Spring 09 lecture 18

Spring 09 lecture 18 - J & M: Complementation tests...

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Plasmid can carry I gene as well…or only I gene (and not operon) Different combinations…. . Also combination of these mutation with mutations in structural genes (as products of structural genes ARE actually measured) Transformation of the bacterial cells with plasmids carrying lac operon (whole or parts) - result is partially diploid cell – MERODIPLOID Purpose: artificially produced 2 sets of “alleles” for lac operon genes, complementation is enabled. Discovered/defined cis- elements and trans factors
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Results of complementation experiments: P- if RNAP cannot bind to the promoter, structural genes will never be expressed Oc Oc is dominant over O+ (WT) - bacteria always cleaves lactose; Oc - constitutive mutation - structural genes constantly expressed Is Is dominant over I+ (WT) - bacteria cannot cleave lactose; Is - noninducible mutation - structural genes are never expressed I- I- is recessive ; I+ (WT) is dominant over I-; I- - constitutive mutation - structural genes constantly expressed
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Genomic DNA : lacI - mutation – produced repressor cannot bind to operator – constitutive mutation; normal β gal always synthesized but plasmid carries normal lacI+ (However, plasmid carries mutated Z gene ) plasmids lacI + gene codes for a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location l synthesis of normal β gal (coded by genomic DNA) will become regulated “Repair” of constitutive mutation lac I- *complementation in action * So, what is actually happening (step by step)?
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Genomic DNA Plasmid DNA lac absent Genom ic DNA Plasm id DNA lac present RNAP binds…. from normal, genomic gene
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Non-inducible mutation lac Is Non-inducible lacI supperrepressor ( lacI s mutant) carried by plasmid can not bind inducer (lactose) All operator sites (O+) eventually occupied by superrepressor l β gal (normal, coded by chromosome) will stop being produced l lacI s mutant encodes a diffusible element that acts in trans by binding to any operator it encounters I mutations affect the expression of genes at both “chromosome” copies in “diploid bacteria” - I codes for the
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This note was uploaded on 08/03/2009 for the course BIOL BIOL308 taught by Professor Miskovic during the Spring '09 term at Waterloo.

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Spring 09 lecture 18 - J & M: Complementation tests...

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