CLONING AND ANALYSIS OF A CDNA-ENCODING A 2-DOMAIN HEMOGLOBIN CHAIN FROM THE WATER FLEA DAPHNIA-MAGNA

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna wa s cloned and its nucleotide (nt) sequence of 1261 bp was determined. T he nt sequence contained 74 bp of the leader sequence, 1047 bp of an o pen reading frame (ORF), and 119 bp of the 3'-untranslated region (UTR ), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenyla tion signal. cDNA-derived amino acid (aa) sequence revealed that D. ma gna Hb chain is synthesized as a secretory precursor with a signal pep tide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues wi th a calculated molecular weight of 36 227, which is composed of two l arge repeated domains, domain 1 and 2. Several key aa that are invaria nt in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (p re-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher t han that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.