PARTIAL CHARACTERIZATION OF GUINEA-PIG CHOLINEPHOSPHOTRANSFERASE CDNA

An alternative molecular biology strategy is needed to characterize ch olinephosphotransferase (CPT) gene because of the complexity of the pr oblem associated with the solubilization of the membrane-bound enzyme without denaturation. We have synthesized five heterologous oligonucle otide probes based on the published yeast CPT gene sequence. Each prob e (24 to 30 mers) was used as either forward or reverse flanking prime rs in combination with lambda gt11 primers to amplify a segment of DNA hom a guinea pig liver 5'cDNA library by polymerase chain reaction (P CR). We detected several clones of varied size (0.1 kb to 2.2 kb) by s ubjecting the PCR products to 1.2% agarose gel electrophoresis. Southe rn blot of a 0.7 kb PCR product did hybridize with a P-32-labeled inte rnal probe. Slot blot hybridization of guinea pig Liver total RNA with the P-32-labeled 0.7 kb PCR product yielded positive transcripts with intensities proportional to the concentration of RNA. Furthermore, a 0.1 kb clone was sequenced and the observed sequence shared 96% homolo gy with the yeast CPT gene sequence. (C) 1997 Academic Press.