Partial characterization of guinea pig cholinephosphotransferase cDNA.
An alternative molecular biology strategy is needed to characterize cholinephosphotransferase (CPT) gene because of the complexity of the problem associated with the solubilization of the membrane-bound enzyme without denaturation. We have synthesized five heterologous oligonucleotide probes based on the published yeast CPT gene sequence. Each probe (24 to 30 mers) was used as either forward or reverse flanking primers in combination with lambda gt11 primers to amplify a segment of DNA from a guinea pig liver 5'cDNA library by polymerase chain reaction (PCR). We detected several clones of varied size (0.1 kb to 2.2 kb) by subjecting the PCR products to 1.2% agarose gel electrophoresis. Southern blot of a 0.7 kb PCR product did hybridize with a 32P-labeled internal probe. Slot blot hybridization of guinea pig liver total RNA with the 32P-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% homology with the yeast CPT gene sequence.
Das
S K
SK
Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208, USA.
Beg
O
O
Mukherjee
S
S
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics*; D015723:Gene Library; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D002798:Diacylglycerol Cholinephosphotransferase