简介：WedescribedtheconstructionofBACcontigsofthegenomeofaindicavarietyofOryzasativa.GuangLuAi4.Anentirerepresentative(Sixfoldcoverageofricechromosomes)andgeneticallystableBAClibraryofricegenomeconstructedinthislabhasbeensystematicallyanalysedbyrestrictionenzymefragmentationandpolyacrylamidegelelectrophoresis.Andalltheimagesthusobtainedweresubjecttoimage-processing,whichconsistedofpreliminarylocationofbands,cooperativetrackingoflanesbycorrelationofadjacentbads.aprecisedensitometricpass,alignmentatthemarkerbandswiththestandard,optionalinteractiveediting,andnormalizationoftheacceptedbands.ThecontigsweregeneratedbasedontheComputerSoftwarespeciallydesignedforgenomemapping.Thenumberofcontigswith600kbinlengthonaveragewas464.ofcontigswith1000kbinlengthonaveragewas107;ofcontigswith1500kbinlengthonaveragewasConstructionofOryzaSativagenomecontigs.23.Therefor,allthecontigswehaveobtainedampuntedupto420megabasesinlength.Consideringthesizeofricegenome(430megabased),thecontigsgeneratedinthislabhavecoverednearly98%ofthericegenome.Wearenowintheprocessofmappingthecontigstochromosomes.

简介：Asimplemethodtocreateachromosome-specificDNAlibrqaryofrice,includingmicrodissection,amplification,charterizationandcloning,isdescribed.Ricechromosome4fromametaphasecellhasbeenisolatedandamplifiedbytheLinkerAdapterPCR(LA-PCR).ThePCRproductswerelabeledasprobeswithDIG-11-dUTPusingtherandomprimingmethod.SouthernblotanalysiswithricegenomicDNAandspecificRFLPmarkersdemonstratedthatthePCRproductswerederivedfromricechromosome4.Alargelibrarycomprisingover100,000recombinantplasmidmicroclonesfromricechromosome4wasconstructed.Colonyhybridizationshowedthat58%oftheclonescontainedsingleorlow-copysequencesand42%containedrepetitivesequences.ThesizeofinsertsgeneratedbyPCRrangedfrom140bpto500bp.ThismethodwillfacilitatecloningofthespecificchromosomeDNAmarkersandimportantgenesofrice.

简介：Thec-erbB-2proto-oncogeneencodesa185kDaproteinp185,whichbelongstoepidermalgrowthfactorreceptorfamily.Amplificationofthisgenehasbeenshowntocorrelatewithpoorclinicalprognosisforcertaincancerpatients.ThemonoclonalantibodyA21whichdirectedagainstp185specificallyinhibitsproliferationoftumorcellsoverexpressingp185,henceallowsittobeacandidatefortargetedtherapy.InordertoovercomeseveraldrawbacksofmurineMAb,wecloneditsVHandVLgenesandconstructedthesingle-chainFv(scFv)throughapeptidelinker.TherecombinantscFvA21wasexpressedinEscherichiacoliandpurifiedbytheaffinitycolumn.SubsequentlyitwascharacterizedbyELISA,Westernblot,cellimmunohistochemistryandFACS.Alltheseassaysshowedthebindingactivitytoextracellulardomain(ECD)ofp185.BasedonthosepropertiesofscFvA21,wefurtherconstructedthescFv-Fcfusionmoleculewithahomodimerformandtherecombinantproductwasexpressedinmammaliancells.Inaseriesofsubsequentanalysisthisfusionproteinshowedidenticalantigenbindingsiteandactivitywiththeparentantibody.Theseanti-p185engineeredantibodieshavepromisedtobefurthermodifiedasatumortargetingdrugs,withaviewofapplicationinthediagnosisandtreatmentofhumanbreastcancer.