The aim ofthis study was to evaluate the applicability of synthetic oligonucleotides to the study of the expression and regulation of neuropeptide messenger RNA in neuroendocrine cells. As a model system, neurotensin gene expression was studied in the rat pheochromocytoma cell line, PC12, which demonstrates cumulative increases in neurotensin production in response to combinations of nerve growth factor, dexamethasone, forskolin and lithium. DNA oligonucleotide probes complementary to the rat neurotensin messenger RNA sequence were synthesized and labeled with [32P] by T4 polynucleotide kinase and terminal deoxynucleotidyl transferase. In Northern blot analysis, two mRNA bands of 10 kb and 15 kb were visible in total RNA preparations ofthe stimulated PC12 cells but not in the unstimulated cell extract. In sim hybridization histochemistry showed a strong signal in about 30% of the stimulated PC12 cells. The tailed probe gave a stronger signal than the 'kinased' probe. The tailed oligonucleotide probe with a sense sequence did not show any hybridization. The unstimulated PC12 cells were unlabeled by either probe. The polymerase chain reaction (PCR) using the same oligonucleotides (with sense and antisense directions) as primer pairs specifically amplified the neurotensin sequence in the stimulated PCI2 cell RNA after it was reverse-transcribed into complementary DNA: a low level of neurotensin gene message was also detected in the unstimulated PC12 cells after 30 cycles of the PCR. Our findings suggest that oligonucleotides play a role in both quantitative and morphological studies of neuroendocrine gene expression at the cellular level.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)