Cloning, characterization, and expression of the gene for the catalytic subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. Identification of highly conserved and unique isoforms generated by alternative splicing.
The nematode Caenorhabditis elegans expresses substantial amounts of several forms (Mr values = 39,000-41,000) of the catalytic subunit (C) of cAMP-dependent protein kinase. Approximately 65% of the total cAMP-dependent phosphotransferase activity is recovered in particulate fractions of homogenates prepared from asynchronous populations of C. elegans. The C subunit is expressed at a low level in cytosolic and particulate compartments during embryogenesis. As the nematodes progress from late embryonic stages to the newly hatched, first larval (L1) stage, C subunit content increases 15-fold. High levels of C subunits are observed in several subsequent larval and adult stages of development. Since the relative abundance of C subunit mRNA changes little with development, it appears that control of C expression is exerted the translational and/or post-translational levels. cDNAs for two types of C have been cloned and sequenced. The derived amino acid sequence of a major isoform (CeCAT alpha, 358 residues) is highly homologous (82% identical) with the murine C alpha subunit. A second, novel C subunit (CeCAT alpha', 374 residues) has a unique 56-residue carboxyl-terminal region that is generated by the alternative splicing of the C pre-mRNA. The splicing process that yields CeCAT alpha' is unusual because it converts the central portion of an apparent 1-kilobase (kb) intron to an exon. The alternative exon introduces the novel carboxyl terminus and a new translation stop signal, while simultaneously converting the coding sequence for 40 carboxyl-terminal residues in CeCAT alpha into 3'-untranslated nucleotides. The 5' end of the C. elegans C subunit mRNA is produced by the trans-splicing of the C gene transcript to a 22-base pair C. elegans leader sequence originally described by Krause, M., and Hirsh, D. [1987) Cell 49, 753-761). The 20-kb C. elegans C gene is divided into seven exons by introns ranging in size from 54 to 8000 bp. The sizes of the C. elegans C subunit gene, cytoplasmic mRNA (2.5 kb), and subunit protein are similar to the sizes of the murine C alpha gene, mRNA, and polypeptide. However, the nematode and murine C genes differ significantly in the organization of their introns and exons.
Gross
R E
RE
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
Bagchi
S
S
Lu
X
X
Rubin
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002107:Caenorhabditis / Q000201:enzymology / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005091:Exons; D005796:Genes; D007438:Introns; D007527:Isoenzymes / Q000235:genetics*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011494:Protein Kinases / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011494:Protein Kinases