Advantages of E.coli and lactose utilization systemCulture large numbers of bacteria allow isolation of rare mutants.Lactose genes are not essential for survivalInduction increasesproteinlevel 1ooo-foldmaking mutantidentificationeasy.Colorchangesusingβ-galactosidase substrates (e.g.,ONPG,X-Gal) make measurement of expression levels efficient.Plateβ-galactosidaseassay using X-GalsubstrateLiquidβ-galactosidaseassayusingONPGsubstrate17-11
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 17-11 Liquid -galactosidase galactosidase assay using ONPG substrate Plate -galactosidase galactosidase assay using X-Gal substrate Advantages of Advantages of E. coli E. coli and lactose utilization system and lactose utilization system Culture large numbers of bacteria allow isolation of rare mutant Culture large numbers of bacteria allow isolation of rare mutants. Lactose genes are not essential for survival. Lactose genes are not essential for survival. Induction increases protein level 1000 Induction increases protein level 1000-fold making mutant identification fold making mutant identification easy. Color changes using Color changes using -galactosidase galactosidase substrates (e.g., ONPG, X substrates (e.g., ONPG, XGal) make measurement of expression levels efficient. Gal) make measurement of expression levels efficient
The1965NobelPrizeforPhysiologyorMedicineFrancois JacobAndreLwoffJacques MonodWorking in the PasteurInstitute in ParisRevealed coordinate repression and induction of three genes bystudying lactose-utilization mutants.Proposed the Operon Theory of gene regulation17-12
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 17-12 François Jacob Jacques Monod André Lwoff Working in the Pasteur Institute in Paris The 1965 Nobel Prize for Physiology or Medicine The 1965 Nobel Prize for Physiology or Medicine Revealed coordinate repression and induction of three genes by Revealed coordinate repression and induction of three genes by studying lactose studying lactose-utilization mutants. utilization mutants. Proposed Proposed the Operon Theory of gene regulation. of gene regulation
Identification of lactose-utilization genesJacgues Monod and his collaborators isolated many Lacmutants unable to grow on lactose.Three genes, lacZ, lacY, and lacA, were identified in a tightlylinkedcluster.laczlacylacAβ-galactosidase Permease Transacetylase3SFig.15.417-13
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 17-13 Identification of lactose-utilization genes Jacques Monod and his collaborators isolated many Lac Jacques Monod and his collaborators isolated many Lac mutants unable to grow on lactose. mutants unable to grow on lactose. Three genes, Three genes, lacZ, lacY, and lacA, were identified in a tightly were identified in a tightly linked cluster. linked cluster. Fig. 15.4
Experimental evidencefora repressor proteinMutants of laclgene:Synthesizeβ-galactosidase and lacpermeaseall thetime,evenin the absence ofinducer. Constitutive mutants:Mutants that synthesize certain enzyme all thetime,irrespective ofenvironmental conditionsSuggest that lacl encodes a repressor-cells would need laclprotein productto prevent expression of lacZ and lacYin theabsence ofinducer.17-14
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 17-14 Experimental evidence for a repressor protein Experimental evidence for a repressor protein Synthesize Synthesize -galactosidase galactosidase and lac permease permease all the time, even all the time, even in the absence of inducer. in the absence of inducer. Constitutive mutants Constitutive mutants: Mutants that synthesize certain enzyme all the utants that synthesize certain enzyme all the time, irrespective of environmental conditions. time, irrespective of environmental conditions. Suggest that Suggest that lacI encodes a encodes a repressor repressor – cells would need cells would need lacI protein product to prevent expression of protein product to prevent expression of lacZ and lacY in the absence of inducer. absence of inducer. Mutants of Mutants of lacI gene:
PaJaMo experimentStart withalacI-laczWheninducerisadded,synthesismutant.ofβ-galactosidasecontinues.TransferlacI andlaczWith no inducer,genes into the cells on aβ-galactosidasesynthesis stops;piece ofDNA.lacitisbeingexpressedproducingaB-galactosidase waslacrtlaczrepressor.genes introduceddetectedimmediatelyafterDNA transfer.and theSynthesisofsynthesis stopped in anβ-galactosidasebeginsshortly after lacrand laczthour.geneswereintroduced;laczgene isbeingexpressed.-If add lactose,β246701358galactosidasekeepsTime(hours)accumulating.17-15Fig. 15.5
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 17-15 PaJaMo experiment experiment Start with a Start with a lacI lacZ mutant. mutant. Transfer Transfer lacI+ and lacZ+ genes into the cells on a genes into the cells on a piece of DNA. piece of DNA. -galactosidase galactosidase was detected immediately after detected immediately after DNA transfer, and the DNA transfer, and the synthesis stopped in an synthesis stopped in an hour. If add lactose, If add lactose, - galactosidase galactosidase keeps accumulating. accumulating. Fig. 15.5