The Primary Structure of Hemoglobin D from the Aldabra Giant Tortoise, Geochelone gigantea

Shishikura, Fumio

doi:10.2108/zsj.19.197

Cited by 11 publications

(18 citation statements)

References 46 publications

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“…For preparation of genomic DNAs from 100% ethanol-fixed red blood cells of adult giant tortoises and the Chaco tortoises, DNA samples were prepared as described previously 6,7) and stored at -80°C.…”

Section: Preparation Of Genomic Dnamentioning

confidence: 99%

“…Before sequencing the PCR amplified fragments with a BigDye Terminator v. 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), the degenerate oligonucleotide primers were tailed with the pUC/M13 forward or pUC/M13 reverse sequencing primer tail (forward 17-mer: 5'-GTAAAACGACGGCCAGT-3', Sigma-Aldrich; and reverse 17-mer: 5'-CAGGAAA CAGCTATGAC-3', Promega, Tokyo, Japan). PCR amplification and the thermal profile including denaturation of the first 5 cycles were described in the previous reports 6,7) . Table 1 for nucleotide sequences of the primers (F1-F9, R1-R6).…”

Section: Primer Design and Pcr Conditionsmentioning

confidence: 99%

“…Here, the author aims to provide scientific information on the origin and the divergence time of the two giant tortoise species, including a close relative to the Galapagos giant tortoises, the Chaco tortoise (Geochelone chilensis; synonym: Chelonoidis chilensis). To analyze intra-genus relationships, the author first constructed a molecular tree based on the primary structure of the globin subunits α A , α D , and β [5][6][7] . Both giant tortoise species were shown to diverge around 15-21 mya 6,7) , which represents a significant delay following the break-up of Gondwana, which separated into Africa and South America during the Cretaceous period 65-135 mya 8,9) .…”

Section: Introductionmentioning

confidence: 99%

“…To analyze intra-genus relationships, the author first constructed a molecular tree based on the primary structure of the globin subunits α A , α D , and β [5][6][7] . Both giant tortoise species were shown to diverge around 15-21 mya 6,7) , which represents a significant delay following the break-up of Gondwana, which separated into Africa and South America during the Cretaceous period 65-135 mya 8,9) . Hence, the author provides genetic relationships, in particular divergence times of the three Geochelone species, based on globin genealogy.…”

Section: Introductionmentioning

confidence: 99%

“…On the contrary, very little is known about intron nucleotide sequences of reptilian globin 6) . The author reports all intron nucleotide sequences of the globin genes, including α A , α D , and β in the above three species, and provides the following two molecular trees: 1) a tree based on a complete sequence of the two introns and three exons compared with a tree based on only the three exons (a protein coding sequence); 2) a tree based on only intron nucleotide sequences.…”

Section: Introductionmentioning

confidence: 99%

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Globin-intron Sequences Imply Molecular Relationships Between the Galapagos Giant Tortoise and the Aldabra Giant Tortoise

Shishikura

2013

Nichidai Igaku Zasshi

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The author previously established a molecular tree based on the cDNA-derived primary structures of globins in two giant tortoises, Geochelone nigra and G. gigantea. The divergence time was estimated to be 15-21 million years ago. In the present study, the author reexamined the divergence time of these two species using another source of genetic information-globin-introns-including those from the Chaco tortoise (G. chilensis), a close relative of G. nigra. The previously determined divergence time was supported by the findings of this intron study. However, the inter-relationships based on the intron nucleotide sequences of the globins from the three Geochelone species remain controversial, because it is difficult to determine which of the three is the ancestral species. In addition, the nucleotide sequences reveal the following interesting characteristics: (1) an abnormal GC dinucleotide sequence located at the 5'-splicing site of the second intron of α D globins instead of a consensus GT-this finding is common to all studied Geochelone species; (2) a repeated sequence 5'-GCCCCGCGCCCCGC-3' found only in the first intron of the G. nigra α A globin gene, is a unique feature distinguishing the Galapagos giant tortoise from the other Geochelone tortoises that have non-repeated GCCCCGC sequences.

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Section: Preparation Of Genomic Dnamentioning

confidence: 99%

Section: Primer Design and Pcr Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Globin-intron Sequences Imply Molecular Relationships Between the Galapagos Giant Tortoise and the Aldabra Giant Tortoise

Shishikura

2013

Nichidai Igaku Zasshi

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Side‐necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA‐derived primary structures and X‐ray crystal structures of Hb A

2011

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Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical β-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P4₁2₁2), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6₁22), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 Å for form A and 2.35 Å for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 Å. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.

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Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

Anwised

Kabbua

Temsiripong³

et al. 2013

Protein J

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The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.

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The Primary Structure of Hemoglobin D from the Aldabra Giant Tortoise, Geochelone gigantea

Cited by 11 publications

References 46 publications

Globin-intron Sequences Imply Molecular Relationships Between the Galapagos Giant Tortoise and the Aldabra Giant Tortoise

Globin-intron Sequences Imply Molecular Relationships Between the Galapagos Giant Tortoise and the Aldabra Giant Tortoise

Side‐necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA‐derived primary structures and X‐ray crystal structures of Hb A

Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

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