title: Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.
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.
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Kristen M
authors: KM
authors: Pluchino
authors: Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, United States of America.
authors: Dominic
authors: D
authors: Esposito
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Janna K
authors: JK
authors: Moen
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Matthew D
authors: MD
authors: Hall
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: James P
authors: JP
authors: Madigan
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Suneet
authors: S
authors: Shukla
authors: Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, United States of America.
authors: Lauren V
authors: LV
authors: Procter
authors: Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, United States of America.
authors: Vanessa E
authors: VE
authors: Wall
authors: Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, Molecular Information Theory Group, Frederick, MD, United States of America.
authors: Thomas D
authors: TD
authors: Schneider
authors: Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors: Ian
authors: I
authors: Pringle
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Suresh V
authors: SV
authors: Ambudkar
authors: Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors: Deborah R
authors: DR
authors: Gill
authors: Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors: Steven C
authors: SC
authors: Hyde
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Michael M
authors: MM
authors: Gottesman
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation; D012808:Sigma Factor / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D018076:DNA, Complementary; D012808:Sigma Factor; C095122:multidrug resistance protein 3; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases