308 Lecture 19

308 Lecture 19 - J & M: Proof through complementation...

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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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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 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 Genomic DNA Plasmid DNA lac present RNAP binds…. from normal, genomic gene
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Non-inducible mutation lac I s Non-inducible lacI supperrepressor ( lacI s mutant) carried by plasmid can not bind inducer (lactose) All operator sites (O+) eventually occupied by superrepressor β gal (normal, coded by chromosome) will stop being produced 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 regulatory factor (repressor protein) which can diffuse (hence “ trans ”) throughout the cell and bind to operators regardless of their chromosomal location
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Constitutive mutation O c Constitutive lacI + product (repressor) cannot bind to O c β gal coded by chromosomal gene
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This note was uploaded on 10/31/2011 for the course BIOL 308 taught by Professor Miskovic during the Winter '09 term at Waterloo.

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308 Lecture 19 - J & M: Proof through complementation...

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