Gene Organization of the Mouse Pro-Hormone and Pro-Protein Convertase PC1

Abstract

Using a probe consisting of either the 5′ end sequence or the full-length cDNA sequence of the mouse prohormone convertase PC1 (mPC1), we isolated from a λEMBL3 mouse genomic library two clones that coded for the 5′ and 3′ ends of the mPC1 gene. The complete gene organization was obtained by combining the results of the sequence of these clones and those of the characterization of polymerase chain reaction-amplified genomic segments. The single-copy mPC1 gene, confirmed by Southern analysis, spans at least 42 kb and is composed of 15 exons and 14 introns of various sizes. The exon lengths varied between 77 to about 1,600 bp, with the longest exon representing the 3′ end of the gene. The intron sizes are between 0.4 and 6.5 kb in length. The active sites Asp■, His■, and Ser■, the catalytically important Asn[unk], and the RGD-containing domain are each found on separate exons. The general organization of the 5′ end and catalytic domain of the mouse PC1 gene is very similar to that reported for the other pro-protein convertases genes, namely human fur, human PC2, and mouse PC4. However, the four genes differ considerably in their 3′ end structure. Primer extension and 5′ RACE analysis demonstrated that the mPC1 mRNA contains multiple transcription initiation sites of which major ones are found at either 211, 209, or 207 bp from the 5′ end of the initiator methionine. Analysis of the sequence of the available 850-bp promoter segment revealed no functional TATA and CCAAT boxes. However, within this segment we noted the presence of two AP-1, Sp1, and cAMP responsive element (CRE) sequences, an interferon consensus sequence (ICS), and three POU proteins (e.g., GHF-1) binding elements. In tissues and cells. Northern blot analysis demonstrated the presence of two major mRNA transcripts of sizes 3 and 5 kb. The cDNA structure of rat PC1 demonstrated that these two transcripts arise by alternative choice of polyadenylation sites and in the mouse these two alternative sites are found on exons 14 and 15, respectively. Accordingly, we show that exon 14 is found in both the 3- and 5-kb transcripts but exon 15 is only found in the 5-kb mRNA. Using a 3′ end probe specifically hybridizing with the 5-kb mRNA, we show that in the mouse pituitary neurointermediate lobe the 3-kb form is negatively regulated by dopamine, while the 5-kb form is not.