Figure 5
The CaMKIIβ splice-variants in mature rat brain essentially all contain exon v4. (a) RNA Seq indicated that most hippocampal CaMKIIα transcripts lack exon v3N and that some hippocampal CaMKIIβ transcripts lack exon v1 or v5 sequences (with percentages indicated), but all contained exons v2, v4, and v6. A BamHI restriction site in β exon v4 can be utilized for a PCR/restriction strategy to identify transcripts lacking the exon v4 sequence: After a first round PCR, restriction digest with BamHI eliminates all templates that contain exon v4 for amplification in a second round of PCR with nested primers. As a positive control, a parallel PCR/restriction approach with SacII should identify the known variant βe that lacks exon v1. (b) A first round RT-PCR generated a band of the expected size (lane 1); ~7% of this band was resistant to digestion with SacII, indicating lack of exon v1 (lane 2); resistance to BamHI was not detected, indicating no lack of exon v4 (lane 3). (c) A second round of PCR after SacII digest yielded PCR products that were largely resistant to SacII (lane 4); a second round of PCR after BamHI digest yielded some BamHI resistant PCR product (lane 5), however, cloning and sequencing of these products did not reveal any products that lacked exon v4 (some had mutations in the BamHI site, while others were non-specific products unrelated to CaMKIIβ.
