Available online at www.sciencedirect.com European Journal of Protistology 49 (2013) 40–49 Variations and evolution of polyubiquitin genes from ciliates Xihan Liua,b, Fei Shia, Jun Gonga,c,∗ aYantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China bLaboratory of Protozoology, Ocean University of China, Qingdao 266003, China cCollege of Life Sciences, South China Normal University, Guangzhou 510631, China Received 2 January 2012; received in revised form 2 May 2012; accepted 5 May 2012 Available online 9 June 2012 Abstract Polyubiquitin genes from seven ciliate species were amplified, cloned and sequenced. It is estimated that Strombidium sulcatum, Euplotes vannus, E. rariseta and Anteholosticha manca have a polyubiquitin gene of 3 repeats, and A. parawarreni, Paramecium caudatum and Pseudokeronopsis flava 4 repeats. The newly obtained ubiquitins mostly differ from that of humans by 1–5 residues in amino acid sequences. A neighbor-joining tree constructed based on monomeric ubiquitin genes supports the monophyly of an assemblage comprising the litostomateans and some oligohymenophoreans, but not the class Spirotrichea. The monomers from the same species are generally placed together and highly supported for the class Litostomatea, the genera Paramecium and Ichthyophthirius, but not for other species. The non-synonymous/synonymous rate ratio (dN/dS) at the protein level are less than 1, and the synonymous nucleotide differences per synonymous site (pS) from intraspecific comparisons are fairly high (0.02–0.72). These results indicate that ciliates have not only the conserved, but also some quite divergent, polyubiquitin genes and confirm that the polyubiquitin genes in ciliates evolve according to the birth-and-death mode of evolution under strong purifying selection. © 2012 Elsevier GmbH. All rights reserved. Keywords: Ciliates; Ubiquitin; Polyubiquitin gene; Molecular evolution; Phylogenetics Introduction The evolution of ubiquitin genes is of great interest due to their important functions in protein degradation and in the Ubiquitin is a highly conserved 76 amino-acid (aa) protein control of numerous processes including cell-cycle progres- found in all eukaryotes (Durner and Boger 1995; Nercessian sion, signal transduction, transcriptional regulation, receptor et al. 2009; Wolf et al. 1993). The ubiquitin protein sequences down-regulation and endocytosis (Finley and Chau 1991; are almost identical among animals, plants, and fungi, with Hershko and Ciechanover 1998; Seufert and Jentsch 1992). only 3 or 4 amino acid differences (identity about 94.7% or The ubiquitin genes are fusion genes of a multigene family 95%) (Hauser et al. 1991). A similar level of identity was that falls into two types: the tandem repeats of the ubiquitin also found for protozoa (Jentsch et al. 1991). Ubiquitin rivals monomer (polyubiquitin), and a single monomer followed histone H4 as the most highly conserved protein (Baker and at the 3 -terminus by an open reading frame that encodes a Board 1991; Callis et al. 1995; Sharp and Li 1987). ribosomal protein. In both cases genes are simultaneously co-transcribed and co-translated, and monomeric ubiquitin units are produced by post-translational proteolysis (Callis et al. 1995; Hershko and Ciechanover 1998; Tachikui et al. ∗Corresponding author at: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China. 2003). The numbers of ubiquitin monomers in polyubiqui- E-mail addresses: [email protected], [email protected] (J. Gong). tin genes vary among loci and species (Krebber et al. 1994; 0932-4739/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2012.05.001 X. Liu et al. / European Journal of Protistology 49 (2013) 40–49 41 Sharp and Li 1987; Swindle et al. 1988; Tachikui et al. 2003). Laboratory of Protozoology, Ocean University of China. A It has been proposed that the polyubiquitin genes undergo clone of each species was maintained in the laboratory at either concerted evolution (Nenoi et al. 1998; Sharp and Li room temperature, with rice grains added to the seawater 1987), i.e. genes from different genomic loci in one species medium as a food source to enrich bacteria. Species identifi- are more related to each other than with the homologous cation follows published references (Chen et al. 2011; Shao genes from another species, or birth-and-death evolution, a et al. 2011; Song et al. 2009). form of independent progress by means of gene duplication with some repeat genes staying in the genome for a long time DNA extraction, amplification and sequencing or becoming nonfunctional (Nei et al. 2000; Nei and Rooney 2005). Polyubiquitin insertion, with one or two amino acids One or more cells of each species clone were isolated using inserted between each monomer, was used successfully to a micropipette under a dissecting microscope. After washing clarify the relationship of Cercozoa and Foraminifera to other three times with 0.22-␮m-filtered seawater cells were trans- eukaryotic lineages (Archibald et al. 2003; Bass et al. 2005). ferred to a PCR microfuge tube with a minimum volume Ciliophora is perhaps the most morphologically differen- of water. Genomic DNA was extracted using REDExtract- tiated taxon among protists (Fleury et al. 1992; Larson et al. N-Amp Tissue PCR Kit (Sigma, St. Louis, MO) according 1991; Lynn 2008), and the genomic organization of ciliates to the manufacturer’s protocol, modified such that only 1/10 is unique among eukaryotes (Prescott 1994). Zufall et al. of the suggested volume for each solution was used (Gong (2006) compared the evolutionary rates of 6 genes (Actin, ␣-, et al. 2007). Polyubiquitin genes were amplified with primers ␤-Tubulin, EF1␣, Histone H4, and HSP90) of ciliates with (UBIQ1: 5-G GCC ATG CAR ATH TTY GTN AAR AC- animals and fungi, and proposed that genome architecture, 3, target motif MQIFVK; IUB2: 5-G ATG CCY TCY TTR including nuclear dimorphism and chromosome fragmenta- TCY TGD ATY TT-3, target motif KIQDKEGI; Archibald tion, leads to elevated rates of protein evolution in ciliates. et al. 2003). This primer set generates a ladder of ubiqui- This lead us to speculate how the most conserved protein and tin gene products ranging from a half-monomer fragment its genes vary in ciliates. to increasing numbers of tandem repeats of the polyubiqui- Currently only a few polyubiquitin gene sequences of cil- tin tract. PCR conditions were: 5 min initial denaturation at iates, mainly for model species, have been published. The 94 ◦C, followed by 35 cycles of 45 s at 92 ◦C, 1 min at 50 ◦C, presence of ubiquitin genes in ciliates was first demonstrated and 1.5 min at 72 ◦C, with a final extension of 5 min at 72 ◦C in Tetrahymena pyriformis (Neves et al. 1988). A polyubiq- (Archibald et al. 2003). To minimize sequence errors, the Ex uitin identified in T. thermophila is almost identical to that Taq (TaKaRa, Japan) which shows 4-fold higher fidelity com- found in T. pyriformis, both of which are composed of 5 iden- pared with the conventional Taq, with an error rate of about tical protein monomers and differ from human ubiquitin in 2.2 × 10−6, was used for PCR amplification. The purification 4 amino acids (positions 16, 19, 24 and 28) (Guerreiro and of PCR products, cloning and sequencing were performed Rodrigues-Pousada 1996; Neves et al. 1991). A polyubiquitin according to previous reports (Huang et al. 2010; Zhang et al. gene in the macronucleus of Euplotes eurystomus comprises 2010). Multiple positive bacterial clones were sequenced for 3 identical monomers that have different amino acids in posi- each species. tions 19, 24 and 28 (Hauser et al. 1991). Nevertheless, it was also noted that another polyubiquitin gene in T. pyriformis contains repeats which differ strikingly from that in humans Phylogenetic and sequence analyses (identity 85.5 and 89.5%) (Neves et al. 1990). In this study, we cloned and sequenced the polyubiquitin Nucleotide sequences were translated to amino acid genes from seven ciliate species. Our aims were to analyze sequences using GeneDoc 2.6.002 (Nicholas et al. 1997) the organization and variability of the genes and the proteins with translation tables of ciliate nuclear and euplotid nuclear among ciliate species and lineages, to explore the possibility genetic codes. Polyubiquitin genes of ciliates available of using ubiquitin for higher classification of ciliates, and from GenBank (Table 1) were downloaded and aligned to examine the evolutionary mode of polyubiquitin genes in with newly sequenced genes using the ClustalW pro- ciliates. gram (Larkin et al. 2007). A neighbor-joining (NJ) tree of nucleotide sequences was constructed with MEGA 4.0 (Tamura et al. 2007) based on p-distance. Sequences from Material and Methods amitochondrial protist Trichomonas vagilis were selected as outgroups. Sample source Selective pressure was measured by the non- synonymous/synonymous rate ratio (dN/dS) at the protein Seven marine ciliates, Anteholosticha manca, A. parawar- level. The values of ω = 1, <1, and >1 indicate neutral evolu- reni, Euplotes vannus, E. rariseta, Paramecium caudatum, tion, purifying selection, and positive selection, respectively Pseudokeronopsis flava, and Strombidium sulcatum, were (Yang 1997, 2007). The protein sequences were aligned obtained from the Ciliate Species Collection at the using ClustalW program (Larkin et al. 2007), and then 42 X. Liu et al. / European Journal of Protistology 49 (2013) 40–49 Table 1. Polyubiquitin gene/protein sequences from GenBank used in this study. Group Species GenBank Accession Number Reference DNA Protein Ciliophora Tetrahymena thermophila U46561 AAC47430 Guerreiro and Rodrigues-Pousada (1996) XM 001018500 XP 001018500* Eisen et al. (2006) Tetrahymena vorax AF003089 AAB61405 Green (unpublished) Tetrahymena pyriformis X61053 CAA43387 Neves et al. (1991) CAA84814* Neves et al. (1990) Ichthyophthirius multifiliis GL983930 EGR30931 Coyne et al. (2011) Paramecium tetraurelia XM 001451092 XP 001451129 Aury et al. (2006) Polyplastron multivesiculatum AJ965270 CAI83754 McEwan et al.