This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Calmodulin is a cytosolic, calcium binding protein that 'senses' intracellular fluctuations of free Ca++ and transduces the calcium second messenger by initiating appropriate cellular responses, including the activation of individual enzymes or specific biochemical cascades. We have cloned and sequenced the entire tomato calmodulin gene family, which consists of 6 distinct genes. Sequence examination revealed that one of these calmodulin genes is alternatively spliced to produce two different mRNA variants, which include a standard cytosolic calmodulin protein, and a modified calmodulin containing a C-terminal, 25 amino acid long nuclear localization signal (NLS), which is encoded by a separate exon. Exons encoding the standard calmodulin protein, and the NLS domain are separated by a 2100 bp long intron. An exhaustive database search revealed an unpublished calmodulin cDNA entry from Petunia (an ancestral Solanaceae family member) that also contains a C-terminal NLS-domain. We isolated Petunia genomic DNA and used PCR to amplify the calmodulin-NLS sequence. Unexpectedly, our results revealed that the NLS domain is continuous with the standard calmodulin coding sequence, and is not contained in a separate downstream exon (as it is in tomato). We are using this experimental system to gain insight into how 'new' genes evolve, how gene products are modified through exon shuffling, and the role of alternative splicing in regulating gene expression. Continuing work will involve isolation and examination of calmodulin genes in other cultivated and wild Solanaceae family members (e.g., potato, pepper, eggplant, tobacco, nightshade).
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