An active non-LTR retrotransposon with tandem structure in the compact genome of the pufferfish Tetraodon nigroviridis

Abstract

The fish retrotransposable element Zebulon encodes a reverse transcriptase and a carboxy-terminal restriction enzyme-like endonuclease, and is related phylogenetically to site-specific non-LTR retrotransposons from nematodes. Zebulon was detected in the pufferfishes Tetraodon nigroviridis and Takifugu rubripes, as well as in the zebrafish Danio rerio. Structural analysis suggested that Zebulon, in contrast to most non-LTR retrotransposons, might be able to retrotranspose as a partial tandem array. Zebulon was active relatively recently in the compact genome of T. nigroviridis, in which it contributed to the extension of intergenic and intronic sequences, and possibly to the formation of genomic rearrangements. Accumulation of Zebulon together with other retrotransposons was observed in some heterochromatic chromosomal regions of the genome of T. nigroviridis that might serve as reservoirs for active elements. Hence, pufferfish compact genomes are not evolutionarily inert and contain active retrotransposons, suggesting the presence of mechanisms allowing accumulation of retrotransposable elements in heterochromatin, but minimizing their impact on euchromatic regions. Homologous recombination between partial tandem sequences eliminating active copies of Zebulon and reducing the size of insertions in intronic and intragenic regions might represent such a mechanism.

Figure 1

Genomic structure of Zebulon elements in T. nigroviridis. The position of 5′ truncations is shown by broken arrows. Most of them are from single sequences and could therefore not be assigned either to the upstream or to the downstream copy; they were arbitrarily positioned in the downstream copy. Trace sequences (http://www.ncbi.nlm.nih.gov/blast/tracemb.html) reflecting the genomic site before insertion are shown, and their percentage of nucleotide identity with the site sequences directly flanking Zebulon insertions is given. Horizontal arrows show the direction of gene transcription. Putative flanking target site duplications are shown. (RT) Reverse transcriptase; (CCCHC) putative CXCX₃CX₈HX₅C zinc finger-like domain.

Figure 2

Sequence comparison between upstream–downstream junctions in tandem arrays (A) and between 3′ ends (B) of Zebulon elements shown in Fig. 1. The part of the tandem array sequences shown extend from the stop codon of the upstream copy to the putative start codon of the downstream copy.

Figure 3

Consensus sequence of the Zebulon retrotransposon of T. nigroviridis. (A) Complete consensus sequence. Amino-acid residues forming the putative amino-terminal (C)CCHC zinc finger domain are boxed. (B) Restriction enzyme-like domain.

Figure 4

Southern blot analysis of the distribution of Zebulon in fish. Genomic DNA was cut with HindIII (does not cut in Zebulon). The probe used is pG16H23. Origin of fishes is given in Crollius et al. (2000) and Volff et al. (2000).

Figure 5

Phylogenetic relationship between Zebulon and the nematode site-specific retrotransposon NeSL-1. Phylogeny was performed using together the reverse transcriptase and REL domains (Burke et al. 2002). The tree (neighbor-joining) is unrooted. Branches with <50% support have been collapsed. Bootstrap values using neighbor-joining (1000 replicates, first values) and maximum parsimony analyses (100 replicates, third values), as well as reliability values for maximum likelihood analysis (quartet puzzling, 10,000 puzzling steps, second values) are given. Accession numbers are the same as in Malik et al. (1999) and Burke et al. (2002).

Figure 6

Chromosomal localization of Zebulon in the genome of T. nigroviridis by FISH. Weak, scattered spots have been removed by electronic thresholding in order to retain only major regions of accumulation. The genomic areas in which Zebulon preferentially localizes (a1) mostly correspond to heterochromatic, DAPI-positive regions as shown in this over-denaturated metaphase (a2). Double FISH between Zebulon (DIG-labeled pG16H23, b1) and Rex3 (biotin-labeled, b2), a non-LTR retrotransposon abundant in the genome of T. nigroviridis (C. Fischer, L. Bouneau, and C. Ozouf-Costaz, unpubl.) shows superimposed signals (b3) corresponding to common regions of accumulation in DAPI-positive regions (b4).