Analysis of tyrosine hydroxylase gene transcription using an intron specific probe.
The nuclear run-on assay is the most commonly used technique to determine transcription rates of specific genes such as tyrosine hydroxylase. Its application to studies in the nervous system is problematic, however, as a result of limitations in sensitivity and the loss of anatomical integrity. We observed that the relative levels of tyrosine hydroxylase intron 2-containing RNA using a ribonuclease protection assay in the adrenal medulla changed in response to pharmacological treatments consistently with changes shown by the nuclear run-on assay. Our results indicate that measures of tyrosine hydroxylase primary transcript levels offer an alternative to the nuclear run-on assay and validate the application of intron-specific in situ hybridization as a means of assessing the relative transcriptional activity of the tyrosine hydroxylase gene. Similar quantitative results were obtained using intron-specific in situ hybridization with oligonucleotide probes specific for rat tyrosine hydroxylase intron 2. Furthermore, we observed that intron-specific in situ hybridization could be used to measure tyrosine hydroxylase transcription rates in the locus coeruleus, providing resolution at the level of single neurons. Thus, measuring the levels of tyrosine hydroxylase intron 2 provides a sensitive measure of tyrosine hydroxylase transcription rate that can be applied to the study of brain catecholaminergic neurons.
Chang
M S
MS
Department of Molecular Genetics and Biochemistry, University of Pittsburgh, PA 15261, USA.
Hahn
M K
MK
Sved
A F
AF
Zigmond
M J
MJ
Austin
M C
MC
Sherman
T G
TG
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D006651:Histocytochemistry; D017403:In Situ Hybridization; D007438:Introns; D008125:Locus Coeruleus / Q000201:enzymology; D008297:Male; D015335:Molecular Probes; D009693:Nucleic Acid Hybridization; D051381:Rats; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D015335:Molecular Probes; D014446:Tyrosine 3-Monooxygenase; D012260:Ribonucleases