Scapharca inaequivalvis A and B miniglobin genes: promoter activity of the 5' flanking regions and in vivo transcription.

Globin genes of the bivalve mollusk Scapharca inaequivalvis have the two intron/three exon organization typical of vertebrate and many invertebrate globins, with introns in highly conserved positions. Sequence studies on the A and B globin genes of the mollusk tetrameric hemoglobin gave evidence for the existence of 'minigenes' spanning part of the first and second intron, in-frame with the heme binding domain encoded by the central exon. Putative promoter and regulatory sequences flanking these minigenes were identified in the 3' regions of intron I. Here we report cloning and functional analysis of these regions ( approximately 400bp) and their promoter activity, which was assessed in K562 cells by transient transfection, was established. Moreover, in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) on total cytoplasmatic RNA demonstrated that the A and B minigenes are transcriptionally active in the erythrocytes of S. inaequivalvis. Thus, the present results lead to the conclusion that the present-day organization of the globin genes of S. inaequivalvis tetrameric hemoglobin is still reminiscent of an ancestral globin gene before exon shuffling.