Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari |
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Authors: | Kim Seong Ryul Lee Kwang Sik Yoon Hyung Joo Park Nam Sook Lee Sang Mong Kim Iksoo Seo Sook Jae Sohn Hung Dae Jin Byung Rae |
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Institution: | a College of Natural Resources and Life Science, Dong-A University, 604-714, Busan, South Korea;b Department of Agricultural Biology, National Institute of Agricultural Science and Technology, 441-100, Suwon, South Korea;c Department of Sericultural and Entomological Biology, Miryang National University, 627-130, Miryang, South Korea;d Division of Life Science, Gyeongsang National University, 660-701, Jinju, South Korea |
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Abstract: | 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. |
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Keywords: | Insect Mulberry longicorn beetle Apriona germari Ferritin cDNA sequences Phylogeny Insect cells Baculovirus |
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