Analysis of chicken progesterone receptor function and phosphorylation using an adenovirus-mediated procedure for high-efficiency DNA transfer.
The expression of heterologous DNA in mammalian cells is crucial to understanding physiological functions or determining biological properties of encoded proteins. However, expression for biological assay or at levels sufficient for recovery and subsequent physical analysis has been limited by the poor efficiency, variability, or cost of current DNA transfer methodologies. We have modified a DNA transfer procedure which exploits the capacity of replication-deficient adenovirus to infect a wide range of cell types, carrying with it transiently associated DNA. We have established conditions for achieving 80% transfection of CV1 cells and have used this procedure for DNA transfer into several mammalian cell lines and primary cell cultures. We have shown that biologically active avian progesterone receptor may be readily detected, both immunologically and functionally, using less than 1 ng of progesterone receptor-encoding plasmid DNA per 2 x 10(5) cells. We previously reported the identification of four phosphorylation sites in chicken progesterone receptor using oviduct tissue minces labeled with [32P]PO4 under nonequilibrium conditions. We now find, using adenovirus-mediated infection and equilibrium labeling conditions, that the same sites are phosphorylated in receptor expressed in CV1 cells and report that there are no additional major phosphorylation sites in chicken progesterone receptor. The ease, efficiency, sensitivity, and wide applicability of this DNA transfer method should simplify current efforts to study heterologous protein expression in mammalian cells.
Allgood
V E
VE
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhang
Y
Y
O'Malley
B W
BW
Weigel
N L
NL
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D010455:Peptides; D011980:Receptors, Progesterone; D011374:Progesterone; D001616:beta-Galactosidase; D014357:Trypsin