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Lecture 22 - reporter genes- BIOL308.pdf

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University of Waterloo
BIOL 308
Bruce Wolff

Lecture 22Currently the most frequently used are reporter genesCode foreasily assayed gene productsusually enzymesIdea to investigate the strength of the promoter you clone the reporter gene after promoter of interestExpression of the reporter gene is under control of promoter of interestLevel of expressionof measurable easy to assay gene product enzyme is proportional to the strength of the promoterYou can deletemutate a part of the promoter starting from 5 end 5 deletion series and repeat the experimentBuild a mapof the control region with indications of relative importance of sequence in that areaReporter Genes have easily assayed gene productsLac Z product betagalactosidase hydrolyses Xgal substrate into a colorless galactose and blue 4chloro3bromindigoCATchloramphenicol acetyl transferase acetylates antibiotic chloramphenicol CAM Acetylated CAM has different properties then unacetylatedLuciferaseATP and luciferin substrateEmission of lightGFP green fluorescent protein from bioluminescent jellyfish Aequoria Victoria Naturally fluoresces green when exposed to blue light not an enzymeRNAPII PromotersPromoters recognized by RNAP II Class II promotersPromotersequence that must be located in the vicinity of the start point and are required for initiation of transcriptionIn most cases class II promoters promoter regions have two partsCore elements1Also called core promoter or only promoterRegulatory elements2One of them is enhancerCore promoterminimal set of elements requried for accurate in vitro transcription intiation by RNAP II1Necessary for recruitment binding and proper positioning of RNAPIITATA box position at 30Initiator Inr on the transcription start siteDownstream elementDPE downstreamTF II B recognition elementBRERegulatory elements bind regulatory proteins2Influence transcription rate through binding of regulatory proteinsHowever sometimes necessary for initiationof transcription NOTES FROM LECTURES Page 1
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