Analysis of micronuclear, macronuclear and cDNA sequences encoding the regulatory subunit of cAMP-dependent protein kinase of Euplotes octocarinatus:Evidence for a ribosomal frameshift

We have isolated and characterized the micronuclear gene encoding the regul atory subunit of cAMP-dependent protein kinase of the ciliated protozoan Eu plotes octocarinatus, as well as its macronuclear version and the correspon ding cDNA. Analyses of the sequences revealed that the micronuclear gene co ntains one small 69-bp internal eliminated sequence (IES) that is removed d uring macronuclear development. The IES is located in the 5'-noncoding regi on of the micronuclear gene and is flanked by a pair of tetranucleotide 5'- TACA-3' direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macro nucleus. Sequence analysis suggests that the expression of this gene requir es a +1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP-dependent protein kinase type I regulatory subunit of Homo sapiens , and 53% identity with the regulatory subunit R-44 of one of the two cAMP- dependent protein kinases of Paramecium. In addition, it contains two highl y conserved cAMP binding sites in the C-terminal domain. The putative autop hosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R-44 of Paramecium but distinct f rom the consensus motif RRXSZ of other eukaryotic regulatory subunits of cA MP-dependent protein kinases.