Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari.
Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5'-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.
Kim
Seong Ryul
SR
College of Natural Resources and Life Science, Dong-A University, 604-714 Busan, South Korea.
Lee
Kwang Sik
KS
Yoon
Hyung Joo
HJ
Park
Nam Sook
NS
Lee
Sang Mong
SM
Kim
Iksoo
I
Seo
Sook Jae
SJ
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D001517:Coleoptera; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D006458:Hemolymph / Q000378:metabolism; D007313:Insecta; D007501:Iron / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D005293:Ferritins; D007501:Iron