Abstract
Hemoglobin (Hb) purified from the water flea, Daphnia magna, reared under hypoxia was analyzed by two-dimensional gel electrophoresis. The Hb was shown to be composed of six major subunit chain species (designated as DHbA to DHbF). The NH2-terminal amino acid sequences of DHbA, DHbB, DHbC, and DHbF are different from one another, indicating that at least four Hb genes are present in D. magna. The NH2-terminal amino acid sequences of DHbD and DHbE are the same as those of DHbA and DHbB, respectively. The six Hb chains were also found in the animal reared under normoxia in small amounts and with altered composition; the extent of decrease under normoxia was higher in the amounts of DHbC, DHbD, and DHbF than those of others. These results indicate that the Hb genes are differentially regulated by the ambient oxygen concentration. Four Hb genes constituting a cluster in the order, dhb4, dhb3, dhb1, and dhb2, were found on the chromosome of D. magna. The complete nucleotide sequences of the dhb1, dhb2, and dhb3 genes and their cDNAs showed that the genes have a seven-exon, six-intron structure. The structure consists of an intron separating an exon encoding a secretory signal sequence, two large repeated regions of a three-exon, two-intron structure that encode each a domain containing a heme-binding site, and an intron bridging the two repeated regions. The deduced amino acid sequences of the gene products showed higher than 79% identity to one another and showed unique features conserved in D. magna Hb chains. The analysis also suggested that DHbB (or DHbE), DHbF, and DHbC are encoded by the dhb1, dhb2, and dhb3 genes, respectively.
Highlights
Hemoglobins (Hb)1 are widely distributed among eukaryotes and in prokaryotes [1]
Twodomain or multidomain Hb chains are only found in invertebrates, and their physiological roles and structures are interesting from the viewpoint of Hb evolution. cDNA clones encoding two-domain intracellular Hb chains from the clam Barbatia reeveana [5] and Barbatia lima [6] and two-domain extracellular Hb chain from nematode Pseudoterranova decipiens [7] have been isolated and their nucleotide sequences determined
Separation by Two-dimensional Gel Electrophoresis and Determination of the NH2-terminal Amino Acid Sequences of Hb Chains—Hb was purified from D. magna reared under hypoxia
Summary
Hemoglobin(s); PMSF, phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; kb, kilobase pair(s); HIF-1, hypoxia-inducible factor-1. They are mostly large extracellular proteins classifiable by the number of chains and that of globin domains in each chain. Extracellular Hb composed of multiple two-domain chains with relatively normal oxygen binding activity playing important roles in oxygen transport are found in hemolymph of Cladocera, such as water fleas Daphnia magna and Moina macrocopa [10] These animals show a drastic increase in Hb synthesis in response to a decrease in the ambient oxygen concentration, which results in a change in the body color from colorless to red [11], providing an excellent model system for studying the environmental control of gene expression. Heterogeneity of D. magna Hemoglobin Chains two-dimensional gel electrophoresis and by determining the nucleotide sequences of their genes and cDNAs
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