Effects of second messenger system activation on functional expression of tyrosine hydroxylase fusion gene constructs in neuronal and nonneuronal cells.
A genomic clone for rat tyrosine hydroxylase (TH) was isolated and a fragment containing 503 bp upstream of the transcription start site was sequenced. The BamHI/AluI fragment was inserted into a plasmid carrying the coding sequence for bacterial chloramphenicol acetyltransferase (CAT). Another construct with the 5' sequence truncated to -151 bp also was prepared. When these were introduced into several mammalian cell lines, including C6 glioma, BE(2) neuroblastoma, CV-1 or Ltk- fibroblasts, different basal levels of CAT expression were observed. In the fibroblast lines, THCAT constructs were not expressed unless the cells were treated with forskolin or TPA. However, the low basal expression was not correlated to endogenous expression as THCAT constructs expressed comparably in BE(2)C, HeLa, and C6 glioma. Treatment of any of the cell lines with forskolin, TPA, or a combination of the two agents stimulated the expression by at least two-fold in all cell lines and the maximally induced levels were at least 10-fold over promoterless controls. These data indicate that the essential promoter elements as well as those conferring responsivity to cyclic AMP reside within 151 bp of the transcription start site. However, the array of elements regulating cell-type expression lie, at least in part, beyond the 500-bp region examined. Further, a role for phosphorylation in the regulation of basal and induced transcription of TH is suggested.
Laboratory of Molecular Neurobiology, Cornell University Medical College, Medical Research Institute Burke, White Plains, NY 10605.
J M
JM
Carroll
K S
KS
Kim
K T
KT
Kim
H M
HM
Goodman
T H
TH
Joh
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005910:Glioma / Q000473:pathology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015290:Second Messenger Systems; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D003907:Dexamethasone; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D013755:Tetradecanoylphorbol Acetate