including the 1972paper "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40:Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon ofEscherichia coli", the 1973 paper "Enzymatic end-to-end joining of DNA molecules" and the 1974paper "Construction of Biologically Functional Bacterial Plasmids in vitro",all of which describedtechniques to isolate and amplify genes or DNA segments and insert them into another cell withprecision, creating a transgenic bacterium. Recombinant DNA technology was made possible by thediscovery, isolation and application of restriction endonucleases by Werner Arber, Daniel Nathans,and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine. Cohen and Boyerapplied for a patent on the Process for producing biologically functional molecular chimeras which

could not exist in nature in 1974. The patent was granted in 1980.

The major growth seen in the

biotechnology industry in recent decades haslargely been driven by the exploitation ofrecombinant DNA techniques. The initialbenefits have been predominantly in thebiomedical area, with products such asvaccines and hormones that have receivedbroad public approval. In the environmental

biotechnology and industrial ecology sectors,biotechnology has the potential to makesignificant advances through the use ofgenetically modified (GM) microbial inoculantsthat can reduce agri-chemical usage orremediate polluted environments. Althoughmany GM inoculants have been developed

and tested under laboratory conditions,commercial exploitation has lagged behind.The development of GM rice containingvitamin A precursors (golden rice) is anexample of how this technology can be appliedto develop products that are of societal benefit(Ye et al. 2000). Biotechnology is also poisedto make a major impact in the development ofimproved microbial inoculants for use in

agriculture. Wild-type bacterial and fungalspecies are currently employed as biologicalcontrol agents, biofertilisers or phyto-stimulators. There are limitations that restrictthe efficacy of some of these inoculants. Such

limitations can include poor survival of theinoculants in particular soils, and inconsistentor low production of required metabolites (vanVeen et al. 1997). GM technology has thecapacity to allow the engineering of newstrains in which these problems are overcome.This requires a thorough understanding of themolecular basis of action in wild-type strains(Bloemberg & Lugtenberg 2001; Walsh et al.2001).

The most notable applications of the

recombinant technology having direct impacton humanity have been:

1. Large scale production of therapeuticprotein such as insulin, hormones,vaccine and interleukins usingrecombinant microorganisms.

2. Production of humanized monoclonal

antibodies for therapeutic application3. Production of insect resistant cotton

plant by incorporation of insecticidaltoxin of Bacillus thuringiensis (Btcotton plant).

4. Production of golden rice (rice havingvitamin A) by incorporating three

genes required for its synthesis in riceplant.

5. Bioremediation by the use ofrecombinant organisms and

6. Use of genetic engineering techniquesin forensic medicine.

1. Exploitation of rDNA Technology inIndustrial MicrobiologyIt is genetically possible to "tailor" the

microorganisms for the production of anymicrobial metabolite - vitamin, amino acid orenzyme. Gene cloning extends the genome othe microorganism by allowing the introduction

of novel genes from comparatively unrelatedspecies. The cloning of genes from highereukaryotes, particularly from man and hisdomestic animals, has been seen to offer evengreater industrial potential. Which microbesshould then be used as universal recipients fosuch genes and hence as productionorganisms. The two most ideal are thprokaryote, Escherichia coli and the


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eukaryote, Saccharomyces cerevisiae.

Some of the important products whichgene cloning may make available in nearfuture. The above proteins could be obtainedon large scale through fermentation bymethods, relatively cheaper than theconventional ones. For example, humangrowth hormone was previously extracted fromthe pituitary gland of cadavers and was mostlyin short supply. Now, increase in supplyshould help more patients. Equally important isthe development of new vaccines throughgene-cloning. Genes for single antigens canbe cloned and expressed by bacteria andpurified antigen which has not been deriveddirectly from the pathogenic organism or virusmay be used as a vaccine. In this way,vaccines for viral hepatitis and foot-and-mouth

disease have been developed.

Types of biomolecules produced throughrecombinant DNA technology

y Recombinant HormonesInsulin (and its analogs), growthhormone, follicle stimulating hormone,salmon calcitonin.

y Blood products Albumin, thrombolytics, fibrinolytics,and clotting factors ( Factor VII, FactorIX, tissue plasminogen activator,recombinant hirudin )

y Cytokines and growth factorsInterferons, interleukins and colonystimulating factors (Interferon, , and, erythropoietin, interlukin-2, GM-CSF, GCSF )

y Monoclonal antibodies and relatedproductsMouse, chimeric or humanized; wholemolecule or fragment; single chain orbispecific; and conjugated (rituximab,trastuzmab, infliximab, bevacizumab)

y Recombinant VaccinesRecombinant protein or peptides, DNAplasmid and anti-idiotype (HBsAgvaccine, HPV vaccine)

y Recombinant EnzymesDornase (Pulomozyme), Acidglucosidase (Myozyme), L-iduronidase (Aldurazyme) and UrateOxidase

y Miscellaneous productsBone morphogenic protein, conjugateantibody, pegylated recombinantproteins, antagonist

1.1 Production of enzymesThe commercial production and use of

enzymes is already a well-establishedpart of the biotechnology industry. Enzymesare used in brewing, food processing, textilemanufacture,the leather industry, washing

powders, medical applications, and basicscientific research to name just a fewexamples. In many cases the enzymes areprepared from natural sources, but in recentyears there has been a move towards the useof enzymes produced by recombinant DNAmethods,where this is possible. In addition tothe scientific problems of producing arecombinant-derived enzyme,there areeconomic factors to take into account, and inmany cases the costbenefit analysis makesthe use of a recombinant enzyme unattractive.Broadly speaking,enzymes are either high-volume/low cost preparations for use in

industrial scale operations, or are low-volume/high value products that may have avery specific and relatively limited market.

There is a nice twist to the gene manipulationstory in that some of the enzymes used in theprocedures are now they produced usingrDNA methods.

Many of the commercial suppliers listrecombinant variants of the commonenzymes,suc h as polymerases (particularlyfor PCR) and others. Recombinant enzymescan sometimes be engineered so that theircharacteristics fit the criteria for a particularprocess better than the natural enzyme, which

increases the fidelity and efficiency of theprocess. In the food industry,one area thathas involved the use of recombinant enzymeis the production of cheese. In cheesemanufacture, rennet (also known as rennin,c

hymase,orchymosin) has been used as partof the process. Proteases and lipases arecommonly used to assist cleaning bydegradation of protein and lipid-basedstaining. A recombinant lipase was developedin 1988 by Novo Nordisk A/V (now known asNovozymes). The company is the largestsupplier of enzymes for commercial use in

cleaning applications. Their recombinant

lipase was known as Lipolase,whic h was thefirst commercial enzyme developed usingrDNA technology and the first lipase used indetergents. A further development involved anengineered avriant ofLipolase called LipolaseUltra,whihc gvi es enhanced fat removal at lowwash temperatures.

1.2 Therapeutic products for use inhuman healthcare

Recombinant DNA products for use inmedical therapy can be divided into three main


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categories. Firstly, protein products may beused forreplacement orsupplementation ofhuman proteins that may be absent orineffective in with a particular illness.Secondly, proteins can be used in specificdisease therapy,to alleviate a disease state

by intervention. Thirdly,the production ofrecombinant vaccines is an area that isdeveloping rapidly and which offers greatpromise.

The widespread condition diabetesmellitus (DM) is usually caused by cells in theislets of Langerhans in the pancreas failing toproduce adequate amounts of the hormoneinsulin. Many millions of people worldwide areaffected by DM, and the World HealthOrganization estimates that the globalincidence will double by 2025. Sufferers areclassed as having either insulin dependentDM (IDDM) or non-insulin dependent DM

(NIDDB). Insulin-dependent patientsobviously require the hormone. As DM iscaused by a problem with a normal body

constituent (insulin), therapy falls into thecategory of replacement orsupplementation. Banting and Bestdeveloped the use of insulin therapy in1921,and for the next 60 or so years diabeticswere dependent on natural sources ofinsulin,with the attendant problems of supplyand quality. In the late 1970s and early 1980srecombinant DNA technology enabledscientists to synthesise insulin in bacteria,with the first approvals granted by 1982.

Recombinant-derived insulin is now availablein several forms,and has a major impact ondiabetes therapy. One of the most widelyused forms is marketed under the nameHumulin by the Eli Lilly company. In an early

method for the production of recombinantinsulin,the insulin A- and B-chains weresynthesised separately in two bacterial strains.The insulin A- and B-genes were placed underthe control of the lac promoter,so thatexpression of the cloned genes could beswitched on by using lactose as the inducer.Following purification of the A- and B-

chains,they were linked together by a chemical

process to produce the final insulin molecule. A development of this method involves thesynthesis of the entire proinsulin polypeptidefrom a single gene sequence. The product isconverted to insulin enzymatically.

2. Exploitation of rDNA technology inMedicine and forensic medicine

Studies in bacteria and bacterialviruses have led to methods to manipulate andrecombine DNA in unique and reproducible

ways and to amplify these recombinedmolecules millions of times. Once properlyidentified, the recombinant DNA moleculescan be used in various ways useful inmedicine and human biology. There are manyapplications for recombinant DNA technology.

Cloned complementary DNA has been used toproduce various human proteins inmicroorganisms. Insulin and growth hormonehave been extensively and successfully testedin humans and insulin has been licensed forsale. Mass production of bacterial and viralantigens with recombinant DNA technology islikely to provide safe and effective vaccines forsome disorders for which there is noprevention. The cloned probes for the human- and -globin loci, for specific diseasegenes, such as the Z allele of -antitrypsin,and for random genomic sequences areproving useful for prenatally diagnosing

disorders and preventing their clinicalconsequences. The diagnosis and treatmentof human is area in which genetic

manipulation is beginning to have aconsiderable effect. Many therapeutic proteinsare now made by recombinant DNA methods,and the number available is increasingsteadily. Thus the treatment of conditions byrecombinant-derived products is already wellestablished. Progress in both of these areas isof course closely linked to our increasingknowledge of the human genome, and thusmany new developments in medical andforensic applications will appear as we

decipher genome.2.1 Diagnosis and characterisation

of medical conditionsGenetically based diseases (often

called simply genetic diseases) represent one

of the most important classes of disease,particularly in children. A disorder present at birthis termed a congenital abnormality,andaround 5% of newborn babies will suffer froma serious medical problem of this type. In mostof these cases there will be a significantgenetic component in the aetiology (cause)of the disease state. It is estimated that about

a third of primary admissions to paediatric

hospitals are due to genetically basedproblems,whilst some 70% of casespresenting more than once are due to geneticdefects. In addition to genetic problemsappearing at birth or in childhood,it seems thata large proportion of diseases presenting inlater life also have a genetic cause orpredisposition. Thus medical genetics,in itstraditional non-recombinant form,has alreadyhad a major impact on the diagnosis ofdisease and abnormality. The development of


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molecular genetics and rDNA technology hasnot only broadened the range of techniquesavailable for diagnosis,but has also opened upthe possibility of novel gene-based treatmentsfor certain conditions.

2.2 Diagnosis of infectionIn addition to genetic conditions that

affect the individual,rDNA technology is alsoimportant in the diagnosis of certain types ofinfection. Normally,bacterial infection isrelatively simple to diagnose,once it has takenhold. Thus the prescription of antibiotics mayfollow a simple investigation by a generalpractitioner. A more specific characterisationof the infectious agent may be carried outusing microbiological culturing techniques,andthis is often necessary when the infection doesnot respond well to treatment. Viral infectionsmay be more difficult to diagnose,although

conditions such as Herpes infections areusually obvious. Despite traditional methodsbeing applied in many cases,there may be

times when these methods are notappropriate. Infection by the humanimmunodeficiency virus (HIV) is one case inpoint. The virus is the causative agent ofacquired immune deficiency syndrome(AIDS). The standard test for HIV infectionrequires immunological detection of anti-HIVantibodies. However,these antibodies may notbe detectable in an infected person untilweeks after initial infection,b y which timeothers may have been infected. A test such as

this,where no positive result is obtained eventhough the individual is infected,is a falsenegative. The use of DNA probes and PCRtechnology circumvents this problem byassaying for the actual viral

DNA in the T-lymphocytes of the patient,thuspermitting a diagnosis before the antibodiesare detectable. Other examples of the use ofrDNA technology in diagnosing infectionsinclude tuberculosis (caused by thebacterium Mycobacterium tuberculosis),human papilloma virus (HPV) infection,andLyme disease (caused by the spirochaete

Borrelia burgdorferi).

2.3 Patterns of inheritance Although diagnosis of infection is an

important use of rDNA technology,it is in thecharacterisation of genetic disease that thetechnology has perhaps been most applied inmedicine to date. Before dealing with somespecific diseases in more detail,it may beuseful to review the basic features oftransmission genetics,and outline the range offactors that may determine how a particulardisease state presents in a patient. Since it

was rediscovered in 1900,the work of GregorMendel has formed the basis for ourunderstanding of how genetic characteristicsare passed on from one generation to thenext.We have already seen that the humangenome is made up of some 3 billion base-

pairs of information. This is organised as adiploid set of 46 chromosomes,ar ranged as22 pairs of autosomes and one pair of sexchromosomes. Prior to reproduction,thehaploid male andfemale gametes (sperm and oocyterespectively) are formed by the reductiondivision of meiosis,whic h reduces thechromosome number to 23. On fertilization ofthe oocyte by the sperm,diploid status isrestored,with the zygote receiving onemember of each chromosome pair from thefather,and one from the mother. In males thesex chromosomes are X and Y,in females

XX,and thus it is the father that demteinr esthe sex otfhe child. Traits may be controlledby single genes,or by many genes acting in

concert. Single-gene disease traits are knownas monogenic disorders,whilst those involvingmany genes are polygenic. Inheritance of amonogenic disease trait usually follows a basicMendelian pattern,and can therefore often betraced in family histories by pedigree analysis.A gene may have alleles (different forms) thatmay be dominant (exhibited when the allele ispresent) or recessive (the effect is maskedby a dominant allele). With respect to aparticular gene,indi viduals are said to be

eitherhomozygous (both alleles the same) orheterozygous (the alleles aredifferent,perhaps one dominant and onerecessive). Patterns of inheritance ofmonogenic traits can be associated with the

autosomes, as eitherautosomal dominant orautosomal recessive, or may be sex-linked(usually with the X chromosome,thus showingX-linked inheritance). In addition to thenuclear chromosomes, mutated genesassociated with the mitochondrial genome cancause disease. As the mitochondria areinherited along with the egg,these traits show

maternal patterns of inheritance.

2.4 Treatment using rDNAtechnology gene therapy

Once genetic defects have beenidentified and characterised,the possibility oftreating the patient arises. If the defectivegene can be replaced with a functional copy(sometimes called the transgene,as intransgenic) that is expressed correctly,thedisease caused by the defect can beprevented. This approach is known as genetherapy,and is one of the most promising


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aspects of the use of gene technology inmedicine. There are two possible approachesto gene therapy: (i) introduction of thetransgene gene into the somatic cells of theaffected tissue,or (ii) introduction into thereproductive (germ line) cells. These two

approaches have markedly different ethicalimplications. Most scientists and cliniciansconsider somatic cell gene therapy anacceptable practice,no more morally troublesome than taking anaspirin. However,tink erring with thereproductive cells,with the probability ofgermline transmission,is akin to altering the genepool of the human species,whic h is regardedas unacceptable by most people. Thus geneticengineering of germ cells is an area that islikely to remain off limits at present. There areseveral requirements for a gene therapyprotocol to be effective.

Firstly,the gene defect itself will havebeen characterised,and the gene cloned andavailable in a form suitable for use in a clinical

programme. Secondly,there must be a systemavailable for getting the gene into the correctsite in the patient. Essentially these are vectorsystems that are functionally equivalent tovectors in a standard gene cloning protocol their function is to carry the DNA sequenceinto the target cells. This also requires amechanism for physical delivery to thetarget,whic h may involve inhalation,injectionor other similar methods. Finally,if theserequirements can be satisfied,the inserted

gene must be expressed in the target cells if anon-functional gene is to be corrected.Ideally,the faulty gene would be replaced by afunctional copy. This is known as genereplacement therapy,and requires

recombination between the defective gene andthe inserted functional copy. Due to technicaldifficulties in achieving this reliably in targetcells,the alternative is to use gene additiontherapy. In addition therapy there is noabsolute requirement for reciprocal exchangeof the gene sequences,and the inserted genefunctions alongside the defective gene. This

approach is useful only if the gene defect is

not dominant,in that a dominant allele will stillproduce the defective protein,whic h mayovercome any effect of the transgene. Therapyfor dominant conditions could be devisedusing antisense mRNA,in which a reversedcopy of the gene is used to produce mRNA inthe antisense configuration. This can bind tothe mRNA from the defective allele andeffectively prevent its translation.

A further complication in gene therapyis the target cell or tissue system itself. In

some situations it may be possible to removecells from a patient and manipulate themoutside the body. The altered cells are thenreplaced,with function restored. This approachis known as ex vivo gene therapy. It is mostlysuitable for diseases that affect the blood

system. It is not suitable for tissue-baseddiseases such as DMD of CF,in which theproblem lies in dispersed and extensive tissuesuch as the lungs and pancreas (CF) or theskeletal muscles (DMD). It is difficult to seehow these conditions could be treated by exvivo therapy,and therefore the technique oftreating these conditions at their locations isused. This is known as in vivo gene therapy.Features of these two types of gene therapyare illustrated in Fig. 11.6,with bothapproaches having been used with somesuccess.

Getting transgenes into patients-

The biologyof the system must be establishedand evaluated,and then thephysical methodfor Treatment using rDNA technology gene

therapy getting the gene to the site of actionhas to be considered. Deciding on the bestmethod for addressing these two aspects of atherapeutic procedure is one important part ofthe strategy. As with vectors for use in cloningprocedures,vir uses are an attractive option fordelivering genes into human cells. We can usethe term vector in its cloning context,as apiece of DNA into which the transgene isinserted. The viral particle itself is often calledthe vehicle for delivery of the transgene,

although some authors describe the wholesystem simply as a vector system. Three mainviral systems have been developed for genetherapy protocols, based on retroviruses,adenoviruses and adeno-associated

viruses. The advantage of viral systems isthat they provide a specific and efficient way ofgetting DNA into the target cells.

Eg: Gene therapy for adenosine deaminasedeficiency,Gene therapy for cystic fibrosis,

2,5 Human monoclonal antibody

production

Monoclonal antibodies (Mab) are veryspecific immunoglobulin that exhibit a widerange of biological activities. In addition to usein diagnostics, antigen binding sites ofantibody molecules have great potential fordeveloping bioactive peptides Because of theirspecific ligand binding activity, they wereconsidered as the magic bullets ashypothesized by Paul Ehrlich. Hybridomatechnology, which used the fusion ofmyoeloma and B cells, helped in the in vitro


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production of monoclonal antibody. However,this technology developed by Kohler andMilestein was not very helpful as most of theantibodies were of murine origin and have theproblems of their low immunogenicity. Application of recombinant DNA technology

resulted in development of chimeric andhumanized antibody with high efficiency andactivity. Because of their efficacy in cancer,there have been tremendous activities indeveloping monoclonal antibodies for humantherapy. In humans, antibodies are classifiedas member of five family or isotypes. Theseare named as immunoglobulin alpha, (IgA),delta (IgD), epsilon ( IgE), gamma ( IgG) andmu (IgM). Most of the isotypes have molecularweight around 160-190 kD except IgM whosemolecular weight is around 1000kD due to itspentameric nature. The most prevalentantibody in human is IgG and majority of the

therapeutic antibodies are of IgG types. Eventhough antibodies act on wide varieties ofpathways, therapeutic antibodies work on one

of the following four ways (a) asimmunotoxicotherapy where the antibodyprevent or reverse toxic effect of venom, toxin,drug or ligand (b) destruction of target cell,where the antibodies are used to destruct thetarget cell such as lymphocytes, cancer cellsetc. (c) alteration of the cell function and finally(d) antibody mediated drug delivery, where thedrug is conjugated with the antibody forspecific targeting. For the large-scaleproduction of monoclonal antibodies,

expression of monoclonal antibody genes isaccomplished through recombinant DNAtechnology. More than 20 monoclonalantibodies have been approved for humanuses. Table 5 lists some of the important

monoclonal antibodies as a result ofrecombinant DNA technology. It is expectedthat in near future, due to production ofhumanized antibodies using recombinant DNAtechnology, there will be many more Mab inthe market.

3. Exploitation of rDNA Technology inAgriculture

The genetic manipulation of plants has

been going on since prehistoric times, whenearly farmers began carefully selecting andmaintaining seeds from their best sow for thenext season. Plant breeders have crossfertilized related plants to provide nextgeneration plants with new characteristicssuch as higher yield, resistance to diseasesand better nutrient content long before thescience of genetics was developed.Recombinant DNA technology can be used for

insertion of genes in plants not only fromrelated plant species, but also from unrelatedspecies such as microorganisms. This processof creation of transgenic plants is far moreprecise and selective than traditional breeding. Application of recombinant technology is

primarily for the production of transgenicplants with higher yield and nutritional values,increased resistance to stress and pests.Several commercially important transgeniccrops such as maize, soybean, tomato, cotton,potato, mustard, rice etc. have beengenetically modified. During the last couple ofdecades, considerable progress has beenmade to understand the function of genes,isolation of novel genes and promoters as wellas the utilization of these genes for thedevelopment of transgenic crops withimproved and new characters. There are manypotential application of plant genetic

engineering. In fact, in 2002, more than 5.5million farmers worldwide cultivated about 58.7million hectares (about 148 million acres)

crops that were genetically manipulated forherbicide tolerance, insect resistance, delayedfruit ripening and improved oil quality. Application of recombinant DNA technologyhas primarily helped in producing three majortypes of transgenic plant having improvedperformances. These are:

(1) Development of stress tolerant plant(2) Development of plant having improved

yield(3) Transgenic plant as a source of

biopharmaceuticals3.1 Development of stress tolerant

plant(a) Plant resistant to environmental stress:Plants need to cope up with abiotic stresses

such as drought, cold, heat and soils that aretoo acidic or salty to support plant growth.While plant breeders have successfullyincorporated genetic resistance to bioticstresses into many crop plants throughcrossbreeding, their success at creating cropsresistant to abiotic stresses has been morelimited, largely because few crops have close

relatives with genes for resistance to these

stresses. Therefore rDNA technology is beingincreasingly used to develop crops that cantolerate difficult growing conditions.Genetically modified tomato and canola plantsthat tolerate salt levels 300 percent greaterthan non-genetically modified varieties havebeen developed. Other researchers haveidentified many genes involved in cold, heatand drought tolerance found naturally in someplants and bacteria. Scientists in Mexico haveproduced maize and papaya that are tolerant


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to the high levels of aluminum that significantlyimpede crop plant productivity in manydeveloping countries.(b) Herbicide Resistant plant: Many effectivebroad spectrum herbicides do not distinguishbetween weeds and crops, but crop plants can

be modified to make them resistant toherbicides, so as to eliminate weeds moreselectively. For example, the herbicide

RoundupTM

contains the active ingredientglyphosate, which kills plants by binding to theactive site of enzymes calledenolpyruvalshikimate phosphate synthase(EPSP synthase). This enzyme is critical forthe synthesis of aromatic amino acids.Roundup is an extremely effective herbicidebut it kills almost all species of plants,including most crop plants. On the other hand,it is very safe for humans and animals

because they do not have EPSP synthase. Byusing rDNA technology, modified EPSPsynthase gene (that produced enzymes thatwere still functional but were not inhibited byglyphosate) have been introduced into cropplants such as cotton and soyabean. Thesegenetically modified plants were found to behighly resistant to treatment with Roundup.Genes that provide resistance to otherherbicides such as sulfonyl ureas,gluphosinates etc. have also been developedand transferred to produce various transgenicplants.(c) Insect resistant plant: To minimize cropdamage by insects, mites and nematodes,farmers use synthetic pesticides extensivelywhich cause severe effects on human healthand environment. The transgenic technologyprovides an alternative and innovative methodto improve pest control management which iseco friendly, effective, sustainable andbeneficial in term of yield. This involvesgenetic incorporation of toxic gene ( product ofwhich is lethal to insect ) in to the plant. Thiskill the insects without use if dangerousinsecticide thus has double benefit in cropimprovement. The first genes available forgenetic engineering of crop plats for pestresistance were Cry genes (popularly known

as Bt genes) from a bacterium Bacillusthuringiensis. These are specific to particulargroup of insect pests, and are not harmful toother useful insects like butter flies and silkworms. Transgenic crops (e.g. cotton, rice,maize, potato, brinjal, cauliflower, cabbageetc.) with Bt genes have been developed andsuch transgenic varieties proved effective incontrolling the insect pests and it has been

claimed worldwide that it has led to significant

increase in yield along with dramatic reductionin pesticides use. The most notable example isBt cotton (which contains Cry/Acgene) that isresistant to a notorious insect pest Bollworm(Hellicoperpa armigera) and only last year(2002) Bt cotton was adopted in India.

Biotechnology has opened up newavenues for natural protection for plants byproviding new biopesticides, such asmicroorganisms, that are toxic to targeted croppests but do not harm humans, animals, fish,birds or beneficial insects. As biopesticides actin unique ways, they can even control pestpopulation that have developed resistance toconventional pesticides. Using recombinantDNA technology, the gene that makes thesemicroorganisms lethal to certain insects canbe transplanted into the plants on which thatinsect feeds. The plant that once was a foodsource for the insect now kills it, lessening the

need to spray crops with chemical pesticidesto control infestation. One such microorganismis commonly found soil bacterium Bacillus

thuringiensis. The spores of Bacillusthuringiensis (Bt) contain a crystalline protein(Cry) which breaks down to release a toxin,known as delta-endotoxin which is highly toxicto lepidopteran larvae. This toxin binds theintestinal lining and creates pores resulting inan ion imbalance, paralysis of the digestivesystem, and consequent deathof the insect. Bttoxin sprays and powders have been in use formany years. Different Cry genes, also knownas Bt genes have been identified, cloned and

characterized. Effective gene constructs havemade it possible to deliver these genes intoplant tissues so that they are expressed atlevels high enough to kill the insects. The Btgenes are effective against different orders of

insects. Bt cotton and maize which haveincreased resistance to boll worms have beendeveloped and cultivated since 1996. Farmersget benefited by saving costs by using less oftraditional pesticides. However, one of themajor concerns about Bt based transgenics isthe possibility of development of toxin resistantinsects. Efforts are also underway to identify

and transfer other genes from Bt, which can

impart insecticidal properties to the plants.One example in this is transfer of vip gene i.e.vegetative insecticidal proteins, for which thetrials are being conducted in some countries.(d) Disease resistance plant: Plants aresusceptible to viral, bacterial and fungaldiseases. Much progress has been made inevolving transgenic plants resistant to viruses.For example, expression of a gene thatencodes the coat protein of tobacco mosaicvirus (TMV) in transgenic tobacco plants has


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been shown to cause the plants to resist TMVinfection. A number of other viral resistantplants species have been developed includingsquash and potatoes. Genetic engineering ofcrop plants for resistance to fungal andbacterial infections has been more difficult.

However, by studying the protective genesthat are expressed in naturally disease-resistant plants, an encouraging progress hasbeen made. The proteins encoded by these socalled pathogenesis related proteins (PRproteins) can, in some cases, provide limiteddisease protection in transgenic plants. Thereare several strategies for engineering plantsfor viral resistance and these utilizes thegenes from virus itself (e.g. the viral coatprotein gene). The virus-derived resistancehas given promising results in number of cropplants such as tobacco, tomato, potato, alfalfa,and papaya. Some viral resistant transgenic

plants like papaya resistance to papaya ringspot virus have been commercialized in somecountries. Plants respond to pathogens by

inducing a variety of defense responses likepathogenesis-related proteins (PR proteins),enzymes that degrade/destroy fungal cell wall(chitinase), antifungal proteins andcompounds, phytoalexins, etc. Severaltransgenic crop plants showing increasedresistance to fungal pathogens are beingraised with genes coding for the differentcompounds mentioned above.

3.2 Development of plant havingimproved yield

(a) Increasing yieldIn addition to increase crop productivity by

using built-in protection against diseases,pests, environmental stresses and weeds tominimize losses, attempts are being made to

use biotechnology to improve crop yieldsdirectly. Researchers at Japan's NationalInstitute of Agrobiological Resources addedmaize photosynthesis genes to rice toincrease its efficiency of converting sunlight toplant starch and increased yields by 30percent. Other scientists are altering plantmetabolism by blocking gene action in order to

shunt nutrients to certain plant parts. Yields

increase as starch accumulates in potatotubers and not leaves, or oilseed crops, suchas canola, allocate most fatty acids to theseeds. Crops that have better accessibility tothe micronutrients they need are also beingdeveloped. Mexican scientists have geneticallymodified plants to secrete citric acid, anaturally occurring compound, from their roots.In response to the slight increase in acidity,minerals bound to soil particles, such ascalcium, phosphorous and potassium, are

released and made available to the plant.Nitrogen is the critical limiting element for plantgrowth and researchers from many scientificdisciplines are tearing apart the details of thesymbiotic relationship that allows nitrogen-fixing bacteria to capture atmospheric nitrogen

and provide it to the plants that harbor them inroot nodules as given below:

(1) Plant geneticists in Hungary andEngland have identified the plantgene and protein that enable theplant to establish a relationship withnitrogen-fixing bacteria in thesurrounding soil.

(2) Microbial geneticists at the University ofQueensland have identified thebacterial gene that stimulates rootnodule formation.

(3) Collaboration among molecularbiologists in the European Union,

United States and Canada yieldedthe complete genome sequence ofone of the nitrogen-fixing bacteria

species.(4) Protein chemists have documented the

precise structure of the bacterialenzyme that converts atmosphericnitrogen into a form the plant canuse.

(b) Increase in quality of plant productsOne of the most successful research

efforts to change the characteristics of a plantproduce was carried out with tomatoes.

Tomatoes need to be picked while still greenso that they are firm enough to withstandmechanical handling and transport.Unfortunately, they do not develop the sameflavor and texture of vineripened tomatoes.

Softening of tomatoes and many other fruits iscaused by the enzyme pectinase orpolygalacturonase (PGA). This enzymedigests the pectin polysaccharide that cementsthe plant cells together. Softening of the fruit iscaused, in part by this breakdown of pectin. Inorder to reduce the levels of PGA in ripeningtomatoes, researchers placed the PGA gene

in reverse orientation relative to the CaMV 35S

promoter. This results in transcription of anantisense RNA that is complementary to thenormal sense PGA mRNA. Although the exactmechanism is unknown, antisense RNA isable to arrest the translation of theendogenous PGA mRNA in the tomato fruit.Transgenic tomato plants that express anantisense PGA gene only have about 5 to 10%of normal PGA levels. Fruits of these plantshave normal color and flavor but they softenmore slowly and can be picked and processed


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after they are ripe. They also have a highercontent of soluble solids and are thereforebetter than normal tomatoes for processedtomato products. Transgenic lines of potatohaving increased levels of starch also havebeen developed by introducing a gene

construct that expresses a gene from bacteriathat produce an enzyme that enhances starchbiosynthesis. A promoter from a potato genethat encodes the major protein in potato tubershas been used, so that the expression of theintroduced gene is limited to the tuber. Tubersaccumulate approximately 3 to 5% morestarch than normal potatoes and when theyare deep fried absorb less oil and yield chipshaving fewer calories. Some of the other valueadded transgenic crops include:

(a) Golden rice: containing beta caroteneto overcome vitamin A deficiency inregions where rice is the staple food

(b) Canola containing high levels of oleicacids and laurate

(c) Barley containing feed enzymes

(d) tomatoes which does not rot in roomtemperature

(e) Other vegetables and fruits withdelayed ripening as well as modifiedflavour characteristics

Transgenic crops with improved nutritionquality have already been produced byintroducing genes involved in the metabolismof vitamins, minerals and amino acids. Fewexamples of genetic modification of nutritional

quality are described below.Vitamin A: Vitamin A deficiency can lead tonight blindness and skin disorders, amongothers. About 124 million children worldwideare deficient in vitamin A and a quarter of a

million go blind each year due to vitamin Adeficiency. The staple food rice is extremelylow in vitamin A, and therefore theimprovement of vitamin A content is veryimportant. In a remarkable example of geneticengineering, Prof. Ingo Potrykus and Dr. PeterBeyer developed genetically engineered rice(popularly known as Golden Rice), which is

enriched in pro-vitamin A by introducing three

genes involved in the biosynthetic pathway forcarotenoid, the precursor for vitamin A. Theseeds of Golden Rice are yellow in colourbecause of pro-vitamin A is produced in theentire grain.Seed Protein Quality: The Nutritional qualityof cereals and legumes are limited because ofdeficiency of the essential amino acids, i.e.lysine in cereals, and methionine andtryptophan in pulses. Two genetic engineeringapproaches have been used to improve the

seed protein quality. In the first case, atransgene (e.g. gene for protein containingsulphur rich amino acids) was introduced intopea plant (which is deficient in methionine andcysteine, but rich in lysine) under the control ofseed specific promoter. In the second

approach, the endogenous genes are modifiedso as to increase the essential amino acidslike lysine in the seed proteins of cereals.The gas hormone, ethylene is involved in theregulation of fruit ripening. Therefore, ripeningcan be slowed down by blocking or reducingethylene production. This can be achieved byintroducing ethylene forming gene(s) in a waythat will suppress its own expression in thecrop plants. Such fruits ripen very slowly(however, they can be ripen by ethyleneapplication) and are very important for exportto longer distances without spoilage as theyshow longer-self life due to slow ripening. A

notable example of this kind is the Flavr Savrtransgenic tomatoes, which werecommercialized in U.S about 6 year ago.

3.3 Transgenic plant as a source ofbio pharmaceuticals

Plants are among the most efficientbioreactors which produce quantities ofmaterial with sunlight and soil based nutrientsas inputs. Attempts are being made to replacethe traditional fermentation procedure for theproduction of biopharmaceuticals to plantbased production. The benefits of using plantsare the ability to increase production at lowcost by planting more acres, rather than

building fermentation capacity, lower capitaland operating cost, simplified downstreamprocessing etc. Therapeutic drugs to treatcancer, infectious diseases, autoimmunediseases, cardiovascular diseases and other

conditions and several vaccines canpotentially be grown in plants. Plant transgenictechnology is being used to produce a plantthat will generate a seed that expresses adesired therapeutic protein. This seed canpropagate under the right growing conditionsto yield plants and seed stock for producingthe desired protein. The desired protein can be

extracted from the seed to make a

biopharmaceutical. Plant based therapeuticsare expected to be much more cost effective.For example, Dow Plant Pharmaceuticals isusing corn to grow pharmaceuticals bydesigning and selecting the plant which willcontain the active pharmaceutical within theendosperm seed compartment. Benefits ofproducing the pharmaceuticals in the corninclude long term storage advantage, easierpurification in view of limited number of solubleseed proteins in a corn seeds, low microbial


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load, low proteolytic activity and specializedpromoters to enable expression of the proteinin specific parts of the plants.

3.4 Therapeutic proteins, enzymesand diagnostics

Transgenic plants can also produce a

variety of proteins used in diagnostics fordetecting human diseases and therapeutics forcuring human and animal diseases in large-scale with low cost. The monoclonalantibodies, blood plasma proteins, peptidehormones and cytokinins are being producedin transgenic plants and their parts such astobacco (in leaves), potato (in tubers),sugarcane (in stems) and maize (in seedendosperm). Plants are amazing and cheapchemical factories that need only water,minerals, sun light and carbon dioxide toproduce thousands of sophisticated chemicalmolecules with different structures. Given the

right genes, plants can serve as bioreactors tomodified or new compounds such as aminoacids, proteins, vitamins, plastics,

pharmaceuticals (peptides and proteins),drugs, and enzymes for food industry and soon. Some of the potential and remarkableexamples of this kind are described here.

3.5Edible vaccinesCrop plants offer cost-effective

bioreactors to express antigens which can beused as edible vaccines. The genes encodingantigenic proteins can be isolated from thepathogens and expressed in plants and suchtransgenic plants or their tissues producing

antigens can be eaten forvaccination/immunization (edible vaccines).The expression of such antigenic proteins incrops like banana and tomato are useful forimmunization of humans since banana and

tomato fruits can be eaten raw. The ediblevaccines that are produced in transgenicplants have great advantages like thealleviation of storage problems, easy deliverysystem by feeding and low cost as comparedto recombinant vaccines produced by bacterialfermentation. Vaccinating people againstdreadful diseases like cholera and hepatitis B

by feeding them banana/ tomato, and

vaccinating animals against importantdiseases such as foot and mouth disease byfeeding them sugar beets could be a reality inthe near future.

3.6 Metabolic engineering andSecondaryproducts

Plant biotechnology will lead toimproved plant sources for the production ofvaluable secondary metabolites mentioned inprevious section on cell culture products.Over-expression of the gene which encode for

the first enzyme in a pathway generally resultsin higher levels of the desired end product,and this has been successfully done in theenhancement of taxol production from thetransformed tissue cultures of Taxus sp. Another strategy involves use of

Agrobaccterium rhizogenes to induce theexcessive formation of secondary roots inplants that normally produce useful secondarymetabolites in this region. Transgenic plantscan be used as factories to producepolyhydroxy butyrate (PHB, biodegradableplastic). Genetically engineered Arabidopsisplants produced PHB globules exclusively intheir chloroplasts without effecting plantgrowth and development The large scaleproduction of PHB may be easily achieved intree plants like populus, where PHB can beextracted from leaves. Industry has alreadybegun to explore the production of

biodegradable plastics from transgenic plants.4. Exploitation of rDNA technology inVeterinary

Applications to animal agriculture canbe expected in animal health management,improved crops and feeds, manipulation ofanimal physiology, and genetic improvementof livestock species, Improved diagnosticreagents and vaccines that will improve herdhealth are currently under development. Yieldof crop plants such as corn will be increasedand the nutritional value of these feedsimproved through applications of recombinantDNA technology. Administration of exogenous

hormones synthesized by bacteria holds greatpromise for increasing the yield of milk andpossibly meat. Research on the transfer ofcloned genes into animals has progressedrapidly and has recently been accomplished insheep and swine. Tissue-specific anddevelopmentally regulated expression oftransferred genes now seems possible withdefined gene promoter sequences. Severalapplications of these biotechnologies can beexpected within 5 to 10 yr, whereas othersmay require longer periods of research. The21

stcentury will herald a new era in animal

science research and applications, with

recombinant DNA and gene transfer playingmajor roles.

4.1 In Animal Health ManagementImproved diagnostic kits are being

developed that utilize monoclonal antibodies todetect disease conditions (Kobbe, 1985).These kits will allow veterinarians to diagnosequickly (and economically) and designtreatment regimens on the farm rather thanrelying on more expensive and time-


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consuming laboratory tests. Because thesenew tests often rely on the detection of anantibody produced in response to a diseaseagent, it is difficult to distinguish between theantibodies stimulatedby a vaccine or by the disease itself. Despite

these problems, tests will soon be available forbovine brucellosis, avian bronchitis andporcine pseudorabies. Neonatal calves havebeen protected againstfatal enteric colibacillosis with a monoclonalantibody to the K99 pilus antigen (Sherman etal., 1983).

With the molecular cloning of theFMD(foot-and-mouth disease) genome andanalysis of the viral proteins, it was possible todetermine that only one protein (VP1) wasimmunogenic (Rowlands and Brown, 1985).The VP1 was produced by genetic engineeringtechniques in the form of a chimeric protein

containing part of the trp E protein fromEscherichia coli (Kleid et al., 1981). Thisprotein produced an immune response that

protected cattle and swine from contractingthis disease.

4.2 Improved Crops and Feeds forAnimal Production

An application of rDNA technoloy inplant breeding and crop improvement leads tothe improvement of animal husbandary.

4.3 Manipulation of AnimalPhysiology

Expression of eukaryotic genes inbacteria has led to the production of pure

protein products such as hormones inquantities sufficient for physiologicalexperiments (Seeburg et al., 1983).recombinantly-derived bovine growth hormone(rbGH) can increase milk production by as

much as 41% when injected daily, while notaffecting milk composition (Bauman et al.,1985a). The effects of exogenous GH on thegrowth of meat animals also are being activelyinvestigated. The control and regulation ofmeat animal growth is complex and involves anumber of hormones (Etherton and Kensinger,1984). Recent studies with porcine GH

indicated a 10% increase in growth rate when

administered daily to young swine (Chung etal., 1985).

4.5 Genetic Improvement ofLivestock Species

Recent advances suggest thepossibility of improving livestock species bythe direct transfer of cloned genes to earlyembryos. Since the first reports of successfulgene transfer to mice (Gordon et al., 1980;Brinster et al., 1981; Costantini and Lacy,1981; Gordon and Ruddle, 1981; Wagner et

al., 1981a,b), astounding progress has beenmade (see reviews of Gordon, 1983; Gordonand Ruddle, 1985; Palmiter and Brinster,1985; Petters, 1985). Production of transgenicswine, sheep and rabbits has recently beenreported (Hammer et al., 1985b). These

experiments are possible through refinementsin experimental mammalian embryology thatinclude embryo recovery, in vitro culture, andtransfer (Mapletoft, 1984). Micromanipulationsof embryos have been improved to allow directmicroinjection of individual nuclei within theembryo (Seidel, 1982; Gordon and Ruddle,1983). Continued rapid progress should beexpected.

5. Exploitation of rDNA technology inEnvironment

A vast majority of applications ofenvironmental biotechnology use naturallyoccurring microorganisms (bacteria, fungi,etc.) to identify and filter manufacturing wastebefore it is introduced into the environment.Bioremediation program involving the use ofmicroorganisms are currently in progress toclean up contaminated air, tracks of land,lakes and waterways. Recombinanttechnology helps in improving the efficacy ofthese processes so that their basic biologicalprocesses are more efficient and can degrademore complex chemicals and higher volumesof waste materials. Recombinant DNAtechnology also is being used in developmentof bioindicators where bacteria have been

genetically modified as 'bioluminescors' thatgive off light in response to several chemicalpollutants. These are being used to measurethe presence of some hazardous chemicals inthe environment. Other genetic sensors thatcan be used to detect various chemicalcontaminants are also undergoing trials andinclude sensors that can be used to track howpollutants are naturally degrading in groundwater. For example when gene such as themercury resistance gene (mer) or the toluenedegradation (tol) gene is linked to genes thatcode for bioluminescence within livingbacterial cells, the biosensor cells can signal

extremely low levels of inorganic mercury ortoluene that are present in contaminatedwaters and soils by emitting visible light, whichcan be measured with fiber-optic fluro meters.

Conventional pesticides andfungicides are extensively used in modernagriculture and some are now considered tocontribute to environmental pollution. Inaddition, some of these chemicals arepotentially hazardous, and residues can


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constitute a risk to food quality and humanhealth. The replacement of these with efficientbiological inoculants will make a significantcontribution to sustainable agriculture. Thesecond area where microbial inoculants willhave an impact is the decontamination of soil

and water polluted with heavy metals, organo-chemicals and other toxic man-madepollutants, a process termed bioremediation(reviewed in Gadd 2000; Pieper & Reineke2000). This approach has been furtherdeveloped using plants as delivery/supportsystems for microbial inoculants in therhizosphere (rhizoremediation). Althoughwildtype inoculants can be used to remediatecontaminated sites, there are majorbottlenecks in the process, includingbioavailability of the pollutant. In addition,many man-made pollutants are xenobioticchemicals, containing structural elements not

typically found in nature. Bioremediation ofthese recalcitrant pollutants can be bestachieved through the construction of strains

containing enzymes with new specificities ornew degradative pathways (Timmis & Pieper1999; de Lorenzo 2001). The primaryrequirements for the successful exploitation ofa GM inoculant are the development of aneffective strain that is robust in theenvironment, and securing of the necessaryregulatory approval from the appropriatecompetent authority. With regard to improvingthe efficacy of biocontrol inoculants, the initialfocus has been on understanding the

molecular basis of biocontrol with the aim ofreprogramming genetic pathways. This workestablished that synthesis of biocontrolmetabolites is under complex control in manybacteria. For example, production of

secondary metabolites in Pseudomonasspecies can be subject to quorum sensing,transcriptional and post-transcriptional controls(Aarons et al. 2000; Bloemberg & Lugtenberg2001; Haas et al. 2000). Re-regulation orbypassing these controls can lead to improvedbiocontrol performance under laboratoryconditions (Delany et al. 2000). Approaches to

engineer improved inoculants for

bioremediation have recently been reviewedand often involve the introduction ofheterologous genes into suitable host strains(Timmis & Pieper 1999; Pieper & Reineke2000; de Lorenzo 2001).With inoculantsdeveloped for biocontrol and bioremediation,as well as for other uses in Agbiotech such asbiofertilisation or phytostimulation it isessential to carry out field-based trials in orderto accurately assess the efficacy of the strain.In an open environment, inoculants are

competing and interacting with a diversecommunity of organisms that can haveprofound effects on the survival andperformance of the introduced strain (Sayler&Ripp 2000;Walsh et al. 2001). Furthermore,field-trial studies allow researchers to carry out

important impact assessment, gene flow andpersistence studies (Gilbert et al. 1993; de Leijet al. 1995; Monne-Loccoz et al. 2001)required by regulatory bodies prior to thecommercial release of GM (and in some casesnon-GM) inoculants.

ReferencesJohn P. Morrissey1, Ultan F. Walsh1, Anne

ODonnell1, Yvan Monne-Loccoz2 &

Fergal OGara1(2002)., Exploitation of

genetically modified inoculants for

industrial ecology Applications, Antonie

van Leeuwenhoek81: 59960

Bloemberg GV & Lugtenberg

BJ (2001)

Molecular basis of plant growth

promotion and biocontrol by

rhizobacteria. Curr. Opin. Plant. Biol. 4:

343350.

Robert M. Agriculture,Recombinant DNA,

Gene Transfer and the Future of Animal, J

Anim Sci1986. 62:1759-1768.

Amulya K. Panda, RECOMBINANT DNA

TECHNOLOGY AND BIOTECHNOLOGY,

Application ofBiotechnology.

Fermentation Microbiology and

Biotechnology Charles FA Bryce and

Mansi El-Mansi, Tylor and Francis , USA

(1999).

Mahaffee WF & Kloepper JW (1997)

Bacterial communities of the rhizosphere

and endorhiza associated with field-

grown cucumber plants inoculated with a

plant growth-promoting rhizobacterium

or its genetically modified derivative. Can.

J. Microbiol. 43: 344353.

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