THE HUMAN GENE FOR THE REGULATORY SUBUNIT RI-ALPHA OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE - 2 DISTINCT PROMOTERS PROVIDE DIFFERENTIAL REGULATION OF ALTERNATELY SPLICED MESSENGER RIBONUCLEIC-ACIDS

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately 2 kilob ases (kb) of the 5'-flanking region obtained by isolation and sequenci ng of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. Our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary DNA ind icated that the RI alpha gene may have multiple leader exons giving ri se to alternately spliced messenger RNAs (mRNAs). Reverse transcriptio n of human testis RNA followed by PCR identified two different RI alph a mRNA species (RI alpha 1a and RI alpha 1b) containing distinct 5'-se quences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression ina human B lymphoi d cell line (Reh) and was stimulated only 4- to 6-fold by treatment wi th cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA we re present in untreated Reh cells, but were stimulated 40- to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich an d did not contain any TATA box. Several putative transcription initiat ion sites were identified in front of each leader exon (exons la and I b)by the 5'-rapid amplification of complementary DNA ends technique. T o determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons la and Ib were inserte d in front of a chloramphenicol acetyltransferase reporter gene, and t heir ability to direct transcription were examined. Transfection of th ese constructs into rat GH(4)C(1) cells demonstrated that both constru cts had promoter activities, as evidenced by high levels of chloramphe nicol acetyltransferase activity.