Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/1996
Publication Date: N/A
Citation: N/A Interpretive Summary: This paper describes one recently developed method of isolating pieces of genes for a group of proteins generally referred to as cytochrome P450s. P450 proteins have enzymatic properties and are an integral part of many metabolic and detoxification pathways in all animals, plants and bacteria. They are especially important in the expression of pesticide resistance by insects and in the detoxification of alcohol and other foreign chemicals in humans. Yet little is known about the specific functions of individual proteins (over 100 exist) because it is difficult to isolate sufficient quantities from tissues for analyses and, once isolated, the proteins are often unstable. Isolating P450 genes and, subsequently, reconstituting and expressing them in controlled systems has proven to be the best way to biochemically characterize what specific P450 proteins do. The method described in this paper explains a strategy which has proven successful for the rapid isolation of pieces of genes for many different P450 proteins. The gene fragments obtained may be used to study various biological problems including pesticide resistance, induction by foreign chemicals and hormonal and developmental control.
Technical Abstract: This chapter describes a strategy to isolate fragments of cytochrome P450 genes of unknown sequence via using polymerase chain reaction (PCR). Using P450 Family 4 as the primary example, we describe how to identify gene regions which have good potential for use as annealing sites for PCR primers and describe successful variations on the design of heterologous oligonucleotide primers made to anneal to the same region. We also describe alternative PCR programs and conditions which we have used successfully. Finally, we describe means by which P450 gene fragments may be positively identified and how to include positive controls in the PCR amplification. Helpful commentary which would not be included in a presentation of original data but can be critical to the successful amplification of the desired genes is included where appropriate.