Invention and Mystery in Hypotrich DNA1

ABSTRACT “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas³ Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.     

Volatility of internal eliminated segments in germ line genes of hypotrichous ciliates.

Germ line micronuclear genes in ciliated protozoa contain two types of interrupting sequences. Some genes contain introns, but internal eliminated segments (IESs) are much more prevalent. IESs are AT-rich DNA segments that separate macronucleus-destined segments (MDSs) in micronuclear genes. All IESs are excised and destroyed when a micronucleus develops into a macronucleus after each cell mating. IESs have no discernible function. Therefore, an investigation of the behavior of IESs in evolution has been undertaken to assess their possible significance. The IESs in the micronuclear gene encoding the beta-subunit of the telomere-binding protein (beta-TP) are not conserved in number, position, sequence, or length during the evolution of four oxytrichid ciliates. In contrast, the scrambled pattern of MDSs and IESs of the micronuclear actin I gene has been conserved during evolution; however, the precise positions, sequences, and lengths of the IESs differ among species, and in some organisms the actin I gene contains an additional IES and MDS. Corresponding IESs in the actin I genes among the different organisms have shifted positions by 1 to 14 bp, presumably by a mutation-shifting mechanism, creating differences in the repeat sequences flanking IESs. Thus, conservation of a particular repeat sequence among species is not required for IES excision. The changes in IES number and position in the beta-TP genes among ciliates are in sharp contrast to the stability of the intron position. Therefore, IESs are volatile, hypermutable elements that are inserted, removed, shifted, and modified continuously in the germ line through evolutionary time.     

Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus.

The hypotrichous ciliated protozoa undergo a massive genome rearrangement process after their sexual cycle. One frequent type of rearrangement is the removal of DNA sequences (internal eliminated sequences; IESs) from internal regions of DNA molecules. In this study, we characterized the removal of IESs in Euplotes crassus. Southern hybridization analyses combined with cytological observations indicated that IES removal is an early event in macronuclear development, occurring during the polytene chromosome stage and prior to the chromosome fragmentation process. The results are consistent with IES removal occurring via an intramolecular DNA breakage and rejoining process.     

Internal Eliminated Segments (IESs) of Oxytrichidae1

ABSTRACT Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding β-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.     

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

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.     

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.     

Detection of circular forms of eliminated DNA during macronuclear development in E. crassus

Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.     

Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5'-TA-3' dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5'-TA-3' repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.     

Evolution of the scrambled germline gene encoding α-telomere binding protein in three hypotrichous ciliates

The micronuclear genes encoding α-telomere-binding protein (αTP) in Oxytricha trifallax and Stylonychia mytilus contain multiple internal eliminated segments, or IESs, that divide the gene into multiple parts called macronuclear destined segments, or MDSs. The MDSs have become disordered, or scrambled, during evolution. The scrambled structures of the αTP genes in Oxytricha trifallax and S. mytilus have been compared with the previously published scrambled structure of the αTP gene in O. nova. The scrambled patterns of the αTP gene in the three species are similar but show significant differences. The micronuclear genes in O. nova and S. mytilus consist of 13 IESs and 14 MDSs, but the gene in O. trifallax is divided into three additional MDSs by the presence of three additional IESs, believed to have been inserted into the O. trifallaxαTP gene after divergence of O. trifallax from the other two species. Corresponding IESs among the three species have shifted along the DNA during evolution, presumably by a mutational mechanism that changes the short repeat sequences that flank IESs. The IESs also have changed markedly in length by insertion and/or deletion of nucleotides. Comparison of the putative αTP amino acid sequences in the three species reveals three conserved and three nonconserved domains. The 5′ nontranslated regions of the gene-sized molecules encoding αTP contain several conserved segments, and the 3′ nontranscribed trailer contains one conserved segment. Received: 29 May 1998; in revised form: 3 August 1998 / Accepted: 18 August 1998     

Template-guided recombination for IES elimination and unscrambling of genes in stichotrichous ciliates

The micronuclear versions of genes in stichotrichous ciliates are interrupted by multiple, short, non-coding DNA segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. In some micronuclear genes MDSs are in a scrambled disorder. During development of a micronucleus into a macronucleus after cell mating the IESs are excised from micronuclear genes and the MDSs are spliced in the sequentially correct order. Pairs of short repeat sequences in the ends of MDSs undergo homologous recombination to excise IESs and splice MDSs. However, the repeat sequences are too short to guide unambiguously their own alignment in preparation for recombination. Based on experiments by others on the distantly related ciliate, Paramecium, we propose a molecular model of template-guided recombination to explain the excision of the 100,000-150,000 IESs and splicing of MDSs, including unscrambling, in the genome of stichotrichous ciliates. The model solves the problem of correct pairing of pointers, precisely identifies MDS-IES junctions, and provides for irreversible recombination.     

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.     

Origin, evolution, and excision of internal eliminated segments in germline genes of ciliates

Three hypotheses on the evolutionary/molecular origin of internal eliminated segments (IESs) in the germline of hypotrichous ciliates are discussed in the context of the high rate of mutation accumulation in IESs, shifting of IESs during speciation, and evolutionary scrambling of segments within some hypotrich germline genes. Developmental excision of IESs from the germline in Paramecium suggests that the parental macronucleus may provide nucleic acid sequence information to guide excision of IESs and splicing of macronuclear-destined sequences. In ciliates of the oxytrichid/stylonychid group, such a mechanism could explain the precision of excision of IESs and gene unscrambling. Recently initiated molecular/genetic studies may eventually clarify the role of the parental macronucleus in IES excision and gene unscrambling as well as the molecular mechanisms of these events.     

The evolutionary scrambling and developmental unscrambling of germline genes in hypotrichous ciliates.

Genes in the germline (micronuclear) genome of hypotrichous ciliates are interrupted by multiple, short, non-coding, AT-rich sequences called internal eliminated segments, or IESs. During conversion of a micronucleus to a somatic nucleus (macronucleus) after cell mating, all IESs are excised from the germline genes and the gene segments, called macronuclear-destined segments, or MDSs, are spliced. Excision of the approximately 150 000 IESs from a haploid germline genome in Oxytricha nova requires approximately 150 000 recombinant events. In three of 10 genes the MDSs are scrambled. During macronuclear development the MDSs are unscrambled, possibly by folding of the DNA to allow MDSs to ligate in the correct order. The nine MDSs in the actin I gene of O.nova are scrambled in the random order, 3-4-6-5-7-9-2-1-8, and MDS 2 is inverted. The 14 MDSs in the alphaTP gene of O.nova and Stylonychia mytilus are scrambled in the non-random order, 1-3-5-7-9-11-2-4-6-8-10-12-13-14. The 45 MDSs in the DNA pol alpha gene are non-randomly scrambled into an odd/even series, with an inversion of one-third of the gene. Additional IESs have been inserted into these three genes during evolution of Oxytricha trifallax, slightly modifying scrambling patterns. The non-random scrambled patterns in the alphaTP and DNA pol alpha genes are explained by multiple, simultaneous IES insertions. The randomly scrambled pattern in the actin I gene may arise from an initially non-randomly scrambled pattern by recombination among multiple IESs. Alternatively, IESs inserted sporadically (individually) in a non-scrambled configuration might subsequently recombine, converting a non-scrambled gene into a randomly scrambled one. IESs shift along a DNA molecule, most likely as a result of mutations at MDS/IES junctions. Shifting of IESs has the effect of 'transferring' nucleotides from one MDS to another, but does not change the overall sequence of nucleotides in the combined MDSs. In addition to shifting in position, IESs accumulate mutations at a high rate and increase and decrease in length within a species and during speciation. The phenomena of IESs and of MDS scrambling represent remarkable flexibility of the hypotrich genome, possibly reflecting a process of MDS shuffling that facilitates the evolution of genes.     

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.