Detection of mRNAs encoding distinct isoenzymes of type II calcium/calmodulin-dependent protein kinase using the polymerase chain reaction.

Abstract

A modification of the polymerase chain reaction (PCR) was used to amplify nucleotide sequences encoding the 50-kDa (alpha) or 58- to 60-kDa (beta',beta) subunits of a brain-specific type II calcium/calmodulin-dependent protein kinase (CaM kinase II). Rat brain RNA from different regions and at different postnatal ages was purified, and reverse transcriptase was used to produce cDNA templates. Oligonucleotide primer pairs flanking a unique sequence in the coding region of the beta',beta subunit-specific cDNA or a unique sequence in the 3' noncoding region of the alpha subunit-specific cDNA were used to amplify sequences encoding portions of these subunits by PCR. Adult rat forebrain contained approximately three times as much alpha subunit mRNA as beta',beta subunit mRNA, whereas adult rat cerebellum contained a molar ratio of 1 alpha: 5 beta',beta. Intermediate levels of alpha and beta',beta subunit mRNAs were observed in adult pons/medulla, and in 4- and 8-day neonatal forebrain. This amplification assay was also used to demonstrate the presence of alpha subunit mRNA in cerebellar granule cells and 4-day neonatal forebrain, which was reported to be undetectable by other methods. Cerebellar granule cells contained less alpha subunit RNA relative to whole cerebellum, suggesting that this cell type expresses an isoform of CaM kinase II containing less alpha subunit protein in the holoenzyme. The observed levels of subunit-specific mRNAs were shown to parallel the levels of expressed protein subunits, suggesting that expression of kinase isoforms is transcriptionally regulated. The data also indicate that the conditions used for amplification of CaM kinase II mRNAs are semiquantitative.