You are studying a mouse gene MtoA. You know the protein product of localizes to

Question

You are studying a mouse gene MtoA. You know the protein product of localizes to mitochondria in mice. Your goal is to know whether the mouse MtoA protein is capable of localizing to mitochondria if produced in yeast cells. You decide to clone a gene into a translational fusion vector that will add a jellyfish DNA sequence encoding the GFP protein (238 amino acids) in place of the nomal stop codon. Unfortunately, you can't find any restriction enzyme recognition sequences in the MtoA gene that would allow you to clone your DNA into the multiple cloning site.

You make the plasmids in a test tube, then transform them into E. coli. You grow the E. coli and extract the plasmid. - What 2 methods could you use to determine if the plasmid has the MtoA-GFP fusion?

Must the plasmid contain a promoter that works in jellyfish? - If yes, state which specific gene on the plasmid will be controlled by that promoter.

Must the plasmid contain a promoter that works in E. coli? - if yes,  state which specific gene on the plasmid will be controlled by that promoter.

Explanation / Answer

From the study, the mouse MtoA protein localizes to mitochondria and similar localization has to be detected in yeast cells.

To determine if plasmid have the MtoA-GFP fusion construct, first only those E.coli would be used that have been successfuly transformed with the plasmid construct , which inturn has selectable markers. If E.coli expresses GFP, then fluorscence base techniques like FACS (Fluorscence activated cell sorting) can be used to see if the transfomed cells express GFP,cells would be sorted based on a fluorescence signal.Also this plasmid may have GFP under the control of an additional promoter.

PCR amplification of the MToA, GFP fusion construct, followed by agarose gel electrophoresis can also determine presence of MtoA-GFP fusion construct.This can be affirmed by DNA sequencing.

The plasmid may not contain a promoter that works in jellyfish. This is because the plasmid has to be transformed in E.coli and expressed in E.coli, so the promoter must conform to E.coli's transcription and translation machinary.

The plasmid must contain a promoter that works in E.coli, since plasmid with MtoA-GFP construct is transformed in E..coli.MtoA gene expression should be controlled by that promoter. Since MToA is fused to GFP, GFP reports expression of MtoA expression.

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