* Institute of Molecular Genetics
Institute of Zoology, Johannes Gutenberg University, Mainz, Germany
Correspondence: E-mail: hankeln{at}uni-mainz.de.
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Abstract |
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Key Words: Ciona intestinalis hemoglobin neuroglobin cytoglobin hemocyanin intron
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Introduction |
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In vertebrates, oxygen transport to the tissues is mediated by several different classes of hemoglobins (Dickerson and Geis 1983), whereas myoglobin functions as an oxygen store in the muscle (Wittenberg 1992), besides being involved as an enzyme in nitric oxide metabolism (Flögel et al. 2001). Recently, two novel classes of vertebrate globins have been described. Neuroglobin (Burmester et al. 2000) is primarily expressed in neuronal cells of the central nervous system and in the retina, where it may sustain aerobic metabolism under conditions of high oxygen demand (Sun et al. 2001; Reuss et al. 2002; Schmidt et al. 2003). Cytoglobin (Burmester et al. 2002; Trent and Hargrove 2002) is expressed in virtually all human tissues, but its physiological role is still unclear.
The sea squirt Ciona intestinalis is of special interest for the understanding of vertebrate evolution because the tunicates (Urochordata) are phylogenetically positioned at the base of the vertebrate tree (Wada and Satoh 1994). Recently, Dehal et al. (2002) reported the draft genome sequence of Ciona. In their discussion of the sea squirt gene repertoire, the authors suggested that the Ciona genome lacks globin genes and proposed that the sea squirt uses hemocyanins for oxygen transport. However, here we report the identification of four different globins in Ciona.
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Materials and Methods |
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C. intestinalis adult specimens were obtained from the Meeresbiologische Forschungsanstalt Helgoland, Germany. Total RNA was prepared from adult tissue (Sambrook and Russell 2001, pp. 7.4), and 5 µg of RNA were converted into cDNA by Superscript II reverse transcriptase following the supplier's protocol (Invitrogen). One-tenth of a cDNA reaction was used for standard PCR, employing globin genespecific oligonucleotide primers that had been derived from the in silicopredicted coding regions of the Ciona globin genes. Alternatively, PCR was performed on a Ciona cDNA clone pool obtained from the German resource center RZPD (Ciona larval library 684; www.rzpd.de) and on a cDNA library made from Helgoland adult Ciona specimen provided by K. Weber (Max Planck Institut für biophysikalische Chemie, Göttingen, Germany). PCR amplificates were either sequenced directly or after cloning into plasmid vectors (pGEM T-easy, Promega) on both strands by the dye terminator cycle sequencing chemistry (Applied Biosystems). Sequencing reactions were loaded onto an Applied Biosystems Prism 3730 capillary sequencer by GENterprise GmbH, Mainz. Sequences were further manipulated using Lasergene programs (DNAstar Inc.).
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Results and Discussion |
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The phylogeny of Ciona globins relative to vertebrate and nonvertebrate globins was investigated using established amino acid sequence alignments (Burmester et al. 2000, 2002) and various tree reconstruction methods. The plant globins were considered as outgroup. In all types of analyses, the globins of Ciona form a well-supported monophyletic clade (fig. 3). However, due to a lack of sufficient phylogenetic signal in the globin sequences, the exact branching orders in the tree of the metazoan globins could not be resolved with sufficient confidence, giving rise to low bootstrap support values and posterior probabilities. Nevertheless, the vertebrate globins form a common clade distinct from both Ciona and the other invertebrate globins (fig. 3).
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Dehal et al. (2002) put forward the idea that copper-containing hemocyanins are used for oxygen transport in Ciona. This would be surprising because hemocyanins have so far only been found in molluscan or arthropod species. The hemocyanins of these two phyla represent two separate protein families that derived independently from distinct types of tyrosinases (Burmester 2001). Although there are in fact two genes in Ciona that display significant similarities to the arthropod hemocyanin superfamily (Burmester 2002), phylogenetic analyses indicate that respiratory hemocyanins evolved only within the arthropod phylum (Immesberger and Burmester, unpublished data). At least three more Ciona genes resemble the molluscan hemocyanins and other members of the large tyrosinase superfamily. However, neither phylogenetic nor structural analyses (single domain proteins in Ciona in contrast to seven or eight domains in molluscan hemocyanins) support a role of these Ciona proteins as respiratory proteins (data not shown). Although the exact role of the Ciona globins in the oxygen metabolism of tunicates requires further studies, it should be considered extremely unlikely that Ciona uses hemocyanins for respiration as suggested (Dehal et al. 2002).
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Acknowledgements |
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Footnotes |
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