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LECTURE 2 OUTLINE
GENETIC ANALYSIS OF GENE REGULATION
Negative and Positive Regulation
-negatively and positively regulated operons behave very
differently in genetic tests
Negative Regulation
Positive Regulation
-inactivation of regulator leads
-inactivation of regulator leads
to CONSTITUTIVE expression to NON-INDUCIBLE
-constitutive mutations are
expression
RECESSIVE to wildtype
-non-inducible mutations are
RECESSIVE to wildtype
-gain-of-function mutations lead -gain-of-function mutations lead
to NON-INDUCIBLE
to CONSTITUTIVE expression
expression
- constitutive mutations are
DOMINANT to wildtype
-non-inducible mutations are
DOMINANT to wildtype
Identification of Cis and Trans Acting Factors
Cis-Acting Factor
-DNA sequence that functions
exclusively as a DNA sequence
-affects only DNA to which it is
physically linked
-eg. promoter/operator
Trans-Acting Factor
-diffusible gene product
-can affect other DNA
molecules
-eg. negative or positive
regulator
-mutations cannot be
-mutations can be
complemented
complemented
-mutations may be recessive or
-mutations may be recessive or
dominant
dominant
regulatory proteins are trans-acting factors that recognize cisacting elements to control gene expression
NEGATIVE REGULATION – THE LAC OPERON
-Jacob & Monod’s analysis of negative regulation of the lac
operon in the 1950s established the framework for all other gene
regulation studies
-Nobel prize 1965
The lac operon
-bacterial genes organized in clusters according to function,
OPERONS
-expressed as part of a single POLYSCISTRONIC transcript,
subject to coordinate control
-enzymes for lactose metabolism are INDUCIBLE
-paradigm determined from genetic analyses of lac mutants
-based on COMPLEMENTATION ANALYSIS
-introduce F’ factors carrying mutated genes (plasmid onto which
lac- mutations have been recombined) to create MERODIPLOIDS
-determine dominance vs. recessivenss
-identify complementation groups/genes
Identification of the structural genes
-one group of lac mutants unable to grow on lactose (Lac-)
-STRUCTURAL GENES identified by complementation analysis
-identify genes involved by complementation analysis
Strain
F’ lacZ Y /lacZ+Y+
F’ lacZ+Y+/lacZ1-Y+
F’ lacZ2-Y+/lacZ+Y+
F’ lacZ2-Y+/lacZ1-Y+
+ +
Phenotype
+
Lac
Lac+
Lac+
Lac-
-most mutants are recessive to wildtype therefore must inactivate
genes required for lactose utilization -2 complementation
groups/genes lacZ and lacY
Identification of the regulator LacI
1) dominant non-inducible Lac- mutants
-one group of mutants were dominant Lac- mutants
-these mutations make cell Lac- even if another good copy of lac
operon is present
-lacIs
Strain
+ + +
lacI Z Y
lacIsZ+Y+
F’ lacI+Z+Y+/lacI+Z+Y+
F’ lacIs Z+Y+/lacI+Y+Z+
Phenotype
+
Lac
LacLac+
Lac-
-lacIs mutations alter LacI such that it binds the operator even in
the presence of inducer and prevents expression of the operon
2) recessive constitutive mutants
-one group of mutants make the cells Lac+ constitutive, rather than
Lac-complementation tests determined that these mutants are recessive
Strain
+ + +
lacI Z Y
lacI-Z+Y+
F’ lacI+Z+Y+/lacI+Z+Y+
F’ lacI-Z+Y+/lacI+Z+Y+
Phenotype
Inducible Lac+
Constitutive Lac+
Inducible Lac+
Inducible Lac+
-complementation tests between recessive constitutive mutants
determined that they are all in lacI - the mutations knock-out LacI
3) dominant constitutive mutants
-one group of mutants make the cells Lac+ constitutive, rather than
Lac-complementation tests determined that these mutants are
dominant
Strain
+ + +
lacI Z Y
lacI-dZ+Y+
F’ lacI+Z+Y+/lacI+Z+Y+
F’ lacI-dZ+Y+/lacI+Z+Y+
Phenotype
Inducible Lac+
Constitutive Lac+
Inducible Lac+
Constitutive Lac+
-lacI-d mutations alter LacI such that it binds the operator even in
the presence of inducer and prevents expression of the operon
Identification of cis-acting sites
i)
promoter mutants
-another group of lac mutants unable to grow on lactose could not
be complemented
-these mutants lie in the lac promoter region
-an F’ carrying a p- mutant, but wildtype copies of lacZ and lacY
can not be complemented in a strain carrying either mutant lacZ or
lacY genes on the chromosome CIS-ACTING REGULATORY
SITE
Strain
+
+
+
p lacZ lacY
Phenotype
Lac
+
p-lacZ+lacY+
Lacp+lacZ-lacY+
Lacp+lacZ+lacYLac+
+ + +
+ +
F’ p lacZ Y /p lacZ Y
Lac+
F’ p-lacZ+Y+/p+lacZ-Y+
LacF’ p-lacZ+Y+/p+lacZ+YLacF’ p-lacZ+Y+/p+lacZ+Y+
Lac+
-these mutations prevent RNAP from initiating transcription at the
lac promoter
ii) lacOc mutants
-another group of mutants resembled the lacI- mutants, they were
constitutive
-these mutants, however, could not be complemented (cis) and
were also dominant
Strain
Phenotype
lacO lacZ
Inducible Lac+
lacOclacZ+
Constitutive Lac+
F’ lacO+lacZ+/lacO+lacZ+
Inducible Lac+
F’ lacOclacZ+/lacO+lacZ+
Constitutive Lac+
-mutations were in LacI repressor binding site or OPERATOR
-prevent binding of LacI, so expression is always on
+
+
Genotype Phenotype
+IPTG -IPTG
wild type
+
LacZLacPLacOc
+
+
LacI+
+
LacI(-d)
+
+
LacI(s)
LacOcLacI(s)
+
+