NfCR1, the First Non-LTR Retrotransposon Characterized in the Australian Lungfish Genome, Neoceratodus forsteri, Shows Similarities to CR1-Like Elements NICKOLCHE SIRIJOVSKI, CATHERINE WOOLNOUGH, JENNIFER ROCK, AND JEAN M.P. JOSS n Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109 Australia ABSTRACT The genomes of lungfish, together with those of some urodele amphibians, are the largest of all vertebrate genomes. It has been assumed that the bulk of the DNA making up these large genomes has been derived from repeat elements, like the noncoding DNA of those genomes that have been sequenced (e.g., human). In an attempt to characterize repeat sequences in the lungfish genome, we have isolated, by restriction enzyme digestion of genomic DNA, sequences of a repeat element in Neoceratodus forsteri, the most primitive of the living lungfishes. The fragments sequenced from the EcoRI and BglII digests were used to perform genome walking PCR in order to obtain the full sequence of the repeat element. This element shares homology with the non-LTR (LINE) element, Chicken Repeat 1 (CR1), described for several vertebrates and some invertebrates; we have called it N. forsteri CR1 (NfCR1). NfCR1 shares all the domains of other CR1 elements but it also has several unique features that suggest it may no longer be active in the lungfish genome. It occurs in both full-length and 5 0 -truncated versions and in its present "inactive" form represents B0.05% of the lungfish genome. J. Exp. Zool. (Mol. Dev. Evol.) 304B:40–49, 2005. r 2005 Wiley- Liss, Inc. INTRODUCTION The genomes of extant lungfish, together with some urodele amphibians, are larger than those of any other group of vertebrates. With 105 pg of DNA/diploid nucleus (Rock et al., ’96), the genome of Neoceratodus forsteri is very large, although it is approximately half that of the other two genera of living lungfish, Lepidosiren and Protopterus (Ca- valier-Smith, ’85; Gregory, 2002). These very large genomes are associated with large cell size (Cavalier-Smith, ’78; Gregory, 2001), and this association has been used to deduce whether lungfish have always had a large genome. By examining the size of osteocytes from fossil lungfish, Thomson (’72) found that the large cell size of living lungfish is a relatively recent phenomenon. He concluded that the large genome of recent lungfish has evolved since the Devonian. As a first step in attempting to better understand how the lungfish genome has become so large, we are examining the composition of the large lungfish genome. We have addressed the occur- rence of large repeat sequences and as a first result have sequenced a Chicken Repeat 1 (CR1)- like transposable element from the genome of the lungfish, N. forsteri. CR1 transposable elements belong to the long interspersed element (LINE) nonterminal repeat (non-LTR) class of retroelements. It is believed to have a very ancient origin in the eukaryotic genome. CR1s have been found in all classes of jawed vertebrates from fish to mammals (Kajikawa et al., ’97; Nobuhisa et al., ’98; Haas et al., 2001; Poulter et al., ’99; Vandergon and Reitman, ’94). Related elements have also been described from several invertebrate species including nematode Grant sponsor: Macquarie University Research Development Scheme; Grant number: A000217 N. Sirijovski’s current address: University of Lund, Sweden C. Woolnough’s current address: University of NSW, Australia J. Rock’s current address: University of Wales, Bangor, U.K. n Correspondence to: Jean M.P. Joss, Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109 Australia. E-mail: jjoss@rna.bio.mq.edu.au Received 15 July 2004; Accepted 24 September 2004 Published online 18 November 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.b.21022 r 2005 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 304B:40–49 (2005)