Prescott's Microbiology
Prescott's Microbiology
11th Edition
ISBN: 9781260211887
Author: WILLEY, Sandman, Wood
Publisher: McGraw Hill
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Chapter 18, Problem 2AL
Summary Introduction

EMSA stands for electrophoretic mobility shift assay and it is the most common method used to study DNA-protein interactions. This method is also termed as a gel shift assay. This is because the DNA binding protein is added to the purified target DNA mixture and its mobility is slowed down using an agarose gel. The DNA-protein complex is found to be shifted to a higher molecular weight when compared to the DNA without the protein. Usually, the magnitude of the shift is related to the DNA-protein ratio. Thus, the largest shift would be seen when sufficient protein is added.

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The lac operon of E. coli controls the expression of genes that code for enzymes involved in lactose metabolism.  Mutations to various regions of the lac operon have been discovered which affect both the control of gene expression and the function of the gene products.  These mutations include: I - = produces a repressor protein that cannot bind to the operator region IS = produces a repressor protein that cannot be removed from the operator region OC = cannot bind a repressor protein Z - = produces a defective (non-functioning) β-galactosidase enzyme + = wild type   Detail the functionality of the lac operon under the following genetic and cellular conditions.  You do not need to consider the role of CAP in your analysis.      Genes Repressed (yes/no) Constitutive (yes/no) IS  O+  Z+       IS  OC  Z+
The lac operon of E. coli controls the expression of genes that code for enzymes involved in lactose metabolism.  Mutations to various regions of the lac operon have been discovered which affect both the control of gene expression and the function of the gene products.  These mutations include: I - = produces a repressor protein that cannot bind to the operator region IS = produces a repressor protein that cannot be removed from the operator region OC = cannot bind a repressor protein Z - = produces a defective (non-functioning) β-galactosidase enzyme + = wild type     Detail the functionality of the lac operon under the following genetic and cellular conditions.  You do not need to consider the role of CAP in your analysis.      Genes Repressed (yes/no) Constitutive (yes/no) I+  OC  Z+       I+  O+  Z+ (Lactose   present)     I-  O+  Z+
You have isolated different mutants (reg1 and reg2) causing constitutive expression of the emu operon (which has genes emu1 and emu2). One mutant contains a defect in a DNA-binding site, and the other has a loss-of-function defect in the gene encoding a protein that binds to the site Say you don’t know which mutant has a defect in the site and which one has a mutation in the binding protein. To figure it out, you construct the two partial diploid strains (i and ii below), and you then assay the levels of the Emu1 and Emu2 proteins in these two strains. F’ (reg1- reg2+ emu1- emu2+) / reg1+ reg2+ emu1+ emu2- F’ (reg1+ reg2- emu1- emu2+) / reg1+ reg2+ emu1+ emu2-   What proteins do you predict will be expressed for strains i and ii if reg2 encodes the regulatory protein and reg1 is the regulatory site?
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