Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 1 de 1
Filter
Add more filters










Database
Language
Publication year range
1.
Assay Drug Dev Technol ; 1(1 Pt 2): 147-60, 2003 Feb.
Article in English | MEDLINE | ID: mdl-15090141

ABSTRACT

Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Genetic Vectors , Nicotiana/enzymology , RNA Viruses/genetics , Yarrowia/enzymology , Cells, Cultured , Chromatography, Thin Layer , Cytochrome P-450 CYP4A/biosynthesis , Cytochrome P-450 CYP4A/genetics , Cytochrome P-450 Enzyme System/genetics , Gas Chromatography-Mass Spectrometry , Gene Expression , Microsomes/enzymology , Phenotype , Plant Leaves/enzymology , Plant Leaves/ultrastructure , Nicotiana/ultrastructure , Transcription, Genetic
SELECTION OF CITATIONS
SEARCH DETAIL
...