Institut fu ¨r Zoomorphologie und Zellbiologie der Heinrich-Heine-Universita ¨t Du ¨sseldorf, Du ¨sseldorf, Germany The actin gene of Hypsibius klebelsbergi (Eutardigrada) – complete sequence and comparison with actin from related and non-related taxa Jochen D’Haese,Assita Traore-Freitag,Ernst Kiehl,Thiruketheeswaran Prasath and Hartmut Greven Abstract The actin gene of tardigrades was sequenced and analysed using a k ZAP Express cDNA library from the eutardigrade Hypsibius klebelsbergi previously constructed by us. We obtained the complete actin coding sequence of one isoform (1128 bp; 375 amino acids; MW 41 674 Da) together with parts of the 3¢- and 5¢- UTR region. Comparison of the 12 incomplete actin sequences of Hypsibius dujardini incorporated in GenBank indicates that this H. klebelsbergi actin sequence probably represents the most abundant muscle isoform. Ten of the H. dujardini clones show minor differences in codon usage and identical amino acid compositions to the H. klebelsbergi actin. Only two clones show amino acid variations in one and five positions, respectively, but show identical amino acids at their N-terminus. A considerable similarity between the 5¢- and 3¢-UTR regions of both tardigrade species was recognized. The H. klebelsbergi actin exhibits an overall high sequence similarity to the vertebrate b-actin. A comparison of muscle actins from various vertebrates as well as Ecdysozoa and non-Ecdysozoa revealed a more pronounced similarity of the tardigrade actin to arthropods and annelids and not to nematodes. Key words: Tardigrada – Hypsibius klebelsbergi – actin – sequence comparison Introduction Actin is one of the most highly conserved cytoskeletal proteins in eucaryotic cells, which generally are encoded by multigene families. Such families are suggested to have arisen by an initial gene duplication and divergence of a common ancestral gene (e.g. Maeda and Smithies 1986). In most organisms, these genes code for several muscle and non-muscle isoforms of actin, which slightly differ, especially at their N-terminus. Actin filaments are formed by polymerization of globular actin subunits consisting of 374–376 amino acids. In vertebrates, three isoelectric variants of actin are present, a-actin in muscle cells and b- and c-actin in non-muscle cells (cytoplasmic actin). In ÔinvertebratesÕ muscle actin is similar to the vertebrate b-actin (e.g. Vandekerckhove and Weber 1984). The large differences between arthropod muscle actins and vertebrate muscle isoforms suggest an independent origin from cytoplasmic isoforms (Mounier et al. 1992). Muscle actin genes have been proved as a useful tool for investigating the chordate lineage. In vertebrates 27 of 375 amino acid positions differentiate muscle and non-muscle actins and are referred to as diagnostic amino acids (Vandekerckhove and Weber 1984; Kovilur et al. 1993). An ÔinvertebrateÕ taxon poorly studied in this respect is the Tardigrada. Analysis of 18S ribosomal RNA (rRNA) sequences clearly supports their monophyly. Relationship to other taxa, however, is a matter of debate. rRNA analysis appears to unite tardigrades with the Onychophora and Euarthropoda to the Panarthropoda (Nielsen 2001). The Panarthropoda are either considered as sister group of (some) Cycloneuralia, especially nematodes, and both constitute the taxon Ecydsozoa (e.g. Garey 2001; Giribet 2003; Colgan et al. 2008) or Panarthropoda form together with the Annelida the traditional assemblage Articulata (e.g. Scholtz 2002). It was also suggested that Ecdysozoa is the sister group of the Annelida (Nielsen 2003). Other molecular markers such as 28S rRNA, myosin II, histone H3 and elongation factor I alpha appear to support the existence of a clade Ecdysozoa (for summary and further references, see Colgan et al. 2008). When constructing the cDNA of the eutardigrade Hypsibius klebelsbergi, we partially sequenced the actin gene of this species and found a considerable similarity of nucleic ⁄ amino acids not only with actin fragments of several eutardigrades (Macrobiotus sp., Ramazzottius oberhaeuseri, Hypsibius dujardini) but also with the actin of Drosophila melanogaster. However, the lack of complete actin sequences allowed only for limited comparison with related and non-related taxa (Kiehl et al. 2007). We now obtained the complete nucleotide sequence of one actin gene and its deduced amino acid sequence from the H. klebelsbergi cDNA library (Kiehl et al. 2007). In addition, we analysed the various fragmentary sequences of the actin gene from the Expressed Sequence Tag (EST) library of the eutardigrade Hypsibius dujardini (Doye`re, 1840) trimmed to regions of good quality (Blaxter H, Daub J, Maroon H, Thomas F, Whitton C, Aboobaker A, unpublished – http:// www.nematodes.org/tardigrades/tardibase_texts/construction. html) and incorporated in GenBank (Benson et al. 2007; TardiBase, Daub et al. 2003). Together with the actin sequences available for many other organisms a more com- prehensive comparative study was possible. Material and Methods The actin sequence was obtained by using the cDNA library of H. klebelsbergi (Kiehl et al. 2007) together with forward and reverse primers of conserved sequence regions of actin and specific primers of the expression vector kTriplEx2 (T 7, 5TripEx, 5Triplepostslip1). Actin fragments were amplified by using the primers Act U235 and Act L667 (Fretz and Spindler 1999). and the general actin primers Actup and Actdown (Viray master, Chicago 1993). After screening of the gene library, one clone containing the full-length actin gene was selected to avoid interference with other actin isoforms (Table 1). All primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). PCR reactions were performed as described (Kiehl et al. 2007). PCR-products were separated in 1.2% agarose gels in TBE Corresponding author: Jochen D’Haese (dhaese@uni-duesseldorf.de) Contributing authors: Assita Traore-Freitag, Ernst Kiehl (tipula@aoi. com), Thiruketheeswaran Prasath (pras_t@gmx.de), Hartmut Greven (grevenh@uni-dusseldorf.de) Ó 2011 Blackwell Verlag GmbH Accepted on 1 October 2010 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2010.00604.x J Zool Syst Evol Res (2011) 49 (Suppl. 1), 84–89