Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification.
Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.
GERMETRAD, Laboratoire de Biochimie et Radiobiochimie, SMAB UPRES EA 2160, Université de Nantes, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France.
Jean-Pierre
JP
Durand
Françoise
F
Goudard
Jacques
J
Pieri
Jean-Michel
JM
Escoubas
Nathalie
N
Schreiber
Jean-Paul
JP
Cadoret
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D020869:Gene Expression Profiling; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010125:Ostreidae / Q000235:genetics*; D010802:Phylogeny; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D035925:Iron-Regulatory Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D005293:Ferritins