Abstract
The cAMP response element (CRE) mediates cAMP responsiveness in many eukaryotic genes (Roesler, W. J., Vandenbark, G. R., and Hansen, R. W. (1988) J. Biol. Chem. 263, 9063-9066). The tyrosine hydroxylase gene (TH) contains a single copy of a consensus CRE at -45 to -38 base pair (bp) upstream of the transcription initiation site. Deletional and mutational analyses of the upstream 2400-base pair region of the rat TH gene using transient transfection assay demonstrated that the CRE was essential for both cAMP-mediated induction and basal transcription of the TH gene. Another domain between -365 and -151 bp, containing the AP1 site, contributed to transcription to a smaller degree. Thus, the CRE appears to play an important dual role as a basal promoter element and an inducible enhancer for TH transcription. Interactions between the DNA binding factors in nuclear extract and CRE-containing oligonucleotides were investigated by gel retardation and competition assays. Oligonucleotides corresponding to the CRE regions of the TH or somatostatin gene gave rise to a pair of distinct protein-DNA complexes with identical mobilities in the gel retardation assay, suggesting that similar nuclear factor(s) might bind to the CREs of the TH and somatostatin genes. This study emphasizes a fundamental role of the CRE in transcriptional activation of the TH gene in catecholaminergic cells.
Highlights
CRE at -45 to -38 basepair(bp)upstreamofthe acute increasesin THactivity or somewhat transcription initiationsite
TheCRE appears to play an Among the important regulators of tyrosine hydroxylase (TH), cAMP has been important dual role as a basal promoter element and shown to stimulate increases in T H activity by promoting an inducible enhancer for TH transcription
Tigated by gel retardation and competition assays. 01- for T H (Lewis et al, 1987). cAMP induces the transcription igonucleotides corresponding to CthReE regions of the of vaariety of genes via a consensus octamer, 5‘fTpgmaerHaclotttroooeersritnt(asas-ort)DmdimnNaatigAtgiooehnsntctebaaositms.nisndTapthylgoei,etxsshneuseewstggCuigatdRehvysEeitdesirnmeoisnfgpettihhtcoaaastlaitzhsmeipemsaoTaiibrlHaoiflfruiatndnineddiussatcimlnisenocea-tnrt-heTilCenGRr AEethtCeoa5Gfl”.,Tuthp1Ce9sAt8sr8-oe3;am’Gm,atothooreesdgtmcaioAtainnnMs, oP1gf9egr9nee0esnp)e.hosTanrshseeegbueeplelraenotmetedceihnnbatyrta(hcCcAattReMrbEizPi)ne,(ddfRsooautenhnsddetal role of theCRE in transcriptionalactivation of the cloned
Summary
Cell Culture-Human neuroblastoma SK-N-BE(2)Ccells were passaged in Dulbecco’smodified Eagle’s medium supplemented with 10% newborn calf serum. PC12 cells were grown in RPMI 1640 medium supplemented with 10% horse serum and 5%fetal calf serum. Each serum was used after heat inactivation. All culture media contained 100 units/ml penicillin and 100 pg/ml streptomycin. The complexes were resolved on nondenaturing 6% polyacrylamide gels. Gels were prewarmed by electrophoresis for 1h at 10 V cm” prior to loading samples. Samples containing bromphenol blue and xylene cyano were electrophoresed for 2 h. Gels were dried and visualized by autoradiography. Was constructed in the pOCAT vector (Carroll et aL, 1991). This construct contains 503 bp of the 5“flanking sequence of the rat TH
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