Structural Organization of the Atlantic Croaker Connexin 32.2 Gene and Its 5' Flanking Region

Gonadotropic hormone stimulates the accumulation of connexin (Cx) 32.2 messenger RNA in ovaries of the marine teleost Atlantic croaker. This effect can be mimicked by protein kinase A (PKA) activators and blocked by PKA inhibitors as well as protein kinase C (PKC) stimulators. However, the mechanisms of Cx32.2 gene regulation are unknown. In this study, we determined the structure of the Cx32.2 gene as a first step toward characterizing the regulatory mechanisms of Cx32.2 gene expression. A cosmid library of croaker genomic DNA was screened with a Cx32.2 complementary DNA probe. One positive clone was subcloned and sequenced. The Cx32.2 gene contained two exons and one intron. The first exon contained a portion of the 5' untranslated region (UTR), and the second exon contained the remaining 5' UTR, the amino acid coding region, and the 3' UTR. The distal 5' flanking region also contained a sequence homologous to a different croaker Cx gene, Cx32.7, but it is unclear if this sequence constitutes a pseudogene or an exon of the Cx32.7 gene. The 5' flanking region of the Cx32.2 gene contained two core cyclic AMP response elements (CRE, CGTCA) and one full-consensus activating protein- (AP)-1 binding site (AGTCAG). The distal core CRE was associated with a sequence that enhances CRE activity (GAGC). A third core CRE site was present in the intron. These findings are consistent with the following hypotheses: the induction of ovarian Cx32.2 mRNA levels by gonadotropic hormone is at least partly mediated by CRE-dependent activation of the Cx32.2 gene, and the inhibition of basal and gonadotropic-hormone-stimulated ovarian Cx32.2 mRNA by PKC is due to negative effects on transcription via the AP-1 transcription factor complex. This study is the first to characterize the structure and putative response elements of the 5' flanking region of fish Cx genes.