Sample answer for 2009 Model-testing part of midterm

BIOC/MCB 568 -- Fall 2009
John W. Little--University of Arizona

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Models are here:

Genetics:

Mutants predicted by each model:

	Model X	Model Y	Model Z
RC	K-      knockout R-      knockout		K-      knockout
RN		K-       knockout A-        knockout	A-      knockout
DC		KS      kinase doesn't need inducer to be active Also acceptable but not necessary: AS      A doesn't need to be phosphorylated to be active	AS      activator doesn't bind kinase
DN	KS      K doesn't bind inducer Also acceptable but not necessary: RS      R doesn't need to be phosphorylated to be active		KS      K doesn't bind inducer

To distinguish:

X vs Y:   X has two classes of RC, Y none
               Y has two classes of RN, X none
               X has (one or two classes of) DN, Y none
               Y has (one or two classes of) DC, X none

X vs Z:   X has two classes of RC, Z has one class
               Z has RN, X doesn't
               X has no DC, Z does
               (Optional but not necessary: X has two classes of DN, Z has one)

Y vs Z:   Y has two classes of RN, Z has one
               Z has RC, Y doesn't
               Z has DN, Y doesn't
               (Optional but not necessary: Y has two classes of DC, Z has one)

 Biochemistry:

Regulator II (RegII) is the protein that binds the target gene. RegI is the other regulatory protein; you can call it kinase since that's what it is in all three models.

X vs Y:          RegII is a repressor in X, an activator in Y. Test with in vitro transcription system with and without RegII [no need to involve RegI or inducer]

In X, kinase is active -inducer, not + inducer; in Y, active +inducer but not - inducer. Test with a kinase assay, using unphosphorylated RegII as substrate.

X vs Z:          RegII is a repressor in X, an activator in Z.

In X, the phosphorylated form of RegII binds DNA, unphosphorylated form does not.
In Z, the unphosphorylated form of RegII binds DNA, phosphorylated form doesn't.

Y vs Z:          In Y, kinase is active +inducer, not -inducer; in Z, it's active -inducer but not +inducer.

In Y, the phosphorylated form of RegII binds DNA, unphosphorylated form does not.
In Z, the unphosphorylated form of RegII binds DNA, phosphorylated form doesn't.

Mutant proteins (note that this part of the question did not ask you to describe the phenotype of the cell, but the biochemical properties of the mutant proteins):

X            K^S DN - kinase is active in the presence of inducer
(Optional: R^S DN - binds DNA without being phosphorylated)

Y            K^S DC - kinase is active in the absence of inducer
(Optional: A^S DC - binds DNA without being phosphorylated)

Z            K^S DN - kinase is active in the presence of the inducer
A^S DC - mutant A can't be phosphorylated by K

Bonus: The simplest way is to make RegII unstable. Optionally, only the phosphorylated form would be unstable. ("Unstable" means degraded by host degradation machinery; often this is a very general system that could not be mutated without being lethal).