A cDNA library of the eutardigrade Hypsibius klebelsbergi Mihelčič, 1959 and analysis of the actin gene

Kiehl, Ernst; Dastych, H.; D’Haese, Jochen; Greven, Hartmut

doi:10.4081/jlimnol.2007.s1.152

Cited by 6 publications

(12 citation statements)

References 19 publications

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“…Two factors have hampered Tardigrada systematics, the small size of individuals (average 500 μm) with limited characters (10 characters common to the whole phylum, Guidetti and Bertolani, 2005), and low nucleic acid concentrations in the cell (Kiehl et al., 2007b; Sands et al., 2008). The consequences of these limitations are evident in the taxon sampling in phylogenetic studies, with the first studies including three or four in‐group taxa, and the latter including 11 (Jørgensen and Kristensen, 2004) to 14 (Kiehl et al., 2007a).…”

Section: Introductionmentioning

confidence: 99%

Phylum Tardigrada: an “individual” approach

et al. 2008

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Phylum Tardigrada consists of 1000 tiny, hardy metazoan species distributed throughout terrestrial, limno-terrestrial and oceanic habitats. Their phylogenetic status has been debated, with current evidence placing them in the Ecdysozoa. Although there have been efforts to explore tardigrade phylogeny using both morphological and molecular data, limitations such as their few morphological characters and low genomic DNA concentrations have resulted in restricted taxonomic coverage. Using a protocol that allows us to identify and extract DNA from individuals, we have sequenced 18S rDNA from 343 tardigrades from across the globe. Using maximum parsimony and Bayesian analyses we have found support for dividing Order Parachela into three superfamilies and further evidence that indicates the traditional taxonomic perspective of families in the class Eutardigrada are nonmonophyletic and require re-working. It appears that conserved morphology within Tardigrada has resulted in conservative taxonomy as we have found cases of several discrete lineages grouped into single genera. Although this work substantially adds to the understanding of the evolution and taxonomy of the phylum, we highlight that inferences gained from this work are likely to be refined with the inclusion of further taxa-specifically representatives of the nine families yet to be sampled.

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Section: Introductionmentioning

confidence: 99%

Phylum Tardigrada: an “individual” approach

et al. 2008

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“…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 λTriplEx2 (T 7, 5TripEx, 5Triplepostslip1).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

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The actin gene of Hypsibius klebelsbergi (Eutardigrada) – complete sequence and comparison with actin from related and non‐related taxa

D’Haese

Traore‐Freitag

Kiehl

et al. 2011

Journal of Zoological Systematics and Evolutionary Research

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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.

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“…Biochemical experiments of tardigrades are hampered due to their small size (average approximately 500 μm) and the lack of a convenient culture system to obtain large amounts of animals for protein isolation. We recently constructed a cDNA library of the eutardigrade Hypsibius klebelsbergi (Kiehl et al, ), a species that lives exclusively on glaciers in cryoconite holes (Dastych et al, ) and obtained the complete nucleotide sequence of one actin gene and its deduced amino acid sequence (D'Haese et al, ). In continuation of these studies, we herein report data about four EF‐hand proteins that trigger by calcium binding the interaction of actin–myosin.…”

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EF‐Hand Proteins and the Regulation of Actin‐Myosin Interaction in the Eutardigrade Hypsibius klebelsbergi (Tardigrada)

Prasath¹,

Greven²,

D’Haese

2012

J. Exp. Zool.

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Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

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A cDNA library of the eutardigrade Hypsibius klebelsbergi Mihelčič, 1959 and analysis of the actin gene

Cited by 6 publications

References 19 publications

Phylum Tardigrada: an “individual” approach

Phylum Tardigrada: an “individual” approach

The actin gene of Hypsibius klebelsbergi (Eutardigrada) – complete sequence and comparison with actin from related and non‐related taxa

EF‐Hand Proteins and the Regulation of Actin‐Myosin Interaction in the Eutardigrade Hypsibius klebelsbergi (Tardigrada)

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