Polygalacturonaseassay ThisenzymeisfamousforbeinginvolvedinthedevelopmentoftheGMOtomatoes(moreinformationfromthelinkatthefootofthispage).Thecellsofthesetomatoeshavebeengeneticallymodifiedtocontainareversedcopyofthegeneforthisenzyme.Thisproducesanti-sensemRNAwhichcombineswiththenormalmRNAforpolygalacturonase,effectivelysupressingitssynthesis.AsaresulttheGMtomatoeshaveonly1%ofusualpolygalacturonaseactivity,andsoftenmuchmoreslowlythanunmodifiedtomatoes-thusextendingtheirshelf-life. Thenormalroleofpolygalacturonaseistohydrolysepectinsduringfruitripening,whichleadstosofteningofthefruit.TheplanthormoneethenehasbeenshowntopromotethetranslationofpolygalacturonasemRNA,raisingthelevelsoftheenzymeinripeningfruit. Someplantpathogenicfungi,suchasPhytophthorainfestans(potatoblight),secretepolygalacturonaseduringtheirattackofhostplantcellwalls. Anotherinterestingfactisthattheregulationoftheproductionoftheenzymeispartlyduetoethene(ethylene),whichinducesthetranslationofpolygalacturonasemRNA,ratherthanregulatingthetranscriptionstep. Theprincipleofthisassayistomixanextractoffruit(orvegetable)withaviscoussolutionofpolygalacturonicacid,andthennotethedecreaseinviscosityduetoitsbreakdownbytheenzyme. Becauseofvariationsinthediameteretc.ofpipettes,itwouldbebesttousethesamepipetteforallreADIngs,carefullyrinsingitoutbetweenthem.Makesurethepipetteisheldvertically,possIBLywitharetortstandandclamp. Preliminaryexperimentswitharipetomatohaveshowna50%decreaseinviscosityafterincubationfor1hour. (1)Sodiumacetatebuffer-40mMsolutionmadeup,thenadd1MHCldropwise,whilstpHismonitoredwithapHmeter. (2)Makeupa3.2%(w/v)ofpolygalacturonicacid(sodiumsalt),bymixingthepowderwithwarmdistilledwater,stirringitthoroughlywithaglassrodandthenplacingthemixtureinaboilingwaterbathfor10minutes-stirringoccasionally.Thenfilterthewarmsolutionorcentrifugeittoprovideaclear,viscoussolutionofpolygalacturonicacidfortheseexperiments.