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1. Genetic Recombination
By: Bijaya Kumar Uprety

2. Contents
•Transformation,conjugation,transduction, protoplastfusion.
•Genecloningandtheirapplications.
•Developmentofhybn'domaformonoclonalantibodies.
•StudyofdrugsproducedbybiotechnologysuchasActivase,Humulin,Humatrope,andHBetc.

3. Auxotroph:A mutated microorganism having nutritional requirements that differ from those of unmutated microorganisms from the same strain.
Cloning vector:genetic element into which genes can be recombined and replicated
Conjugation:transfer of genes from one prokaryotic cell to another by a mechanism involving cell-to-cell contact and a plasmid
Diploid: a eukaryotic cell or organism containing two sets of chromosomes
Electroporation:the use of an electric pulse to induce cells to take up free DNA
Gene disruption:use of genetic techniques to inactivate a gene by inserting within it a DNA fragment containing an easily selectable marker. The inserted fragment is called a cassette, and the process of insertion, cassette mutagenesis
WORKING GLOSSARY

4. Genetic map:the arrangement of genes on a chromosome
Genome:the total complement of genes of a cell or a virus
Genotype:the precise genetic makeup of an organism
Hybridization:formation of a duplex nucleic acid molecule with strands derived from different sources by complementary base pairing
Molecular cloning:isolation and incorporation of a fragment of DNA into a vector where it can be replicated
Haploid:a cell or organism that has only one set of chromosomes
Mutagens:agents that cause mutation
Mutant:an organism whose genome carries a mutation
Mutation:an inheritable change in the base sequence of the genome of an organism

5. Nucleic acid probe:a strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from mixture of other nucleic acids
Phenotype:the observable characteristics of an organism
Plasmid:an extra chromosomal genetic element that has no extracellular form
Point mutation:a mutation that involves one or only a very few base pairs
Polymerase chain reaction (PCR):a method used to amplify a specific DNA sequence in vitro by repeated cycles of synthesis using specific primers and DNA polymerase
Recombination:the process by which parts or all of the DNA molecules from two separate sources are exchanged or brought together into a single unit.

6. Restriction enzyme:an enzyme that recognizes and makes double-stranded breaks at specific DNA sequences
Shotgun cloning:making a gene library by closing random DNA fragments
Site-directed mutagenesis:a technique whereby a gene with a specific mutation can be constructed in vitro
Synthetic DNA:a DNA molecule made by a chemical process in a laboratory
Transduction:transfer of host genes from one cell to another by a virus
Transformation:transfer of bacterial genes involving free DNA

7. INTRODUCTION
•Geneticrecombinationistheprocessbywhichgeneticelementsfromtwoseparatesourcesarebroughttogetherinasingleunit.
•Atmolecularlevel,recombinationcanbethoughtofasthemovementofgeneticinformationfromonemoleculeofnucleicacidtoanother.
•ThegeneticexchangeoccurringbetweenhomologousDNAsequencesfromtwodifferentsourcesistermedasgeneralrecombination.Forthistohappen,identicalsequencesonthetworecombiningmoleculesarerequired.
•Theprocessofgeneticexchangewhichoccursineukaryotesduringsexualreproduction(meiosis)isanexampleofthistypeofgeneticrecombination.

8. •Figure 1.Duringmeiosis, homologous recombination can produce new combinations of genes as shown here betweensimilar but not identical copies of human chromosome 1

9. Molecular Events in General Recombination
•Atmolecularlevel,recombinationhasbeenstudiedonlyinprokaryotesandviruses.
•Theprocessistoocomplicatedtobeanalysedintheeukaryotes.
•Inbacteria,generalrecombinationinvolvestheparticipationofaspecificproteincalledtherecAprotein.
•TherecAproteinisspecifiedbytherecAgene.TherecAproteinishelicalinstructureanditwrapsitselfaroundtheDNAhelix,facilitatingrecombination.
Note:RecAproteinRecAgenemutationinRecA decreasedlevelofgeneticrecombination.

10. •BacteriawhicharemutantinrecAshowmarkedlydecreasedlevelsofgeneralrecombination.
•AnoverallmolecularmechanismofgeneralrecombinationbeginswithanickinoneofDNAmoleculeswhichleadstotheformationofashortsingle-strandedsegment.
•Thehelix-destabilizingproteinscombinesatthissiteandaidsinopeningupoftheDNAdoublehelix.
•TherecAproteinbindstothesingle-strandedfragmentandpositionsitinsuchawaythatannealingoccurswithacomplementarysequenceintheadjacentduplex,simultaneouslydisplacingtheresidentstrand.
Note:nickinoneofDNAstrandbindingofhelixdestabilizingproteinsrecAproteinsbindstosinglestrandedfragmentdisplacementofresidentstrand.

11. •ExchangeofgeneticmaterialoccursbetweenhomologouschromosomesleadingtotheformationofrecombinantDNAstructures.
•ThemeansbywhichDNAfragmentsareintroducedintotherecipientare: transformation,transductionandconjugation.

12. Asimplifiedversionofonemolecularmechanismofrecombinationisshowninfigureinthenextslide)
HomologousDNAmoleculespairandexchangeDNAsegments.
Themechanisminvolvesbreakageandreunionofpairedsegments.Twooftheproteinsinvolved,asingle- strandedbinding(SSB)proteinandtheRecAprotein.
Notethattherearetwopossibleoutcomes,dependingonwhichstrandsarecutduringtheresolutionprocess. Inoneoutcometherecombinantmoleculeshavepatches,whereasintheotherthetwoparentalmoleculesappeartohavebeencutandthensplicedtogether.

14. In order to detect physical exchange of DNA segments, the cells resulting from recombination must be phenotypically different from the parents.
Detection of Recombination
Strains that lack some selectable characteristic that the recombinants will possess. For instance, the recipient may not be able to grow on a particular medium on which the genetic recombinants selected can.
Various kinds of selectable and nonselectable markers (such as drug resistance, nutritional requirements, and so on) may be used.

15. (1) Transformation, which involves donor DNA free in the environment
(2) Transduction, in which the donor DNA transfer is mediated by a virus
(3) Conjugation, in which the transfer involves cell-to- cell contact and a conjugative plasmid in the donor cell
Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

16. Genetic Transformation
•Griffith’s Experiment:
Griffith'sexperiment,conductedin1928byFrederickGriffith,wasoneofthefirstexperimentssuggestingthatbacteriaarecapableoftransferringgeneticinformationthroughaprocessknownastransformation.
Griffithusedtwostrainsofpneumococcus(Streptococcuspneumoniae)bacteriawhichinfectmice–atypeIII-S(smooth)andtypeII-R(rough)strain.TheIII-Sstraincoversitselfwithapolysaccharidecapsulethatprotectsitfromthehost'simmunesystem,resultinginthedeathofthehost,whiletheII-Rstraindoesn'thavethatprotectivecapsuleandisdefeatedbythehost'simmunesystem.UntilGriffith'sexperiment,bacteriologistsbelievedthatthetypeswerefixedandunchangeable,fromonegenerationtoanother.

17. •Inthisexperiment,bacteriafromtheIII-Sstrainwerekilledbyheat, andtheirremainswereaddedtoII-Rstrainbacteria.Whileneitheraloneharmedthemice,thecombinationwasabletokillitshost. GriffithwasalsoabletoisolatebothliveII-RandliveIII-Sstrainsofpneumococcusfromthebloodofthesedeadmice.GriffithconcludedthatthetypeII-Rhadbeen"transformed"intothelethalIII-Sstrainbya"transformingprinciple"thatwassomehowpartofthedeadIII-Sstrainbacteria.
•Today,weknowthatthe"transformingprinciple"GriffithobservedwastheDNAoftheIII-Sstrainbacteria.Whilethebacteriahadbeenkilled,theDNAhadsurvivedtheheatingprocessandwastakenupbytheII-Rstrainbacteria.TheIII-SstrainDNAcontainsthegenesthatformtheprotectivepolysaccharidecapsule.Equippedwiththisgene,theformerII-Rstrainbacteriawerenowprotectedfromthehost'simmunesystemandcouldkillthehost.Theexactnatureofthetransformingprinciple(DNA)wasverifiedintheexperimentsdonebyAvery,McLeodandMcCartyandbyHersheyandChase.

19. •Transformationisaprocesswherecertain‘competent’bacteriaareabletotakeupfreeDNAreleasedbyotherbacteria.
•TheDNAistakenuponlyinrelativelysmallamountandcanbeacquiredonlyinasingleevent.
•Onlycertainstrainsarecompetentandthisabilityseemstobeaninheritedpropertyoftheorganism.
•Competenceincertainbacteriaisgovernedbycertainproteinswhichincludemembrane-associatedDNA-bindingprotein,cellwallautolysin,andvariousnucleases.

20. •IncreasedtransformationefficiencyincertainspeciesofbacteriamaybeduetothedeficiencyincertainDNases,whichnormallydestroyincomingDNA.
•ThenatureofthecellsurfacealsoplaysanimportantroleindeterminingwhetheracellcantakeupDNA.
•DuringtransformationcompetentbacteriafirstbindDNAreversiblyandsoonthebindingbecomeirreversible.
•CompetentcellsbindmuchmoreDNAthanthenon- competentcells(asmuchasthousandtimesmore).
•ThetransformingDNAisboundatthecellsurfacebyaDNA-bindingprotein.

21. •TheDNAiseitherincorporatedcompletelyintothecellorisdegradedbythehost’snucleaseenzyme,whereonestrandisdegradedandtheotherstrandistakenupbythehostcell.
•TheincorporatedDNAgetsassociatedwithacompetence- specificprotein,whichremainsattachedtotheDNAsegmentpreventingiffromthenucleaseattackuntilitreachesthehostchromosomewheretherecAproteintakesover[recAisaproductofrecgenes(recombinationgenes).]
•TheDNAisthenintegratedintothegenomeoftherecipientbyarecombinationalprocessandrecAproteinsarereleased.

22. Gene Recombination in Bacteria
•Microorganismscarryoutseveraltypesofgenerecombination,themostcommonofthembeing, generalrecombination.
•Thisresultsinareciprocalexchangeofgenesbetweenapairofhomologouschromosomes.
•Itcanoccuratanyplaceinthechromosomeanditresultsfromthebreakageandreunionofchromosomesleadingtocrossingover.
•Theproductsofrecgenessuchasrec-Aproteinplayanimportantroleinrecombination.

23. •Inbacterialtransformation,anonreciprocaltypeofrecombinationtakesplace.
•ApieceofgeneticmaterialisinsertedintothechromosomethroughtheincorporationofasinglestrandtoformastretchofheteroduplexDNA.
•Anothertypeofrecombination,importantintheintegrationofvirusgenomesintobacterialchromosomes,issite-specificrecombination.
•TransfectionisaprocesswherebacteriacanbetransformedwithDNAextractedfromabacterialvirusratherthanfromanotherbacterialcell.

24. •Transfectionhasbecomeausefultoolforstudyingthemechanismoftransformationandrecombinationbecause,thesmallsizeofthephagegenomeallowstheisolationofanearlyhomogeneouspopulationofDNAmolecules.

26. (a)Binding of free DNA by a membrane- bound DNA binding protein.
(b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.
(c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.
The introduction of DNA into cells by mixing the DNA and the cell
Transformed cell

27. The mechanism of bacterial transformation

28. Agrobacterium tumefaciensMediated Gene TransferWhat isAgrobacterium tumefaciens?
ØBacterial plant pathogen found in the soil that results in tumorous growths and/or roots to develop in infected plants (“Agrobacteriumtumefaciens” 2001)
ØThis infection is known asCrown Gall Disease(Deacon 2002)
ØThe bacteria transfers a tumor-inducing (Ti) plasmid located in a section of its DNA (known as T-DNA) into the nucleus of an infected plant cell.
ØThe newly introduced Ti-plasmid is incorporated into the plant genome and is consequently transcribed (Sforza2002)
ØThe T-DNA that is integrated into the plant genome contains cancer-causing oncogenic genes and genes that synthesize opines which are excreted by infected Crown Gall cells and are a food source forAgrobacteriumtumefaciens(González-Cabrera 1998)

29. Transduction
•TransductionisaprocesswhereDNAistransferredfromcelltocellthroughtheagencyofviruses.
•Suchgenetictransfersfromthedonortotherecipientthroughthevirusescanoccurintwoways,onebeinggeneralizedtransductionwheredefectivevirusparticlesrandomlyincorporatefragmentsofthehostDNA(virtuallyanygeneofthedonor)andtransferittotherecipientcell.
•ThesecondisspecializedtransductionwheretheDNAofatemperatevirusexcisesincorrectlyandbringsadjacenthostgenesalongwithit,andonlygenesclosetotheintegrationpointofthevirusaretransduced.

30. •Theefficiencyofgeneralizedtransductionisverylowcomparedtothatofaspecializedone.
•Transductionhasbeenfoundtooccurinavarietyofbacteria. Allphagesdon'tparticipateintransductionandallthebacteriaarenottransducible.
•Butthisphenomenonissufficientlywidespreadanditplaysanimportantroleingenetictransferinnature.
•Transductionhasbeenfoundtooccurinavarietyofprokaryotes,includingcertainspeciesoftheBacteria: Desulfovibrio,Escherichia,Pseudomonas,Rhodococcus, Rhodobacter,Salmonella,Staphylococcus,andXanthobacter, aswellasthearchaeanMethanobacteriumthermoautotrophicum.

31. Generalised transduction
•DiscoveredbyZinderandLederberg.
•ThiswasfirstextensivelystudiedinthebacteriumSalmonellatyphimuriumwithphageP22
•Whenthepopulationofsensitivebacteriaisinfectedwiththephage,eithertemperateorvirulent,theeventsofthephagelyticcyclemaybeinitiated.
•Inalyticinfection,thehostDNAoftenbreaksdownintovirus- sizedpiecesandsomeofthesepiecesbecomeincorporatedinsidevirusparticles.
•Uponlysisofthecell,theseparticlesarereleasedwiththenormalvirusparticles,sothatthelysatecontainsamixtureofnormalandtransducingvirusparticles.

32. •Whenthislysateisusedtoinfectapopulationofrecipientcells,mostofthecellsbecomeinfectedwithnormalvirusparticles.
•However,asmallproportionofthepopulationreceivestransducingparticleswhoseDNAcannowundergogeneticrecombinationwiththehostDNA.
•Sinceonlyasmallproportionoftheparticlesinthelysateisofthedefectivetransducingtype,theprobabilityofadefectivephageparticletransferringaparticulargeneisquitelowandusuallyonlyaboutonephageparticleinonemilliontransducesagivenmarker.

33. Generalized transduction

34. In above figure in text box 2 instead of reproduction its replication…………….

35. Specializedtransduction:occursonlyinsometemperateviruses;DNAfromaspecificregionofthehostchromosomeisintegrateddirectlyintothevirusgenome- usuallyreplacingsomeofthevirusgenes.
Generalizedtransduction:hostDNAderivedfromvirtuallyanyportionofthehostgenomebecomesapartoftheDNAofthematurevirusparticleinplaceofthevirusgenome.

36. Specialized transduction
•Incontrasttogeneralized(non-restricted)transduction,whichresultsintransferofanygenefromdonortorecipientbacterialcell,specialized(restricted)transductionisthatwhichleadstothetransferofonlyspecific(restricted)genesfromdonortorecipientcell.
•Specializedtransductionismediatedbythosetemperatebacteriophages(e.g.,lambda(l)phage,mu(m)phageandf80phage)thatusuallyincorporate(integrate)theirDNAintothebacterialchromosome.
•Thephage-DNAiscalledprophageinitsintegratedstatewiththebacterialchromosome;thebacteriumhavingaprophageissaidtobelysogenic,andthisphage-host-relationshipiscalledlysogeny.

37. •Lysogenictemperatephagesspontaneouslyswitchoverfromlysogenictolyticstateatalowrate(aboutonein195celldivisions) innature,ortheymaybeinducedtodosobyirradiationwithultravioletlight.
•Duringthistransition,theprophageisusuallyexcisedpreciselyfromthespecificsiteofintegrationinitsexactlyoriginalform.Butoccasionally,itmayexciseimpreciselysothatittakeswithitthatspecificportionofbacterialchromosomewhichliesclosetothesiteofprophageinsertionandleavesaportionofitsownDNAremainingintegratedwithinthebacterialchromosome.
•Suchprophageiscalledspecializedtransducingprincipleandispackagedintoadevelopingphageparticleinsidethehostbacterialcell.Phageparticlesodevelopediscalledspecializedtransducingphageandisreleasedafterthehostbacterialcellundergoeslysis.

38. •Onlythosespecializedtransducingphagesareviablethatcontainanamountofgreaterthan73%andlessthan110%ofthephage-DNA.Whenaviablespecialized-transducing-phageinfectsanewbacterialcell,itsspecialized-transducingprinciplethatalreadycontainsspecificportionofbacterialchromosomeinsertsintotherecipientbacterialchromosomethusmakingthelatterdiploidforthatspecificbacterialgene(partialdiploidorheterogenoteormerogenote).Sincethespecializedtransducingphageis'defective'phageasithaslostsomegenesduringtheexcision,itfunctionsinrecipientbacterialcellonlywhenthelatterisalreadyinfectedbyanotherphage(termedashelperphage)thatcontainsthemissinggenes.

42. Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together for the actual transfer of DNA.
Bacterial conjugation (mating) is a process of genetic transfer that involves cell-to-cell contact.
Conjugation

43. Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipientcell, which does not.
Thegenes that control conjugation are contained in the traregion of the plasmid. Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili,
Thepilimakespecificcontactwithareceptorontherecipientandthenretract,pullingthetwocellstogether. Thecontactsbetweenthedonorandrecipientcellsthenbecomestabilized,probablyfromfusionoftheoutermembranes,andtheDNAisthentransferredfromonecelltoanother.

44. •Conjugationinvolvingthetransferofanentireplasmidisthemostcommonform.TheF+plasmid(Fisfertilityfactor)undergoesrollingcirclereplication,meaningthatitisreplicatedasalinearsinglenucleosideratherthanacompletecircularstrandofDNAasoccursinreplicationofthechromosome.AfterpassingthroughthepilusintotheF-recipientcell, thecomplementarynucleosideissynthesizedandtheplasmidislinkedintocircularformbyligase.
•Hfr(highfrequencyrecombination)conjugationoccurswhenaplasmidfromtheF+donor,previouslyrecombinedwiththechromosometoproduceanewHfr+cell,ispartiallycopiedalongwithchromosomalgenesbyrollingcirclereplicationandpassedtotherecipientcell.TheDNAfragmentiscompletedandrecombinedwiththerecipientchromosome, butsincealloftheplasmidDNAwasnottransferred,therecipientremainsanF-cellandcannotparticipateinfurtherconjugationwithothercells.

50. Protoplast fusion
•Protoplastsarethecellsofwhichcellwallsareremovedandcytoplasmicmembraneistheoutermostlayerinsuchcells.
•Protoplastcanbeobtainedbyspecificlyticenzymestoremovecellwall.
•Protoplastfusionisaphysicalphenomenon,
•Duringfusiontwoormoreprotoplastscomeincontactandadherewithoneanothereitherspontaneouslyorinpresenceoffusioninducingagents.Byprotoplastfusionitispossibletotransfersomeusefulgenessuchasdiseaseresistance,nitrogenfixation,rapidgrowthrate,moreproductformationrate,proteinquality,frosthardiness,droughtresistance,herbicideresistance,heatandcoldresistancefromonespeciestoanother.
•Protoplastfusionanimportanttoolsinstrainimprovementforbringinggeneticrecombinationsanddevelopinghybridstrainsinfilamentousfungi.
•Protoplastfusionhasbeenusedtocombinegenesfromdifferentorganismstocreatestrainswithdesiredproperties.Thesearethepowerfultechniquesforengineeringofmicrobialstrainsfordesirableindustrialproperties.

51. •In bacteriology, a protoplast may be defined as —‘the sphere remaining after Gram-positive bacteria have had their cell contents lysed; and the bacterial cell wall constitutes are absent’.
•Productionofhybridplantsthroughthefusionofprotoplastsoftwodifferentplantspecies/varietiesiscalledsomatichybridizationandsuchhybridsareknownassomatichybrids.
•Thetechniqueofsomatichybridizationinvolvesthefollowingfoursteps:
1.Isolationofprotoplasts
2.Fusionoftheprotoplastsofdesiredspecies/varieties
3.Selectionofsomatichybridcells,and
4.Cultureofthehybridcellsandregenerationofhybridplantsfromthem.

52. •Significance of Protoplasts Fusion : The various cardinal significance of protoplast fusion are, namely :
(1) For hybridization between genera’ or species that are incapable to cross by the normal and conventional method of sexual hybridization, and
(2) Significance fully realized in plant kingdom by virtue of the fact that the hybrid cells are capable of being inducted to regenerate into whole plants consequently.
•Two types of protoplast fusion:
Spontaneous fusion
Induced fusion

53. Somatic hybridization technique1. isolation of protoplast2. Fusion of the protoplasts of desired species/varieties3. Identification and Selection of somatic hybrid cells4. Culture of the hybrid cells5. Regeneration of hybrid plants

54. Spontaneous fusion
•Protoplastduringisolationoftenfusespontaneouslyandthisphenomenoniscalledspontaneousfusion.
•Simplyphysicalcontactissufficienttobringaboutthespontaneousfusionamongthesimilarparentalprotoplasts.
•Duringtheenzymetreatmentfortheisolationofprotoplast,itisfoundthatprotoplastsfromadjoiningcellsfusethroughtheirplasmodesmatatoformamultinucleateprotoplast.

55. •Spontaneousfusionisstrictlyintraspecificandgiverisetohomokaryon.
•However,oncetheprotoplastsarefreelyisolated, theydon’tfusespontaneouslywitheachother.
•Anexceptionistheprotoplastfrommicrosporocytesofsomeplantsoflilyfamilywherefreelyisolatedprotoplastfusespontaneously.Thistypeofspontaneousfusionhasbeenusedtoproduceintergenericfusione.g.thespontaneousfusionofmicrosporocyteprotoplastofLoliumlongiflorumandTrilliumkamtschalicum.

56. Induced fusion
•Fusionoffreelyisolatedprotoplastsfromdifferentsourceswiththehelpoffusioninducingchemicalagentsisknownasinducedfusion.
•Normally,isolatedprotoplastsdon’tfusewitheachotherastheiroutersurfacecarriesnegativechargesaroundthemandtheyrepeleachother.
•Sothistypeoffusionneedsafusioninducingagentorsystemwhichactuallyreduceselectronegativityoftheisolatedprotoplastsandallowthemtofusewitheachother.

57. Various techniques for induced fusion
(a)Inanimals:Sendaivirus(inactivated)—isrequiredtoinitiatetheprocessofinducedfusion,and
(b)Inplants:
•PEGtreatment;
•NaNO3treatment;
•Calciumiontreatment;
•andelectricalimpulse—areneededtoachievethephenomenonofinducedfusion.

58. PEG treatment
•Polyethyleneglycol(PEG)inducedprotoplastfusionisthemostcommonlyusedasitinducesreproduciblehighfrequencyfusionaccompaniedwithlowtoxicitytomostcelltypes.
•Theprotoplastmixtureistreatedwith28-50%PEGfor15-30min,followedbygradualwashingoftheprotoplaststoremovePEG;protoplastfusionoccursduringthewashing.
•Thewashingmediummaybealkaline(pH9-10)andcontainahighCa2+ionconcentration;thisapproachisacombinationofPEGandhighpH-highCa2+treatments,andisusuallymoreeffectivethaneithertreatmentalone.

59. •PEG is negatively charged and may bind to cations like Ca2+, which , in turn, may bind to the negatively charged molecules present in plasmalemma (plasma membrane).
•During the washing process, PEG molecules may pull out the plasma lemma components bound to them.
•This would disturb plasma lemma organisation and may lead to the fusion of protoplasts located close to each other.

60. Calcium ion treatment
•BhojwaniandRazdan*(1983)devisedamethodinvolvingcentrifugation(spinning)oftheprotoplaststakenupinafusion-inducingsolution(0.05MCaCl2.2H2Oin0.4MmannitolatpH10.5)for30minutesat50°C,
•Afterwhichthetubeswereincubatedatwater-bathmaintainedat37°Cforadurationrangingbetween40-50minutes,whichcausedfusionofprotoplaststotheextentof20-50%.
•However,themethodprovedtobesuperiorincomparisontoothermethodsincertaincases,whereasthehighpH(10.5) turnedouttobetootoxicinotherinstances.

61. Electrofusion Technique
•Itisamoreselectiveandlessdrasticapproachwhichutilizeslowvoltage(65-80Vcm-1) electricpulsestoaligntheprotoplastsinasinglerowlikeapearl-chain.
•Thealignedprotoplastscanbemoved,withamicromanipulator,andpairsofprotoplastsmaybeisolatedinindividualmicroelectrofusionchambers.
•Thepairsofprotoplastscanbefusedbyaverybriefpulseofhighvoltage(500-1000Vcm-1).

62. •Alternatively,theprotoplastsmaybesubjectedtomasselectrofusion.
•Insuchcasethepopulationofprotoplastsissubjectedtohighvoltageaftertheyarebroughtclosetoeachotherbythelowvoltagecurrent.
•Thehighvoltagecreatestransientdisturbancesintheorganisationofplasmalemma,whichleadstothefusionofneighbouringprotoplasts.
•Theentireoperationiscarriedoutmanuallyinspeciallydesignedequipment,calledelectroporator.

63. Fusion induced by Sodium or Potassium Nitrate
•In this method, equal densities of protoplast from two different sources are mixed and then centrifuged at 100 g for 5 mins to get a dense pellet.
•This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to resuspend the protoplast pellet.
•The suspended protoplasts are kept in waterbath at 350C for 5 mins and again centrifuged at 200g for 5 mins.
[g = (1.118 ×10-5) R S2]; where g is relative centrifugal force, R is radius of rotor and S is speed of centrifuge in revolution per minute (RPM).

64. •Thepelletisonceagainkeptinwaterbathat300Cfor30mins.
•Fusionofprotoplasttakesplaceatthetimeofincubation.
•Thepelletisagainsuspendedby0.1%sodiumnitratefor5-10mins.
•Theprotoplastsarewashedtwicewithliquidculturemediumbyrepeatedcentrifugation.
•Finally,theprotoplastsareplatedinsemisolidculturemedium.
•Thefrequencyoffusionisnothighinthismethodandsodiumnitrateistoxictocellathighconcentrationyetthisisoneoftheusefultechniqueforprotoplastsderivedfrommeristematiccells.

66. What is cloning????
•Cloninginbiologyistheprocessofproducingsimilarpopulationsofgeneticallyidenticalindividualsthatoccursinnature.
•CloninginbiotechnologyreferstoprocessesusedtocreatecopiesofDNAfragments(molecularcloning),cells(cellcloning), ororganisms.

67. What is DNA cloning?
•When DNA is extracted from an organism, all its genes are obtained
•In gene (DNA) cloning a particular gene is copied (cloned)

68. •To"cloneagene"istomakemanycopiesofit.
•Actofmakingmanyidenticalcopiesofgene.
•Genecanbeanexactcopyofanaturalgene.
•Genecanbeanalteredversionofanaturalgene.
Theterm“genecloning”coversawiderangeoftechniquesthatmakeitpossibletomanipulateDNAinatesttubeandalsotoreturnittolivingorganismswhereitfunctionsnormally.
What is Gene cloning?

69. Restriction Enzymes
•Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”.
•Cut up “foreign” DNA that invades the cell.
•Type II and III restriction enzymes cleave DNA chains at selected sites.
•Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.
•An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.

70. Type II restriction enzyme nomenclature
•EcoRI –Escherichia colistrain R, 1stenzyme
•BamHI –Bacillus amyloliquefaciensstrain H, 1stenzyme
•DpnI –Diplococcus pneumoniae, 1stenzyme
•HindIII –Haemophilus influenzae, strain D, 3rdenzyme
•BglII –Bacillus globigii, 2ndenzyme
•PstI –Providencia stuartii 164, 1stenzyme
•Sau3AI –Staphylococcus aureusstrain 3A, 1stenzyme
•KpnI –Klebsiella pneumoniae, 1stenzyme
Why the funny names?

71. Figure 1.1The basic steps in gene cloning.

72. •EversincetheBritishscientists,in1997,carriedoutthesuccessfulcloningofsheep(namedDOLLY)bymeticulouslytransferringthenucleusfromanudder-cellofanadultsheeprightintothecytoplasmofanenucleatedfertilizedegg.
•Subsequently,theresulting‘egg’wasneatlytransplantedintotheuterusofasurrogatemotherwhereiniteventuallydevelopedjustlikeanormalzygote
•Ultimatelyintoa‘normallamb’thathasnowgrownintoanormaladultsheep.
•Therefore,onemayrightlyconcludeandinfer,basedontheaboveactualrealisticexperimentalevidences,that—
•‘whencompleteanimalsaredulyaccomplishedfromthesomaticcellsofananimal’—itisusuallytermedas‘animalcloning’.

73. Cloning of Dolly (eg of animal cloning)

74. Cloning of dolly

75. Cloning process
(i) DNA—cloning,
(ii) Cloning larger DNA fragments in specified cloning vectors,
(iii) Cloning Eukaryotic DNAs in bacterial plasmids,
(iv) Cloning Eukaryotic DNAs in phage genomes,
(v) Cloning cDNAs
(vi) Expression cloning.
(vii) Amplifying DNA : The Polymerase Chain Reaction (PCR)

76. DNA-cloning
•TheDNAcloningisnothingbutabroadbasedtechniquewherebylargeamountofaparticularlyDNA-segmentareproduced.
•Usually,theDNAsegmentwhichistobeclonedisfirstlinkedtoavectorDNA,thatservesasavehicleforcarryingforeignDNAintoasuitablehostcell,suchasthebacteriumEscherichiacoli.
•Thevector(i.e.,E.coli)essentiallycontainssequenceswhichinturnpermitstobereplicatedwithinthehostcell.InordertocloneDNAswithinbacterialhoststwotypesofvectorsarecommonlyemployed,namely:
(a)TheDNAsegmenttobeclonedinintroducedintothebacterialcellbyfirstjoiningittoaplasmidandsecondly,causingthebacterialcellstotakeuptheplasmidfromthemedium,and
(b)TheDNAsegmentisjoinedtoaportionofthegenomeofthebacterialviruslambda(λ)whichissubsequentlyallowedtoinfectacultureofbacterialcells.Thus,ahugequantumofviralprogenyareproduced,eachofwhichcontainstheforeignDNAsegment.

77. •ByfollowingeitherofthetwomethodsstatedabovetechniquestheDNAsegmentoncegetsinsideabacterium,itwillundergothereplicationprocesswiththebacterial(orviral)DNAandpartitionedtothedaughtercells(orprogenyviralparticles).
•Inthismanner,theactualnumberofbacterialcellswhichareactuallyformed.
•Besides,cloningmayalsobeemployedasaversatilemethodtoisolateinapureformanyspecificDNAfragmentamongstarelativelylargeheterogeneouspopulationofDNAmolecules.

79. Cloning larger DNA fragments in specified cloning vectors
•Ithasbeenobservedthatneitherplasmidorlambdaphage(λ)vectorsareadequatelysuitableforcloningDNAswhoselengthismorethan20-25kb. ThishasrevitalizedtheinterestofresearcherstolookintothedevelopmentofseveralothervectorswhichmightfacilitatetoclonemuchlargersegmentsofDNA.However,themostimportanttothesevectorsaretermedasyeastartificialchromosomes(YACs).
•YACsarenothingbutartificialversionsofanormalyeastchromosome. TheynormallycompriseofalltheelementsofayeastchromosomewhichareabsolutelynecessaryforthespecificstructuretobereplicatedduringS-phaseandsubsequentlysegregatedtodaughtercellsduringmitosis, including:
•Oneofmoreoriginsofreplication,
•Havingtelomersattheendsofthechromosomes,and
•Acentromeretowhichthespindlefibersmaygetattachedduringchromosomeseparation.

80. •Invariably,theYACsaredesignedinsuchafashionsoastoprovideessentially:
(a)AgenewhoseencodedproductpermitsthoseparticularcellshavingtheYACtobeselectedfromthosethatlacktheelement,and
(b)TheDNAfragmenttobeclonedlikeothercells,subsequentlytheyeastcellsshallpickupDNAfromtheirrespectivemediumthatcatersforthepathwherebyYACsareintroduceddirectlyintothecells.
•IthasbeenobservedthatDNAfragmentsclonedinYACsrangetypicallyfrom100kbto1,000kbinlength.Example:‘Therestrictionenzymeusuallyrecognizestheeight-nucleotidesequenceGCGGCCGC,whichinturnspecificallycleavesmammalianDNAintofragmentsapproximatelyonemillionbasepairslong’.FragmentsofthislengthcannowbeintroducedconvenientlyintoYACsandsubsequentlyclonedwithinhostyeastcells.

81. Fig. Strategy for using YAC

82. Cloning eukaryotic DNA in bacterial plasmid
•AforeignDNAintendedtobeclonedisstrategicallyinsertedintotheplasmidtogivebirthtoarecombinantDNAmolecule.
•However,theplasmidusedforDNAcloningareexclusivelythemodifiedversionsofthoseoccurringinthebacterialcells.
•Consequently,thebacterialcellsareabletotakeupDNAfromtheirmedium.Thisparticularphenomenonistermedas‘transformation’andformsthebasisforcloningplasmidinbacterialcells.
•Fig.2.8.representstheDNAcloningusingbacterialplasmid. FirstofalltherecombinantplasmidseachcontainingadifferentforeignDNAinsertareaddedtoabacterialculture(E.coli)whichhasbeenpreviouslytreatedwithCa2+ions.

83. •ThesebacteriaaregainfullystimulatedtotakeupDNAfromtheirrespectivesurroundingmediumuponexposuretoabriefthermal-shocktreatmentyieldingplasmidDNA(purified).
•Secondly,humanDNAarealsoobtainedinthepurifiedform. SubsequenttreatmentofhumanDNAandplasmidDNAwithEcoR1resultintothecleavageofhumanandbacterialDNAintovarioussizedfragments.
•Now,thesesmallfragmentsjointogethertoyieldrecombinantDNAswithDNAligaseandthusgiverisetotheplasmids.ThesepopulationofplasmidsinvariablycontainvarioussegmentsofhumanDNA.
•Incubation of these plasmids with E. coli cells under controlled experimental parameters ultimately yields plasmid that are free from E coli.

84. •Ithasbeenobservedthatonlyaverysmallpercentageofthecellsarecompetenttopickupandretainoneofthecombinantreplicatemolecules.
•Onceitistakenuptheplasmidundergoesreplicationautonomouslywithintherecipientandissubsequentlypassedontoitsprogenyduringcelldivision.
•Theisolatedrecombinantplasmidscanthenbetreatedwiththesamerestrictionenzymesusedintheirformation,thatreleasestheclonedDNAsegmentsfromtheremainderoftheDNAwhichservedasthevector.Thus,theclonedDNAcanbeseparatedfromtheplasmid.

86. Cloning eukaryotic DNAs Phage genome
•Abacteriophase,ormorecommonlyaphageisavirusparticlewhichinfectsabacterialcell.
•Infact,aphageparticlenormallycomprisesoftwoessentialcomponents;first,aphageheadthatcontainsthegeneticmaterialandsecondly,atailthroughwhichthegeneticmaterialisinjectedintothehostcell.
•Interestingly,oneofthemostbroadlyexploredofthethesephageparticles, termedBacteriophageLambda[orbacteriophage(λ)],hasmoreorlessturnedouttobeacommonlyemployedcloningvector.
•Thegenomeoflambdaisalinearanddouble-strandedDNAmoleculehaving50kblength.
•Inusualpractice,themodifiedstrain(mutant)employedinmostcloningexperimentscontainstwocleavagesitesfortheenzymesEcoR1thatultimatelyfragmentsthegenomeintothreelargesegments.
•However,thetwooutersegmentsessentiallycontainallinformationsrequiredfortheinfectiousgrowth,whereasthemiddlefragmentcouldberejectedconvenientlyandreplacedsuitablybyapieceofDNAupto25kbinlength.

87. •ThetwooutersegmentsofthebacteriophageundergosplicingwitheukaryoticfragmenttoresultintotheformationofrecombinantDNA. Consequently,therecombinantDNAmoleculescanbepackagedintophageheadsinvitroandinturnthesegeneticallyengineeredphageparticlemaybeemployedtoinfecthostbacteria.
•Oncegainingentryintothebacteria,theeukaryoticDNAsegmentisadequatelyamplifiedalongwiththeviralDNAandsubsequentlypackagedintoanaltogethernewgenerationofvirusparticlethatarereleasedwhenthecellundergoeslysis.
•Thereleasedparticlethusobtainedinfectnewcells,andwithoutanylossoftimeeitheraplaque(Azoneoflysisorcellinhibitioncausedbyvirusinfectiononalawnofcells)oraclearspotinthe‘bacteriallawn’isvisibledistinctlyatthesiteofinfection.Eachplaque,whichisnothingbutazoneoflysis,possessesmillionsofphageparticle,eachcarryingasinglecopyofthesameeukaryoticDNAsegment.)

88. Fig. Sequence for cloning DNA fragments in Lambda (I) phage.

89. Cloning cDNA
•Thecentraldogmaofmolecularbiologyoutlinesthatinsynthesizingproteins,DNAistranscribedintomRNA,whichistranslatedintoprotein.Onedifferencebetweeneukaryoticandprokaryoticgenesisthateukaryoticgenescancontainintrons(interveningsequences)whicharenotcodingsequences(incontrastwithexonswhicharecodingsequences),andmustberemovedfromtheRNAprimarytranscriptbeforeitbecomesmRNAandcanbetranslatedintoprotein.Prokaryoticgeneshavenointrons,sotheirRNAisnotsubjecttosplicing.
•Oftenitisdesirabletoexpresseukaryoticgenesinprokaryoticcells.AsimplifiedmethodofdoingsowouldincludetheadditionofeukaryoticDNAtoavector, sometimesaprokaryotichost,whichwouldthentranscribetheDNAtomRNAandthentranslateittoprotein.However,aseukaryoticDNAhasintrons,andsinceprokaryoteslackthemachinerytosplicethem,thesplicingofeukaryoticDNAmustbedonepriortoaddingtheeukaryoticDNAintothehost.ThisDNA, whichwasmadeasacomplementarycopyoftheRNAandhasnointrons,iscalledcomplementaryDNA(cDNA).ToobtainexpressionoftheproteinencodedbytheeukaryoticcDNA,prokaryoticregulatorysequenceswouldalsoberequired(e.g.apromoter).

90. •ThecloningthathasbeendescribedherewillworkforanyrandompieceofDNA. ButsincethegoalofmanycloningexperimentsistoobtainasequenceofDNAthatdirectstheproductionofaspecificprotein,anyprocedurethatoptimizescloningwillbebeneficial.OnesuchtechniqueiscDNAcloning.
•TheprinciplebehindthistechniqueisthatanmRNApopulationisolatedfromaspecificdevelopmentalstageshouldcontainmRNAsspecificforanyproteinexpressedduringthatstage.Thus,ifthemRNAcanbeisolated,thegenecanbestudied.
•mRNAcannotbecloneddirectly,butaDNAacopyofthemRNAcanbecloned.(Inthisregard,thetermcDNAisshortfor"copyDNA".)ThisconversionisaccomplishedbytheactionofreversetranscriptaseandDNApolymerase.Thereversetranscriptasemakesasingle-strandedDNAcopyofthemRNA.
•ThesecondDNAstrandisgeneratedbyDNApolymeraseandthedouble-strandedproductisintroducedintoanappropriateplasmidorlambdavector.

91. •InordertoclonecDNAs,firstofallasizablepopulationofmRNAisisolated;
•secondly,itisemployedasatemplatetoprovideasingle- strandedDNAcomplement;
•thirdly,theresultingproduct(singlestranded)isdulyconvertedtoadoublestrandedpopulationwiththehelpofaDNApolymerase;
•andfourthly,theyarefinallycombinedwiththedesiredvector. ItisquiteevidentthatessentiallymRNApopulationstypicallyconsiststhousandsofaltogetherdifferentspecies,andaswithexperimentsemployinggenomicDNAfragments,theclonesshouldbeinvariablyscreenedtoisolateonlyoneparticularsequencefromaheterogeneouspopulationofrecombinantmolecule.

92. Fig: Sythesis whereby cDNA get cloned in plasmid.
itmaybeobservedthatwhenpolypeptide(A)andmRNAareannealed,itprovidesasmallsegmentofprimerattachedtopoly(A)tothetailofmRNA.Now, withthehelpofreservetranscriptasetheprimertopoly(A)getsfullydeveloped.AlkalihelpsintheseparationofDNAandRNAstrandstogiverisetofullydevelopedprimeralone, whichontreatmentwithRNApolymerase1yieldsthecombinedproduct.TheresultingproductwhendigestedwithS1nucleasetwoseparatestrandsoftheprimerandpoly(A)areobtained. Lastly,integratecDNAintotheplasmidvectorthatwillproduce
abacteriumwhereinDNAcanbecloned.

93. Expression cloning
•ExpressioncloningisatechniqueinDNAcloningthatusesexpressionvectorstogeneratealibraryofclones,witheachcloneexpressingoneprotein.
•Thisexpressionlibraryisthenscreenedforthepropertyofinterestandclonesofinterestrecoveredforfurtheranalysis.
•AnexpressionvectorisarelativelysmallDNAmoleculethatisusedtointroduceandexpressaspecificgeneintoatargetcell.Oncetheexpressionvectorisinsidethecell,theproteinthatisencodedbythegeneisproducedbythecellulartranscriptionandtranslationmachinery.
•Usuallytheultimateaimofexpressioncloningistoproducelargequantitiesofspecificproteins.
•Expressionvectorsareusedformolecularbiologytechniquessuchassite- directedmutagenesis.Ingeneral,DNAvectorsthatareusedinmanymolecularbiologygenecloningexperimentsneednotresultintheexpressionofaprotein.

94. •Expressionvectorsareoftenspecificallydesignedtocontainregulatorysequencesthatactasenhancerandpromoterregions,andleadtoefficienttranscriptionofthegenethatiscarriedontheexpressionvector.
•Expressionvectorsarebasictoolsforbiotechnologyandtheproductionofproteinssuchasinsulinthatareimportantformedicaltreatmentsofspecificdiseaseslikediabetes.
•Expressionvectorsareusedformolecularbiologytechniquessuchassite- directedmutagenesis.Ingeneral,DNAvectorsthatareusedinmanymolecularbiologygenecloningexperimentsneednotresultintheexpressionofaprotein.
•Expressionvectorsareoftenspecificallydesignedtocontainregulatorysequencesthatactasenhancerandpromoterregions,andleadtoefficienttranscriptionofthegenethatiscarriedontheexpressionvector.
•Expressionvectorsarebasictoolsforbiotechnologyandtheproductionofproteinssuchasinsulinthatareimportantformedicaltreatmentsofspecificdiseaseslikediabetes.

95. :::::Note:::::
•AllvectorsusedforpropagationofDNAinsertsinasuitablehostarecalledcloningvectors.
•Butwhenavectorisdesignedfortheexpressionofi.e.productionoftheproteinspecifiedby,theDNAinsert,itistermedasexpressionvector.
•Asarule,suchvectorscontainatleasttheregulatorysequences,i.e.promoters, operators,ribosomalbindingsites,etc,havingoptimumfunctioninthechosenhost.
•Therearemanytypesofcloningvectors.Geneticallyengineeredplasmidsandbacteriophages(suchasphageλ)areperhapsmostcommonlyusedforthispurpose.Othertypesofcloningvectorsincludebacterialartificialchromosomes(BACs)andyeastartificialchromosomes(YACs).
•Inthecaseofexpressionvectors,themainpurposeofthesevehiclesisthecontrolledexpressionofaparticulargeneinsideaconvenienthostorganism(e.g.E.coli).Controlofexpressioncanbeveryimportant;itisusuallydesirabletoinsertthetargetDNAintoasitethatisunderthecontrolofaparticularpromoter.SomecommonlyusedpromotersareT7promoters,lacpromoters(blapromoter)andcauliflowermosaicvirus's35spromoter(forplantvectors).

96. Applications
ProteinProduction-AnexpressionvectorwithproteinproducingDNAisimportedintoacellthroughtheuseofplasmidsintoabacterialcell.Thiscausesthebacterialcelltothenproducesaidprotein.Thismethodcanbeusedinmedicinetofarmproteinsonawide- scalesuchasInsulin,Erythyroprotein,orTissuePlasminogenActivator.

97. Ingenetics,apromoterisaregionofDNAthatfacilitatesthetranscriptionofaparticulargene.Promotersarelocatednearthegenestheyregulate,onthesamestrandandtypicallyupstream(towardsthe5'regionofthesensestrand).
AnenhancerisashortregionofDNAthatcanbeboundwithproteins(namely,thetrans- actingfactors,muchlikeasetoftranscriptionfactors)toenhancetranscriptionlevelsofgenes(hencethename)inagenecluster.
Fig. The creation of an expression vector with which proteins can be grown.

98. Amplifying DNA: The polymerase chain reaction (PCR)
•Gene Amplification through Polymerase chain reaction
•The polymerase chain reaction is one of the powerful gene amplification technique developed by Kary Mullis in 1985.
•It generates microgram quantities (upto billion copies) of DNA copies of the desired DNA (or RNA) segment, present even as a single copy in the initial preparation, in a matter of few hours.
•The PCR utilizes the following:
1. DNA preparation containing the desired segment to be amplified
2. Two nucleotide primers (about 20 bases long) specific, i.e. complementary, to 3’-borders (the sequences present at the 3’-ends of the two strands) of the desired segment,

99. •Thefourdeoxynucleosidetriphosphates[dTTP(deoxythymidinetriphosphate),dCTP(deoxycyctidinetriphosphate),dATP(deoxyadenosinetriphosphate)anddGTP(deoxyguanosinetriphosphate),andaheatstableDNApolymerase,e.g.Taq(isolatedfromthebacteriumThermusacquaticus),pfu(fromPyrococcusfuriousus)andVent(fromThermococcuslitoralis)polymerases.PfuandVentpolymerasesaremoreefficientthantheTaqpolymerase.
Procedure of PCR:
•At the start of PCR, the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxynucleoside triphosphates and the DNA polymerase are mixed together in the reaction mixture that has appropriate quantities of Mg2+
•The following operations are now performed sequentially:
Denaturation:
•The reaction mixture is first heated to a temperature between 90-98 0C (commonly at 94 0C) that ensures DNA denaturation. This is the denaturation step. The duration of this step in the first cycle of PCR is usually 2 min at 94 0C.

100. Annealing:
•Themixtureisnowcooledtoatemperature(generally,between40-600C) thatpermitsannealingoftheprimertothecomplementarysequencesintheDNA.Asarule,thesesequencesarelocatedatthe3’endsofthetwostrandsofthesegmenttobeamplified.Thisstepiscalledannealing.
•Thedurationofannealingstepisusually1minduringthefirstaswellasthesubsequentcyclesofPCR.SincetheprimerconcentrationiskeptveryhighrelativetothatofthetemplateDNA,primer-templatehybridformationisgreatlyfavouredoverreannealingofthetemplatestrands.
PrimerExtension:
•ThetemperatureisnowsoadjustedthattheDNApolymerasesynthesizesthecomplementarystrandsbyutilizingthe3’-OHoftheprimers,thisreactionisthesameasthatoccursinvivoduringreplicationoftheleadingstrandofaDNAduplex.
•TheprimersareextendedtowardseachothersothattheDNAsegmentlyingbetweenthetwoprimersiscopied.
•Thedurationofprimerextensionisusually2minsat720C.

101. •Thecompletionoftheextensionstepcompletesthefirstcycleofamplification;eachcyclemaytakefewminutes.
•ItshouldbenotedthattheextensionofprimercontinuestillthestrandsareseparatedduringthedenaturationstepofthenextPCRcycle.
•Theproductoffirstcycleisthe‘longproduct’.
•Numeroussubsequentcycletakesplacesimilartofirstcyclethusamplifyingthethegeneexponentially.

104. Development of hybridoma for monoclonal antibody (Mas)
•Ahybridomaisahybridcellobtainedbyfusingantibody–producingcellandamultiplemyelomacell.(Multiplemyelomaisacancerofplasmacell.)
•ORItmaybedefinedasahybridcellobtainedbyfusingB-lymphocytewithusuallyatumorcelloftheantibodyformingsystemorofB-lymphocytes(thesearecalledmyelomas).
•ThehybridcellsthusproducedpossesstheabilitytoproduceantibodiesduetotheB-lymphocytegenomeandthecapacityforindefinitegrowthinvitroduetothetumor(myeloma)cellinvolvedinthefusion.

105. •Therefore,hybridomacellsareeitherculturedinvitroorpassagedthroughmouseperitonealcavitytoobtainmonoclonalantibodies.Thisiscalledhybridomatechnology.
•B-lymphocytesareisolatedfromthespleenofananimal,e.g,mouse,whichhadbeenimmunizedwiththeantigenagainstwhichmonoclonalantibodiesaretoberaised(produced).
•Myelomacellsareselectedformainlytwofeatures: (1)thesecellsmustnotproduceantibodiesthemselves,and

106. •(2) they must contain a genetic marker e.g. HGPRT-trait (hypoxanthine –guanine phospho-ribosyltransferase), which permits an easy selection of the resulting hybrid cells.
•When HGPRT-cells are fused with B- lymphocytes, the resulting cell population will consist of (1) hybrid cells (hybridomes), (2) myeloma cells (3) B-lymphocytes.
•This cell population is now cultured in HAT medium containing the drug aminopterin.

107. •Similarly,theB-lymphocytesdonotgrowforlongperiodsoftimeintissuecultureandeventuallydie.
•Incontrast,onlythehybridomacellsproliferateontheHATmediumsincetheB-lymphocytegenomemakesthemHGPRT+andtheyhavethecapabilityforindefinitegrowthfromthemyelomacell.
•Thushybridomas(myeloma+B-lymphocytehybridcells)areselectedbyusingasuitableselectivemediumlikeHAT,whichallowsonlythehybridomastoproliferate.

108. •Thenextstepconsistsofidentificationandisolationofthehybridomacellsproducingantibodiesspecifictotheantigenusedforimmunizationoftheanimals.
[Thehybridomacellsaresuspended,suitablydilutedanddistributedintomicro-wells,onecellineachmicro-well,andallowedtogrow.Thehybridomacellsgrowandsecreteantibodiesintothemedium.Thesupernatantfromeachmicro-wellissampledandassayedforthepresenceofantibodiesspecifictotheantigeninquestionusingoneoftheassaymethodse.g.ELISA.Wellscontainingtheantibodiesspecifictotheantigenareidentifiedandthehybridomacellsfromthemisolatedandclonedtoensurethatahybridomacloneproducesantibodiesofasinglespecificity.]

109. •Oncethedesiredhybridomaclonehasbeenobtained,itismultipliedeitherinvitroorinvivotoobtainmonoclonalantibodies.
•Invivoproductionsysteminvolvesinjectionofhybridomacellsintotheperitonealcavityofisogenicanimals,collectionoftheasciticfluidandseparationoftheantibodiesfromit.
•Invitroproductioninvolvesgrowinghybridomacellsaregrowninvitroinasuitablelargescaleculturesystemandthemonoclonalantibodiesarepurifiedfromthesecultures.

110. HAT medium
•HATmediumisoneoftheseveralselectivemediausedfortheselectionofhybridcells.
•Thismediumissupplementedwithhypoxanthine, aminopterinandthymidine,hencethenameHATmedium.
•Antimetaboliteaminopterinblocksthecellularbiosynthesisofpurinesandpyrimidinesfromsimplesugarsandaminoacids.
•However,normalhumanandmousecellscanstillmultiplyastheycanutilizehypoxanthineandthymidinepresentinthemediumthroughasalvagepathway,whichordinarilyrecyclesthepurinesandPyrimidesproducedfromdegradationofnucleicacids.

111. •HypoxanthineisconvertedintoguaninebytheenzymeHGPRT,whilethymidineisphosphorylatedbythymidinekinase(TK);bothHGPRTandTKareenzymesofthesalvagepathway.
•OnaHATmedium,onlythosecellsthathaveactiveHGPRT(HGPRT+)andTK(TK+)enzymescanproliferate,whilethosedeficientintheseenzymes(HGPRT-and/orTK-)cannotdivide.
•Thus,onemayfuseHGPRTdeficienthumancells(designatedasTK+HGPRT-)withTKdeficientmousecells(denotedasTK- HGPRT+).Theirfusionproducts(hybridcells)willbeTK+(duetohumangene)andHGPRT+(duetomousegene)andwillmultiplyontheHATmedium,whilethemanandmousecellswillfailtodoso.

112. Application of monoclonal antibodies (Mabs)
•Diagnosticapplication:
WhenMabsareusedtodetectthepresenceofaspecificantigenorofantibodiesspecifictoanantigeninasampleorsamples,thisconstitutesadiagnosticapplication.Someexamplesofdiagnosticapplicationsareasfollows:
1.Mabsareavailablefortheunequivocalclassificationofbloodgroupse.g.ABO,Rh, etc.

113. 2. Mabs are applied for a clear and decisive detection of pathogens involved in disease (disease diagnosis).
3. Mabs can be used for the accurate detection of specific chromosomes of a given species.
Therapeutic application:
1. Antibodies specific to a cell type, say, tumor cells, can be linked with a toxin polypeptide to yield a conjugate molecule called immunotoxin. The antibody component of immunotoxin will ensure its binding specifically and only to the target cells and the attached toxin will kill such cells.

114. 2. Mabs can be administered to provide passive immunity against diseases.
3. Mabs are very useful in the purification of antigens specific to pathogens; these purified antigens are used as vaccines.
Immunopurification:
The highly specific interaction of an antibody to the antigen is used to purify antigens present in small quantities as a mixture with several types of molecules; this is known as immunopurification.

115. Drugs produced by biotechnology
•TheEuropeanFederationofBiotechnology(FEB)considers‘biotechnology’as—‘theintegrationofnaturalsciencesandorganisms,cells,partsthereof,andmolecularanaloguesforproductsandservices.’
•NewBiotechnologicalprocessesessentiallyembracealmostallmethodsofgeneticmodificationbyrecombinantDNAandcellfusiontechniques,togetherwiththe‘magictouch’ofthemoderndevelopmentsoftheso-called‘traditional- biotechnologicalprocesses’.
•Interestingly,theseprocesseswill,inmanyinstances,functionatrelativelylowtemperature,willconsumelittleenergy,andwillrelymainlyoninexpensivesubstratesforbiosynthesis.

116. •Althoughtherearealargenumberof‘drugs’thathavebeenevolvedviathebiotechnologicalprocesses.Fewofwhicharelistedbelow:
(i)Altepase[Activase®],
(ii)HumanInsulin[Humulin(R)],
(iii)Humatrope:GrowthHormone,and
(iv)HepatitisB[RecombinantHB(Merck)—aHepatitisBvaccine]

117. Alteplase [ Activase®]
•Activase®(Alteplase)isFDA-approvedfortreatmentofmyocardialinfarction(heartattack),acuteischemicstroke(bloodclotinthebrain)&acutemassivepulmonaryembolism(bloodclotsinthelungs).
•Activaseisoneoftherecombinanttissueplasminogenactivator,orr-tPAandisproducedbyrecombinantDNAtechnology.
•Forcertainpatients,Activasemayimprovethechancesofrecoveryfromstrokewithlittleornodisability.PatientscanreceiveActivaseonlyiftheybegintreatmentwithin3hoursaftertheirstrokesymptomsstartandonlyaftertheyhavehadascantoruleoutbleedinginthebrain.
•Tissueplasminogenactivator(abbreviatedtPAorPLAT)isaproteininvolvedinthebreakdownofbloodclots.Itisaserineproteasefoundonendothelialcells,thecellsthatlinethebloodvessels.

118. •Asanenzyme,itcatalyzestheconversionofplasminogentoplasmin,themajorenzymeresponsibleforclotbreakdown.Becauseitworksontheclottingsystem,tPAisusedinclinicalmedicinetotreatonlyembolicorthromboticstroke.Useiscontraindicated(notadvisable)inhemorrhagicstrokeandheadtrauma.
•tPAmaybemanufacturedusingrecombinantbiotechnologytechniques. tPAcreatedthiswaymaybereferredtoasrecombinanttissueplasminogenactivator(rtPA).Recombinanttissueplasminogenactivators(r-tPAs)includealteplase,reteplase,andtenecteplase(TNKase).
•Storage: Alteplase need to be stored preferably at –20°C or even below in perfectly sealed containers.

119. •Units:TheactivityofalteplasemaybemeasuredintermsofInternationalUnits(IU)byemployingthe2ndInternationalStandardfortheTissuePlasminogenActivatorestablishedin1987,althoughitisanusualpracticetoexpressthedosesbyweight.TheSpecificActivityofalteplaseis580000IUs.mg–1.
•Pharmacokinetics : It has been duly observed that alteplasegets cleared from the plasma, chiefly via metabolism in the liver.
Note: IU is the amount of an enzyme that catalysesthe transformation of 1 micromole of substrate per minute (under defined conditions of pH, concentration, and temperature)

120. Uses and Mechanism of Action :
•Thevariousapplicationsandpossiblemechanismofactionof‘alteplase’areasfollows:
(1)Itisathrombolyticagent,whichisapredominantrepresentativeofasingle-chainformoftheendogenousenzymetissueplasminogenactivatormeticulouslyproducedbytherecombinantDNAtechnology.
Verymuchsimilartotheendogenoustissueplasminogenactivator, itconvertsfibrin-boundplasminogentothecorrespondingactiveformofplasmin,therebycausinginmarkedandpronouncedfibrinolysisanddissolutionofclots.
(2)Alteplaseisemployedverymuchakin(similarinqualityandcharacter)tosteptokinasebothinthemanagementandtreatmentofthrombo-embolicdisorders,specificallythemyocardialinfarctionandvenousthrombo-embolism.

121. (2)Alteplasehasalmostnegligibleeffectuponthecirculating, unboundplasminogen;andhence,maybetermedasafibrin- specificagent.
Itwasperhapsthoughtthatfibrinspecificitycouldbeanabsolutenecessityforminimisingtheprevailingriskofensuinghaemorrhageintimatelyassociatedwiththeapplicationofthrombolytics;althoughthelatestfibrinspecificdrugsusuallygiverisetoappreciablebleedingincomparisontothenon-specificthrombolytics.

122. Humulin : Humulin®
•Humulin:Humulin®isthebrandedproductofthefamouspharmaceuticalmanufacturer,Lilly,containinghuman-insulinanditshostofvariants,beingproducedbyitindifferentcountriesacrosstheglobe.
DescriptionofInsulin:
•Insulinisapancreatic-hormoneessentiallyinvolvedintheregulationofblood- glucoseconcentrationsandalsohavingaspecificroleintheproteinandlipidmetabolism.
•Inusualpractice,thehuman,porcine,bovineormixedporcine-bovineinsulinisadministeredtosuchpatientshavinginsulin-dependentdiabetesmellitusinthemanagementandcontroloftheirblood-glucoseconcentrations.
Itmayalsobeusednecessarilyincertainnon-insulin-dependentdiabetics.Insulinisalsoanessentialcomponentoftheemergencymanagementandcontrolofdiabeticketoacidosis.
Insulinis—‘ahormoneproducedbytheβ-cellsoftheisletsofLangerthansofthepancreasandessentiallycompriseoftwoseparatechainsofaminoacids, theAandBchains,joinedtogetherbytwodisulphidebridges’.

124. Human insulin
•Humaninsulinisadimercomprisingonechainof21aminoacid(Achain)andtheother30aminoacids(Bchain).
•BoththechainsAandBarederivedfromasinglepolypeptidechain,andareheldtogetherbytwodisulphidebridges.
•Generally,theinsulingenecodesforpreproinsulin,apolypeptidecontainingtheAandBpolypeptidesoftheactiveinsulin,andaconnectingpolypeptidethatisabsentfrommatureinsulin.
•InsulinisprocessedfrompreproinsulinviaproinsulinbyenzymaticcleavageoftheconnectingpolypeptidefromtheAandBchains.

125. Production of human insulin
•OneoftheprocessesdevelopedbyElilillyincollaborationwithGenetechinvolvesthefollowinsteps:
•Thegenes(=DNAsequences)forchainsAandBofinsulinweresynthesizedseparately.Eachgenecontainsmethioninecodonat5’endandstopcodonat3’ end.
•ThesegenewereintegratedseparatelywiththelacZαgene(encodingβ-galactosidase)oftwoseparatepBR322plasmids.

126. •TheseplasmidsweretransformedintoE.colistrains.
•ThetransformedbacteriathusproducesthefusionproteinofAchainandBchainseparately.
•TheAandBchainswereseparatedbyamethionineresiduefromtheβ-galactosidasesequenceencodedbylacZα.
•Therefore,theinsulinsequenceswereseparatedfromtheβ- galactosidasesequencesbytreatingthefusionproteinswithcyanogenbromide.
•ThepurifiedchainsAandBwerethenattachedtoeachotherbydisulphidebondstoproduceinsulin.

127. •Thismethodhoweverturnedouttobeaninefficientreaction.
•Therefore,agenerepresentingB,CandAChainswassynthesizedandexpressedinE.coli;inthiscase,theinterveningchainisremovedproteolyticallyfollowingspontaneousfoldingoftheproinsulinmolecule.

128. Hepatitis B [Recombivax HM (Merck) —A Hepatitis B Vaccine]
•TheRecombivaxHB(Merck),ahepatitisBvaccine,isoneofthemostrecentandsignificantdevelopmentsinthefieldofrecombinantDNAtechnology,thatessentiallycompriseofhighlyspecificantibodieswhichactlikemagicbullets.
•Ithasbeendulyobservedthathepatitistendstocauseasevereacuteinfectionandmayultimatelyleadtochronicinfectionandpermanentliverdamage.ItisessentiallycausedbyhepatitisBvirus(HBV);andrecognizedasanenvelopedanddouble-strandedDNAvirus.
•Ithasbeenadequatelyrevealedthroughmeticulousstudiesthatindividualswhoareatthemostvulnerableandgreatestriskforinfectioninclude:IV-drugabusers(e.g.,morphine/heroinaddicts);homosexualmen;HBV-infectedmothers;andaboveallthehealthcareworkers.
•Various steps being followed in a sequential manner with regard to the production of a genetically engineered vaccine e.g., Hepatitis B Vaccine.

130. •Step1:Geneticmaterial(DNA)isextractedfromtheensuinghepatitisvirus.Atthisstagethe‘surfaceproteins’essentiallyprovokeanimmuneresponse.
•Step2:The‘individualgenes’areadequatelyanalyzedandidentified.
•Step3:The‘specificgene’whichcategoricallydirectsproductionofsurfaceproteinislocatedcarefully.
•Step4:InthismostcriticalstepsthegeneisremovedfromtheviralDNAandinsertedintotheplasmidcarefully.
•Step5:Theplasmidsaremeticulouslyinsertedintothecorrespondingyeastcells.
•Step6:Yeastisallowedtogrowviafermentation.Inthismannerthecellsreproduceandgeneratemorequantumofsurfaceprotein.
•Step7:Afteradurationof48hoursthecorrespondingyeastcellsarerupturedtofreetheensuing‘surfaceprotein’.Theresultingmixtureisdulyprocessedsoastoextractthepurify
•thesurfaceprotein.
•Step8:Alargeamountofsurfaceproteinparticles,initspurestform,areobtainedwhich
•ultimatelyprovokeanimmuneresponseeffectively.
•Step9:Theresultingsurfaceproteinsareadequatelymixedwithappropriatepreservationstogetherwithotheringredientstoobtainthevaccine.

131. Humatrope® [Growth Hormone]
•Growthhormoneisananabolichormonesecretedbytheanteriorpituitarywhichstimulatestissuegrowthandanabolism.Itisfoundtoaffectfat,carbohydrateandmineralmetabolism.
•Humatrope(somatropin):
ItisapolypeptidehormoneofrDNAorigin. ManufacturedbyEliLillyandCompany,itisusedtostimulatelineargrowthinpediatricpatientswholackadequatenormalhumangrowthhormone.Ithas191aminoacidresiduesandamolecularweightof22,125daltons.

132. •Itsaminoacidsequenceisidenticaltothatofhumangrowthhormoneofpituitaryorigin(anteriorlobe).HumatropeissynthesizedinastrainofE.colimodifiedbytheadditionofageneforhumangrowthhormone.
•OtherhumangrowthhormoneproducedfromrDNAsinclude;Omnitrope(Sandoz),Nutropin,Norditropin,Genotropin(Pfizer).
•Units:OneAmpouleoftheFirstInternationalStandard(1987) :4.4unitsofthehumangrowthhormone(somatropin)arecontainedin1.75mgoffreeze-driedpurifiedhumangrowthhormone,with20mgofglycine,2mgofmannitol,2mgoflactose,and2mgofsodiumbicarbonate.

133. Pharmacokinetics:
•Somatropiniswell-absorbedaftersubcutaneousorIMinjection.
•AfterIVinjectionithasahalf-lifeofabout20-30minutes; however,aftersubcutaneousorIMadministrationtheprevailingserumconcentrationsusuallydeclinehavingahalf- lifeof3-5hours,onaccountoftherelativelymoreprolongedreleasefromthesiteofinjection.Itisfoundtobemetabolisedintheliverandexcretedinbile.

134. Uses and Mechanism of Action :
•Somatropinisasynthetichumangrowthhormone;andSomatremisitscorrespondingmethionylanalogue.The‘drug’promotesthegrowthofmuscular,skeletal,andothertissues, stimulatesproteinanabolism;andalsoaffectsfatandmineralmetabolism.
•Thehormoneexhibitsadiabetogenicactionuponthecarbohydratemetabolismspecifically.
•Thesecretionisobservedtobepulsatileandsolelydependsupontheneuralandhormonalinfluences,suchas:(a) hypothalamicrelease-inhibitinghormonee.g.,somatostatin, and(b)hypothalamicreleasinghormonee.g.,somatorelin.Infact,therearecertainphysicalandphysiologicalfactorsthatlargelyinfluenceanenhancedsecretionofthegrowthhormone,suchas:sleep,emotionalstress,andhypoglycaemia.