Evolutionary history of Cer elements and their impact on the C. elegans genome

Abstract

We report the results of sequence analysis and chromosomal distribution of all distinguishable long terminal repeat (LTR) retrotransposons (Cer elements) in the Caenorhabditis elegans genome. Included in this analysis are all readily recognizable full-length and fragmented elements, as well as solo LTRs. Our results indicate that there are 19 families of Cer elements, some of which display significant subfamily structure. Cer elements can be clustered based on their tRNA primer binding sites (PBSs). These clusters are in concordance with our reverse transcriptase- and LTR-based phylogenies. Although we find that most Cer elements are located in the gene depauperate chromosome ends, some elements are located in or near putative genes and may contribute to gene structure and function. The results of RT-PCR analyses are consistent with this prediction.

Figure 1

Composite RT/LTR phylogenetic analysis of Cer elements. Shown is an unrooted NJ phylogram of RT (amino acid) and LTR (nucleotide) sequences. RT amino acid alignments were used to establish family structure (black); LTR nucleotide sequences were added to establish subfamily structure (red). tRNA primer binding sites (PBSs) are highlighted to show conservation of tRNA priming across families. Alignments were produced via MacVector (http://www.gcg.com) and ClustalX 1.8 (Thompson et al. 1997). * PBS reported by Frame et al. (2001).

Figure 2

Phylogenetic trees of subfamily structure based on LTR nucleotide sequence data. LTRs from full, fragmented, and solo LTR elements were aligned via ClustalX 1.8 (Thompson et al. 1997), and the NJ method was used to construct trees. Insertions/deletions were ignored. Values on individual branches are bootstrap percentages based on 1000 bootstrap repetitions. Each LTR in the tree is named by the genomic clone in which it was found. For elements with two LTRs, the 3′ LTR is labeled by a lower case “b” following the clone number. Each tree is shown with a scale bar determined by the number of nucleotide substitutions per site between two sequences. (A) Phylogenetic tree displaying substructure within Cer8 and Cer9 families, with Cer7 as the outgroup. The tight branching of the tree demonstrates the high sequence identity shared among Cer9 family members. * indicates the presence of a ∼108 bp insert in the center of Cer9 LTR; ** indicates the presence of a ∼106 bp insert in the center of the Cer9 LTR. Both inserts are >85% identical. (B) Phylogenetic tree displaying substructure within Cer12 and Cer16 families, with Cer7 as the outgroup. Cer12 consists of two subfamilies (Cer12 and 12-1); Cer16 has three subfamilies (Cer16, 16-1, and 16-2). The tight clustering seen in both families represents a high degree of nucleotide identity between elements within a subfamily.

Figure 2

Phylogenetic trees of subfamily structure based on LTR nucleotide sequence data. LTRs from full, fragmented, and solo LTR elements were aligned via ClustalX 1.8 (Thompson et al. 1997), and the NJ method was used to construct trees. Insertions/deletions were ignored. Values on individual branches are bootstrap percentages based on 1000 bootstrap repetitions. Each LTR in the tree is named by the genomic clone in which it was found. For elements with two LTRs, the 3′ LTR is labeled by a lower case “b” following the clone number. Each tree is shown with a scale bar determined by the number of nucleotide substitutions per site between two sequences. (A) Phylogenetic tree displaying substructure within Cer8 and Cer9 families, with Cer7 as the outgroup. The tight branching of the tree demonstrates the high sequence identity shared among Cer9 family members. * indicates the presence of a ∼108 bp insert in the center of Cer9 LTR; ** indicates the presence of a ∼106 bp insert in the center of the Cer9 LTR. Both inserts are >85% identical. (B) Phylogenetic tree displaying substructure within Cer12 and Cer16 families, with Cer7 as the outgroup. Cer12 consists of two subfamilies (Cer12 and 12-1); Cer16 has three subfamilies (Cer16, 16-1, and 16-2). The tight clustering seen in both families represents a high degree of nucleotide identity between elements within a subfamily.

Figure 3

Distribution of Cer full-length, fragmented and solo LTR element sequences in the C. elegans genome. A genomic coordinate value for all Cer elements was calculated (see Methods) and elements plotted to their respective chromosome location. Chromosomes were divided into three regions (left, center, right). All chromosomes except chromosome III display significant clustering outside of the centric genic region. Cer elements are randomly distributed across chromosomes.

Figure 4

Cer element LTRs are part of some C. elegans genes. Green arrows represent Wormbase-predicted gene regions with corresponding identification. Blue arrows depict ESTs concordant to the predicted gene region. Orange boxes are predicted exon regions. Red boxes denote LTR position, and internal arrows indicate direction. The black line and numbers represent position along the genomic clone sequence (F20B4, C56G3, 6R55, F53E10). Black arrows indicate direction and location of forward (f) or reverse(r) PCR primers. For visual simplicity, only introns (i#) discussed in the text are displayed above and between exons. (A) An entire LTR from the 5′ end of a full-length Cer16-2 element is part of the 5′ end of a putative C. elegans gene (6R55.2) of unknown function. (B) The Cer9 LTR overlaps two exons of an aldo/keto reductase homolog in C. elegans (C56G3.2). The LTR is the 3′ end of a fragmented Cer9 element. (C) A Cer16-1 solo LTR is part of intron 1 of a C. elegans gene (F20B4.6) in the glucosyltransferase family. (D) A Cer2 solo LTR constitutes the 3′ end of a putative C. elegans gene (F53E10.5) of unknown function.

Figure 5

PCR/RT-PCR analysis of C. elegans genes containing Cer LTR sequence showing the production of spliced, polyadenylated transcripts from these loci. A negative image is presented for visual clarity. Within a locus, PCR (control) and RT-PCR were performed using the same primer set. DNA (+) and DNA (−) indicate PCR reactions with and without nematode genomic DNA, respectively. RT (+) and RT (−) indicate RT-PCR reactions with and without reverse transcriptase, respectively. M = 100 bp ladder.