Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cl adocera, Moina macrocopa. were cloned and their nucleotide (nt) sequences w ere determined. The amino acid (aa) sequences of both the gene products ded uced from the nt sequences consisted of 348 residues and showed 98% identit y with each other. These sequences together with the NH-terminal aa sequenc es of the Hb chains determined after separation by two-dimensional gel elec trophoresis showed that the Hb chains are synthesized as a secretory precur sor with a signal peptide of 17 aa residues. The aa sequences of M. macroco pa Hb chains shared the following features with those of Daphnia Rb chains. Firstly, the signal peptide is followed by an NH2-terminal extension conta ining a threonine-rich sequence that might play a role in the multimerizati on of subunit chains. Secondly, the identity between the aa sequences of th e first and second domains is exceptionally low. These facts suggest that d uplication of the cladoceran Hb gene occurred before the divergence of fami lies Moinidae and Daphniidae. Analysis of genomic DNA showed that the M mac rocopa Hb genes consist of two large repeated regions. encoding the first a nd second domains of Hb, chains, respectively. The intron-exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure chara cteristic of animal Rb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moi na genes, providing a new example of intron loss. The following elements in the 5' flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, G AAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moi na); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sit es for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia. (C ) 2001 Elsevier Science B.V. All rights reserved.