Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila

Tlr elements are a novel family of ~30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein

Tlr1 is a member of a family of ~20–30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan Tetrahymena. Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the Tetrahymena genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.     

Alternate Junctions and Microheterogeneity of Tlr1, a Developmentally Regulated DNA Rearrangement in Tetrahymena thermophila

ABSTRACT. A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila , Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.     

Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences in Paramecium tetraurelia

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.     

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.     

Tec3, a new developmentally eliminated DNA element in Euplotes crassus

More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from the genome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNA as well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E. crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements have long inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recombinase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, and resides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme ends of this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excised during the polytene chromosome stage of macronuclear development to generate a free circular form with an unusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacks the terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini. The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject to developmental elimination. Models for the origins of these two types of Tec3 elements are presented, along with a discussion of how some members of this new transposon family may have come to be excised by the same machinery that removes other E. crassus IESs.     

Varied truncation and clustering characterize some short repeats identified in micronucleus-specific DNA of Tetrahymena thermophila

There are over 6000 internally eliminated DNA sequences (IESs) in the Tetrahymena genome that are deleted in a programmed fashion during the development of a polyploid, somatic macronucleus from a diploid germline micronucleus. Recently, based on several results, a homology and small RNA-based mechanism has been proposed for the efficient elimination of IES elements. Since the RNAi machinery is proposed to be intimately involved in silencing potentially harmful repeats such as transposons and viruses, characterization of repeats and the conditions for their developmental elimination from the somatic genome is warranted. Three short (500–600 bp) repeat families, members of which had been experimentally identified in IESs, that is, in micronucleus-specific DNA, are examined here using the Tetrahymena genome database. Members of all three families display varied degrees of truncation and are represented in macronuclear sequences. A 200 bp segment of one of the families can appear in the genome on its own, or as part of a 600 bp repeat detected experimentally, or in association with an unrelated 1 kb sequence to form a 1.2 kb repeat that is also frequently truncated. The 1 kb sequence contains a 300 bp section similar to a repeat associated with a non-long terminal repeat-like element and is often found accompanied by several more copies of this shorter repeat. These observations indicate that transposition may have had a role in the evolution of the short repeat families.     

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax

Background

Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells.

Results

Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells.

Conclusions

These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements.     

Developmentally coordinated en masse excision of a highly repetitive element in E. crassus

The E. crassus Tec1 element is present in greater than 10(4) copies in the micronuclear genome but is absent from the macronuclear genome. During formation of a macronucleus from a micronucleus, a majority of the Tec1 elements appear as extrachromosomal circles. The circular and integrated forms of Tec1 have been characterized by restriction mapping to produce consensus maps and by sequence analysis of the element's termini. The circular forms are resistant to BAL31 and have the restriction map expected if the element excises at the end of its inverted repeats. DNA sequence analysis of a circular form confirms that the inverted repeats are in a head-to-head configuration. Excision of Tec1 occurs very early during macronuclear development as the DNA begins to replicate to form polytene chromosomes.     

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.     

Two distinct P element subfamilies in the genome of Drosophila bifasciata

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5′ end and 14 by at the 3′ end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.     

A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ~21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.     

Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus.

The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.