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Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.

We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).