Abstract
The chapter presents a study on cloning of sheep and mouse prostaglandin endoperoxide synthases. The chapter describes the protocol used for preparing a λgtl0 library and for isolating cDNAs coding for prostaglandin endoperoxide (PGG/H) synthase from sheep vesicular gland, Recently a similar approach was used to isolate a cDNA coding for a mouse 3T3 cell PGG/H synthase. Radiolabeled oligonucleotide probes used to identify the PGG/H synthase cDNA clone were designed on the basis of amino acid sequence information obtained from the purified sheep vesicular gland enzyme. PGG/H synthase (E.C.I. 14.99.1) catalyzes two consecutive reactions in prostaglandin biosynthesis, they are: (1) a cyclooxygenase reaction involving transformation of one molecule of arachidonate and two molecules of oxygen to prostaglandin G 2 and (2) a hydroperoxidase reaction in which PGG 2 undergoes a two-electron reduction to yield PGH 2 . PGG/H synthase activity can be regulated by growth factors, tumor promoters, and steroids in a variety of cell types. These observations have prompted the cloning of cDNAs for PGG/H synthase to be used as probes for studying transcriptional regulation of the PGG/H synthase gene. The availability of a cDNA containing the entire coding region of PGG/H synthase has also permitted studies of enzyme function using site-directed mutagenesis.
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