Evolution of serum albumin intron-1 is shaped by a 5′ truncated non-long terminal repeat retrotransposon in western Palearctic water frogs (Neobatrachia)
Introduction
Spliceosomal introns are parts of all eukaryote genomes so far investigated. They are generally composed of quasi-random sequences and lack open reading frames (reviewed by Roy and Gilbert, 2006). Introns are excised (‘spliced’) out of RNA transcripts of coding genes prior to protein synthesis and thus are usually considered non-functional and selectively neutral, but conserved sequence motifs, especially around the intron–exon boundaries, indicate that there are functional constraints linked with the spliceosomal machinery. The formation of secondary structures in pre-mRNA may also be functional (reviewed by Buratti and Baralle, 2004).
The mechanisms of intron evolution are largely unknown. Comparative studies using intron sequences of closely and distantly related taxa may help to identify functional and non-functional structural motifs important for understanding these mechanisms. The western Palearctic water frog (WPWF) group includes at least seven closely related species (Plötner, 2005), most of which originated in the upper Miocene and middle Pliocene/early Pleistocene (Beerli et al., 1996, Plötner et al., in press). It thus provides an opportunity to study intron evolution on a ‘fine scale’, within the last 10 million years (My).
Here we present first results of such a comparative study of intron-1 of the serum albumin gene (SAI-1) of WPWFs. We also describe a non-long terminal repeat (non-LTR) retrotransposon that is embedded in the SAI-1 of the water frog species investigated. This non-LTR retrotransposon was classified as a chicken repeat (CR) 1-like long interspersed nuclear element (LINE). CR1 retrotransposons (Stumph et al., 1981) and related non-LTR retroelements are widely distributed in the genomes of vertebrates and invertebrates (e.g. Eickbush, 1994, Haas et al., 2001, Deininger and Batzer, 2002, Deininger et al., 2003, Shedlock, 2006, Kordiš et al., 2006, Novikova et al., 2007, Shedlock et al., 2007; and literature cited therein). They are increasingly used as characters for phylogenetic reconstructions (e.g., John et al., 2005, Kordiš et al., 2006, Shedlock, 2006, Watanabe et al., 2006, Kaiser et al., 2007, Shedlock et al., 2007, Treplin and Tiedemann, 2007). The primary advantage of retroelement insertions for such studies is the high likelihood that, if two genomes share a mobile element at the same locus, the mobile element and insertion are identical by descent (e.g., Deininger and Batzer, 2002).
We compared the structure and base composition of the SAI-1 and CR1-like sequences and discuss the impact that RanaCR1 has had on the evolution of SAI-1 in WPWF. We also used sequences of both SAI-1 and the inserted CR1-like retroelement to test hypotheses on water frog systematics proposed on the basis of protein electrophoretic data (Beerli et al., 1996) and mitochondrial sequences (Plötner, 1998, Plötner, 2005, Plötner and Ohst, 2001, Plötner et al., 2001, Plötner et al., 2007).
Section snippets
Samples
We analysed SAI-1 and RanaCR1 sequences of 24 WPWFs from 21 localities (Table 1) and one eastern Palearctic water frog (an R. nigromaculata from North Korea, EMBL FN432386).
DNA isolation and primer development
Total genomic DNA was extracted from pieces of muscle tissue taken from ethanol preserved specimens by use of a DNA extraction kit (Qiagen, Hilden, Germany) following the standard protocol for preserved tissues. Primers for PCR amplification and sequencing were designed using the serum albumin cDNA of Rana shqiperica. For
Structure of SAI-1 and RanaCR1
Intron-1 of the serum albumin gene in western Palearctic water frogs and the single eastern Palearctic water frog species (R. nigromaculata) examined has typical exon/intron splice site junction sequences (exon-1/GT for the donor site, AG/exon-2 for the acceptor site). An adenine (an essential element for splicing) is located in a pyrimidine rich region 13–14 nt upstream of the acceptor site. The BDGB program (http://www.fruitfly.org/seq_tools/splice.html) predicted several additional splice
Structure of RanaCR1 and its potential impact on SAI-1 evolution
RanaCR1 is the first non-LTR retroelement detected in the genome of neobatrachian anurans. Like most transposable elements (TEs), RanaCR1 is 5′ truncated probably resulting from abortive RNA reverse transcription, when the reverse transcriptase dissociates from its RNA template before having completed cDNA synthesis (e.g., Silva and Burch, 1989, Eickbush, 1994, Burch et al., 1993, Kajikawa et al., 1997, Poulter et al., 1999). As our data show, length variation of TEs may also be caused by
Acknowledgments
For providing water frog samples, we thank Rainer Günther (Berlin), Spartak N. Litvinchuk (St. Petersburg), and Dirk Schmeller (Saint Girons). Tissue samples from Greece were made available by the Greek Ministry of Rural Development and Food, kindly mediated by Doris Tippmann (Embassy of the FRG, Athens). We are grateful to Nils Hof (Berlin) for technical assistance. Two anonymous reviewers provided constructive criticism. This work was supported by the Deutsche Forschungsgemeinschaft (grants
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