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Published on: May 27, 2016
Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA
Kristen M Pluchino1, Dominic Esposito2, Janna K Moen1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
Abstract:
The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.
Insights
Mouse mdr1a cDNA exhibits genetic instability in bacteria due to a cryptic promoter causing toxic P-glycoprotein (P-gp) expression. A specific mutation (M107L) resolves this toxicity, enabling efficient mdr1a cloning in E. coli.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- P-glycoprotein (P-gp) is a key efflux transporter influencing pharmacokinetics and multidrug resistance.
- Cloning homologous cDNAs is crucial for understanding species-specific P-gp functional differences.
- Mouse mdr1a cDNA shows genetic instability in bacteria, suggesting toxicity.
Purpose of the Study:
- Identify the cause of genetic instability in mouse mdr1a cDNA during bacterial cloning.
- Characterize the cryptic promoter responsible for bacterial expression of mouse P-gp.
- Develop a stable cloning strategy for mouse mdr1a cDNA.
Main Methods:
- Sigma 70 binding site analysis to locate potential bacterial promoter regions.
- Green Fluorescent Protein (GFP) reporter plasmids to test for bacterial protein expression.
- Site-directed mutagenesis to create a non-toxic mdr1a cDNA variant.
Main Results:
- A cryptic promoter within the first 321 base pairs of mouse mdr1a cDNA was identified.
- This promoter drives bacterial expression of mouse P-gp, leading to toxicity.
- A single point mutation (M107L) in the translational start site abolished toxicity, allowing stable cloning.
Conclusions:
- The genetic instability of mouse mdr1a cDNA in E. coli is caused by an intrinsic cryptic promoter.
- The M107L mutant mdr1a cDNA provides a solution for stable and efficient cloning of this gene.
- This mutant facilitates further research into P-gp function and species-specific variations.
