A non-long terminal repeat retrotransposon family is restricted to the germ line micronucleus of the ciliated protozoan Tetrahymena thermophila

Abstract

The ciliated protozoan Tetrahymena thermophila undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle. To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in T. thermophila, the first characterized in the ciliated protozoa. This multiple-copy retrotransposon family is restricted to the micronucleus of T. thermophila. The REP (Tetrahymena non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains. Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family. Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development. The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in T. thermophila evolved as a method of eliminating deleterious transposons from the somatic macronucleus.

FIG. 1.

Genomic organization and partial restriction maps of pMBR-RP1 to pMBR-RP3 and pMBR-RP6 with additional genomic sequence for pMBR-RP6 (see Materials and Methods). The plasmids are numbered from 0 to the end of sequenced regions, with an additional 2,833 bp of micronuclear sequence added to pMBR-RP6 (−2833 to the vertical dotted line where cloned pMBR-RP6 sequence begins). The putative ORFs are transcribed from left to right. The DNA sequence corresponding to each REP element is boxed (see text for details). Abbreviations: H, HindIII; E, EcoRI; R, EcoRV; B, BglII; S, SacI; P, PstI.

FIG. 2.

Multiple sequence alignment of the APE domain of REP1 and REP2-2 with APE domains of several non-LTR retrotransposons (see Materials and Methods for details). Key residues identified for APE activity (45) are indicated by an asterisk. Black and grey shading indicates identical and similar amino acid residues, respectively.

FIG. 3.

Multiple sequence alignment of the RT domain of REP1, REP2-2, REP3, and REP6 with that of several non-LTR retrotransposons (see Materials and Methods for details). RT subdomains are indicated by a corresponding number above the conserved block of amino acid sequence (see text for details). With respect to domain 9, non-LTR retrotransposons are divided into two subgroups based upon the presence or absence of a glycine-rich tetramer (domain 9 versus 9*: see boxed region). Shading of amino acid residues is as in Fig. 2.

FIG. 4.

Multiple sequence alignment of the conserved 54-bp sequence found 3′ of ORF2 of the REP elements. An arrow represents the point within the conserved sequence where there is a small amount of overlap with the repetitive sequence described in Fig. 5. The numbering begins at the stop codon of ORF2 of the respective REP element. The black shading indicates nucleotides that are identical in all five sequences.

FIG. 5.

Multiple sequence alignment of the ≈350-bp imperfectly repeated sequence found immediately downstream of the conserved 54-mer (Fig. 4; included here as a part of REP1, REP2-1, and REP2-2-1). The sequence similarity between the repeats of REP2-1, REP2-2-1, and REP2-2-2 extends further than that of REP1 and REP2-2-3. Nucleotide residues that are identical in all five sequences are shaded black.

FIG. 6.

Southern analysis of restriction-digested T. thermophila macronuclear and micronuclear DNA probed with a DNA fragment complementary to the REP1 RT domain (PstI/BglII fragment of pMBR-RP1). The blot was subsequently stripped and reprobed to verify that equal amounts of macronuclear and micronuclear DNA preparations were used (data not shown). Abbreviations: M, 1-kb ladder (New England Biolabs); MAC, macronuclear DNA; MIC, micronuclear DNA.

FIG.7.

Phylogenetic relationships of the T. thermophila REP element with other non-LTR retrotransposons based upon the RT domain as defined in Malik et al. (40). The tree is neighbor joining with bootstrap values indicated as number out of 1,000. Only bootstrap values of >50% are indicated. The amino acid divergence scale is indicated. The alignment and amino acid sequences were derived as described in Materials and Methods. The non-LTR elements are divided into clades based upon phylogenetic relationships. Clades are indicated by the black bars and are defined in Malik et al. (40).

FIG. 8.

Phylogenetic relationships of the T. thermophila REP element with other non-LTR retrotransposons based upon the APE domain as defined in Malik et al. (40). The tree is neighbor joining with bootstrap values indicated as number out of 1,000. Only bootstrap values of >50% are indicated. The amino acid divergence scale is indicated. The alignment and amino acid sequences were derived as described in Materials and Methods. The non-LTR elements are divided into clades based upon phylogenetic relationships. Clades are indicated by the black bars and as defined in Malik et al. (40).

FIG. 9.

Northern analysis of REP element transcription. Whole-cell RNA was separated on a 1% agarose-formaldehyde denaturing gel, transferred to a nylon filter, and probed with a DNA fragment corresponding to the RT domain of the REP element (corresponding to the EcoRV-BglII fragment of REP2-2 that encompasses the RT domain). The blot was also probed for the 17s rRNA as a loading control to test for RNA integrity as well as PDD1, a development-specific gene (39).