BIO 344 1st Edition Lecture 5 Outline of Last Lecture I. Analyzing papers-- example: Meselson Stahl Papera. Question: mechanism of replicationb. Hypothesisc. Data and resultsII. The Meselson Stahl experimenta. N15 and N14b. conclusionOutline of Current Lecture I. Gene Regulationa. Lac operon as an exampleII. Lac operon-- Metabolizing lactosea. Complementation testb. What gene encodes whatIII. Constitutive vs. uninduciblea. What phenotype is seenIV. Cis or trans actingV. What does the repressor repressThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Current LectureGene Regulation- Different cells contain different genomes to specialize a collection of functional proteins or tissues- Each genome has directed different stable pathways o A mechanism for this (gene regulation) is necessary A genetic “switch”—turning genes on and off- Lac operon exemplifies gene regulation- Jacob and Monod:o conceived of the idea of gene regulation and worked out the basic principles of the lac operono The idea of DNA sequences that affect gene expression but themselves are not expressed was revolutionaryLac Operon—metabolizing lactose- Lac operon codes for proteins required to transport lactose into the cell and break it down- under positive and negative controlo need glucose to be absent and lactose to be present when both conditions are met, the operon is highly expressed- Finding out how lactose is metabolized (i.e. what different genes in the operon are respobsible for) and find out what phenotype these mutants will have and how you will distinguish them from mutants in the glycolytic pathway by seeing how mutants behave (mutants gives a way to find out what causes what, what effects what when they are present)o Beta galactosidase allows galactose and glucose to be broken down from lactoseo Isolate mutants in lactose metabolismo Mutants can be screened by replica printing onto plates containing lactose as thesole carbon sourse Add mutants to lactose and distribute into smaller aliquots Grow mutants on plates that allow wild-type and mutants to grow Replica print to plates containing lactose as the sole carbon source- No growth indicates that the mutant can’t grow on lactoseo Lac- (can’t metabolize lactose)- Lactose is upstream from glucoseo If something can’t grow on glucose OR lactose—it can’t metabolize eithero If growth on glucose but not lactose, we know it can’t metabolize eithero How many genes are present? Say we get 5 mutants—how many genes do they represent?- Complementation test used to define lac genes- Number of complementation groups defines number of geneso See slides for a chart showing thiso If two different mutants conjugate and are -, the mutation is on the same geneo If +, the mutation is on different genes- 2 complementation groups are seeno Lac-1 and lac-3 have same gene mutationo Lac-2, Lac-4, lac-5 have same gene mutation, different from the gene mutation on lac-1 and lac-3o Two genes: lacZ and lacYo Which gene codes beta-galactosidase (lacY or lacZ)? Lactose is taken up by permease and hydrolyzed by beta-galactosidase into glucose and galactose- lacY- and lacZ- (mutant forms) are individually extracted from cells- ONPG= an artificial chromogenic substrate analog to galactaseo Has a similar component to galactase bound to another moleculeo Beta-gal will still cleave o Yellow color indicates increasing time or enzyme concentration (meaning the bond is cleaved)o lacZ codes for the enzyme- How do cells control their expression of their genes in time, space and response to environment? How ill lacZ expression be altered in regulatory mutants?o See slides for graph of lacZ mRNA vs. time after addition of IPTG (a glucose like molecule)o Constitutive= can not turn off/always on (first regulatory mutants ever isolate and easier to see in experiment)o Uninducible= can not turn on/always off Constitutive lac+ mutants will have high lacZ protein production in presence or absence of inducer Uninducible lac mutants will have low lacZ protein production in the presence or absence of inducer Wild-type will have low lacZ protein production in the absence of the inducer and high in the presence of the inducero X-gal = chromogenic substrate analog for beta-gal that does not induce the lac operon With no inducer and x-gal, lac constitutive mutant colony will growo Mutation in a structural gene will typically inactivate the protein encoded by thatgeneo A mutation in a regulatory gene will affect the expression of all the genes controlled by the regulatory gene Pleiotropic phenotypes Lac constitutive mutants are close to but not in lacZ or lacY, so are called lacI (for induction—aka always on)- Does lacI encode a repressor or activator? How can you get a constitutive phenotype from a mutation in a regulatory protein?o Null loss of function mutation—recessive to WT If lacI encodes a repressor, loss of function mutation would result in constitutive phenotype of lac+ (lac constitutive) mutantso Gain of function mutation—dominant to WT If lacI encodes an activator then only gain of function mutations can resultin a constitutive phenotypeo Wild-type Hfr and lacZ-LacI constitutive mutant F- were crossed to make a lacI constitutive/wild-type merodiploid Beta-gal was inducible, Wild-type lacI+ phenotype is seen, which tells us that lacI constitutive is recessive, which tells us that it encodes a repressor- Does lacI work in trans or cis?o See slide with charto Conclusion is that it is trans working and therefore must be a diffusible molecule- Summary and conclusions of evidence thus far:o Lac operon structural genes are regulated by a separate gene, lacI Lac constitutive mutations are pleiotropic- Affect both beta-gal and permease The lacI gene encodes a repressor whose function is to repress both permease and beta-gal- lacI constitutive mutants are constitutive and recessive the regulator is a repressor but is involved in inducing the lac operon- the inducer is a metabolite—allolactase, reversibly inactivates the repressor- What does the repressor repress? What is the binding site?o Repressor binds to an operatoro Operator= a second class of regulatory mutantso What is the phenotype of operator mutants? If lacO is a gene, the system is constitutively on-