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Cloning, sequence, and expression of mouse protoporphyrinogen oxidase
T A Dailey1, H A Dailey, P Meissner
1Department of Microbiology, University of Georgia, Athens 30602-2605, USA.
Archives of Biochemistry and Biophysics
|December 20, 1995
Summary
Researchers cloned and expressed mouse protoporphyrinogen oxidase, the enzyme deficient in variegate porphyria. The cloned gene
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Protoporphyrinogen oxidase (PPOX) is crucial for heme biosynthesis.
- Genetic deficiencies in PPOX cause variegate porphyria.
- Understanding PPOX function is vital for metabolic disease research.
Purpose of the Study:
- To clone, sequence, and express the mouse protoporphyrinogen oxidase gene.
- To characterize the expressed mouse PPOX protein.
- To investigate PPOX gene expression in mouse cells.
Main Methods:
- Cloning PPOX cDNA using complementation of an E. coli mutant with a mouse erythroleukemia (MEL) cell expression library.
- Sequencing the cDNA and 5' untranslated region.
- Expressing the protein in E. coli and analyzing its sensitivity to acifluorfen.
- Northern blot analysis of PPOX mRNA in MEL and hepatoma cells.
Main Results:
- Successfully cloned and sequenced the mouse PPOX cDNA (1814 bp) encoding a 477-amino acid protein.
- The expressed mouse PPOX protein is sensitive to the herbicide acifluorfen.
- Northern blots revealed two major PPOX mRNA species (1.8 and 3.6 kb) in mouse cells.
Conclusions:
- The successful cloning and expression of mouse PPOX provide a tool for further research.
- Characterization of mouse PPOX offers insights into heme biosynthesis and related genetic disorders.
- Understanding PPOX expression patterns is important for studying its role in cellular metabolism.