Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene.
A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.
Kurten
R C
RC
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Levy
L O
LO
Shey
J
J
Durica
J M
JM
Richards
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002467:Cell Nucleus / Q000502:physiology; D000242:Cyclic AMP / Q000502:physiology*; D003596:Cytosine; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D015723:Gene Library; D006107:Granulosa Cells / Q000201:enzymology*; D006147:Guanine; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D016385:TATA Box; D014162:Transfection
D007527:Isoenzymes; D046911:Macromolecular Substances; D006147:Guanine; D003596:Cytosine; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases