You have isolated two different mutants (reg1 and reg2) causing constitutive exp

Question

You have isolated two different mutants (reg1 and reg2) causing constitutive expression of the emu operon (emu1 emu2). One mutant contains a defect in a DNAbinding site, and the other has a loss-of function defect in the gene encoding a protein that binds to the site.

A. Is the DNA-binding protein a positive or negative regulator of gene expression?

B. To determine which mutant has a defect in the site and which one has a mutation in the binding protein, you decide to do generate partial diploids using F? plasmids. Assuming you can assay levels of the Emu1 and Emu2 proteins, what results do you predict for the two strains (i and ii) (see descriptions below) if reg2 encodes the regulatory protein and reg1 is the regulatory site?

i) F? reg1? reg2+ emu1? emu2+ /reg1+ reg2+ emu1+ emu2?

ii) F? reg1+ reg2? emu1? emu2+/reg1+ reg2+ emu1+ emu2 ?

C. What results do you predict for the two strains (i and ii) if reg1 encodes the regulatory protein and reg2 is the regulatory site?

Explanation / Answer

A. The DNA binding site is a positive regulator of gene expression. unless the DNA binding site is efficient, the RNA polymerase cannot bind to it and carryout transcription. Therefore a mutation causing defect in DNA binding protein terminates the transcription and turns off the gene expression.

B. The strain (i) F- reg1- reg2+ emu1- emu2+ /reg1+ reg2+ emu1+ emu2- is useful to identify the mutation of reg1 encoding the regulatory site. This is because when reg1 is absent in cell containing the F- plasmid and this cell undergoes recombination with reg1+ cell, the F- plasmid gains the reg1+ mutation and becomes a F+ plasmid.

(ii) F-reg1+ reg2- emu1- emu2+/reg1+ reg2+ emu1+ emu2 -, This strain (ii) is useful indentifying the mutation in reg2 gene encoding regulatory protein. The F- plasmid with reg2- gene undergoes recombination with a cell contain reg2+ and gains mutation and becomes F+ cell.

C. If the reg1 encodes the regulatory protein strain I is useful to identify mutation in reg1 gene.

If the reg2 gene encodes the regulatory site, strain ii is useful to identify mutation in reg2 gene.

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