PMC2266942_01
PMC2266942_02
PMC2266942_03
PMC2266942_04
Activities of P450scc mutations. COS-1 cells were transfected with either wild-type or mutant F2 plasmid. Pregnenolone secreted into the culture medium was determined by RIA. Empty vector was used as a negative control, and the StAR-independent substrate 22R-hydroxycholesterol was added to the culture medium. Values are the mean +- SEM from four independent transfection experiments, each performed in triplicate.
ukmss-1523-f0001.jpg
PMC2266942_04_ukmss-1523-f0001.jpg
F1
Similarly, when the c835delA mutation identified in Patient 6 was introduced into the P450scc moiety of the F2 plasmid, no activity was detected (Fig. 1).
PMC2266942_05
PMC2266942_06
Minigene experiment. HeLa cells were transfected with empty pcDNA3.1 vector, or vector expressing the wild-type or mutant CYP11A1 minigene construct. Total RNA from the transfected HeLa cells were used for RT-PCR, showing the expected 580 bp product from the wild-type construct but no correctly spliced product from the mutant. PCR of GAPDH RNA was used as an internal control, producing a 247 bp PCR fragment.
ukmss-1523-f0002.jpg
PMC2266942_06_ukmss-1523-f0002.jpg
F2
PCR amplification of the RNA produced from the transcription of the wild-type minigene showed that intron 3 was correctly spliced, yielding a product of the predicted size (580 bp) (Fig. 2).