During macronuclear development in the ciliate Euplotes crassus, the highly repetitive, transposon-like Tec elements possess an unusual chromatin structure. We observed that the Tec element chromatin is highly resistant to salt extraction and behaves like a nuclear matrix/chromosome scaffold-associated structure. Standard matrix/scaffold extraction procedures identified two major proteins: 1) an ~140-kDa protein that seems to be topoisomerase II based on its reactivity with anti-topoisomerase II antibodies, and 2) an 85-kDa protein that we further purified by acid extraction and have shown to be a novel protein by sequence analysis of its gene. The 85-kDa protein (p85) is a developmental stage-specific protein and is located exclusively in the developing macronucleus. Immunolocalization studies of p85 show that it colocalizes with topoisomerase II in chromatin. In addition, in situ hybridization combined with immunofluorescence localization of the proteins indicates that 100% of the Tec elements colocalize with 70% of the p85, whereas no significant colocalization with a total macronuclear sequence-specific probe is observed. p85 is the first developmental stage-specific protein identified as being specifically associated with sequences undergoing elimination in E. crassus.

ABSTRACT The macronuclear genome of the ciliate Euplotes is comprised of millions of small linear DNA molecules that have telomeres on each end. These molecules are generated during the sexual stage of the life cycle, when the new macronucleus is formed by a series of DNA processing events and multiple rounds of DNA amplification. We have used two-dimensional gels to compare the location of the replication origins used during vegetative growth and the two periods during macronuclear development when DNA amplification takes place. When we examined the pattern of ribosomal DNA (rDNA) replication intermediates, we observed almost identical Y arcs regardless of when in the Euplotes life cycle the DNA was isolated. No bubble or bubble-to-Y arcs could be detected. This indicates that replication of the macronuclear rDNA initiates at or near the telomere even when these molecules are being differentially amplified. Since replication rarely initiated from both ends of the rDNA, we examined the direction of replication fork movement to determine which end of the rDNA served as the origin. Fork movement gels indicated that replication initiated at the 5′ end. As transcription also starts near the telomere at the 5′ end, our findings suggest that the telomere and the promoter region cooperate to recruit Euplotes replication initiation complexes.

The Tec1 and Tec2 transposons of the ciliate Euplotes crassus carry a gene for a tyrosine-type site-specific recombinase. The expression of the Tec2 gene apparently uses a programmed +1 frameshift. To test this hypothesis, we first examined whether this gene has evolved under purifying selection in Tec1 and Tec2. Each element carries three genes, and each has evolved under purifying selection for the function of its encoded protein, as evidenced by a dearth of nonsynonymous changes. This distortion of divergence is apparent in codons both 5' and 3' of the frameshift site. Thus, Tec2 transposons have diverged from each other while using a programmed +1 frameshift to produce recombinase, the function of which is under purifying selection. What might this function be? Tyrosine-type site-specific recombinases are extremely rare in eukaryotes, and Tec elements are the first known eukaryotic type II transposons to encode a site-specific recombinase. Tec elements also encode a widespread transposase. The Tec recombinase might function in transposition, resolve products of transposition (bacterial replicative transposons use recombinase or resolvase to separate joined replicons), or provide a function that benefits the ciliate host. Transposons in ciliated protozoa are removed from the macronucleus, and it has been proposed that the transposons provide this "excisase" activity.

The germline genomes of ciliated protozoa are dynamic structures, undergoing massive DNA rearrangement during the formation of a functional macronucleus. Macronuclear development involves chromosome fragmentation coupled with de novo telomere synthesis, numerous DNA splicing events that remove internal segments of DNA, and, in some ciliates, the reordering of scrambled gene segments. Despite the fact that all ciliates share similar forms of DNA rearrangement, there appears to be great diversity in both the nature of the rearranged DNA and the molecular mechanisms involved. Epigenetic effects on rearrangement have also been observed, and recent work suggests that chromatin differentiation plays a role in specifying DNA segments either for rearrangement or for elimination.

ABSTRACT We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E. crassus genes. The beginning and end of the primary transcript were mapped. In order to determine whether conserved sequences that might serve as replication origins were present, the 5′ and 3′ non‐coding sequences from E. crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism. Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin. The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus. To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome. Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are ∼300–600 base pairs, or “subnucleosomal,” in size. In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases. Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products. Characterization of the most micrococcal nuclease–resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus. Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination. The potential role of developmental stage–specific histone variants in this chromatin differentiation is discussed.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

Characterization of the histone H3 genes of the ciliated protozoan Euplotes crassus indicates that one gene functions only during the sexual phase of the life cycle. Maximum expression of this gene, as judged by transcript accumulation, correlates with DNA replications leading to polytenization of the micronuclear chromosomes before massive DNA elimination, which produces a transcriptionally active macronucleus. Transcripts of the other gene accumulate primarily during vegetative growth and in the sexual phase of the life cycle during replication phases not related to polytenization. Although both histone H3 genes encode proteins that are fairly divergent in sequence at the amino terminus, the meiotic/polytene-specific histone H3 contains two insertions in the amino terminus that increase the size of the protein by 15 amino acids. Analysis of micrococcal nuclease digests of chromatin using hybridization probes specific for micronuclear vs. macronuclear sequences indicates that a change in nucleosomal spacing correlates with the maximal expression of the meiotic/polytene-specific histone H3 gene. Thus, we surmise that this unusual histone H3 may play a key role in targeting DNA sequences for either transcriptional activation and retention in the macronucleus or heterochromatization and elimination.

Two 1.5-kb macronuclear chromosomes bearing histone H2B genes from the ciliated protozoan Euplotes crassus were cloned and sequenced. Although the noncoding sequences on these macronuclear chromosomes are very different, the genes encode an identical 113-aa histone H2B protein that has a shortened N-terminus and a highly conserved C-terminus relative to histone H2B proteins in other organisms. Primer extension was used to determine the transcription start points. Northern analysis shows that the abundance of H2B mRNA changes relative to DNA replication periods during the sexual phase of the life cycle. Analysis of 3′ RACE products indicates that the H2B genes are coexpressed.

The transposon-like Tec elements of Euplotes crassus are precisely excised during formation of polytene chromosomes in the developing macronucleus. To determine whether all Tec elements exhibit identical developmental timing of excision, we used polymerase chain reaction to visualize amplification and diminution at numerous randomly selected Tec insertion sites. Two classes of sites are evident. Early replicating sites show one or more rounds of amplification and diminution (corresponding to excision) and frequently occur within macronuclear-destined sequences. Late replicating sites do not undergo diminution until chromosome fragmentation and are predominantly associated with eliminated sequences. We conclude that the previously described clustering of macro-nuclear-destined sequences in the micronuclear genome allows for their differential replication at the polytene stage and results in targeting of these sequences for transcriptional activation and highly specific deletion and chromosome fragmentation processes.

Macronuclear development in Euplotes crassus begins with polytenization of micronuclear chromosomes and is accompanied by highly precise excision of DNA sequences known as internal eliminated sequences and transposon-like elements (Tecs). Quantitation of radiolabeled-precursor incorporation into DNA indicates that DNA synthesis during formation of polytene chromosomes is not continuous and occurs during two distinct periods. We demonstrate that the timing of Tec excision coincides with these replication periods and that excision can occur during both periods even at a single locus. We also show that Tec and internal eliminated sequence excisions are coincident in the second replication period, thus providing further evidence for similarity in their excision mechanism. Inhibition of DNA synthesis with hydroxyurea diminishes Tec element excision, indicating that replication is an important aspect of the excision process.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

During macromolecular development in the ciliated protozoan, Euplotes crassus, > 105 Tec elements are precisely eliminated from the genome in a 2-4 h time interval, generating extrachromosomal circular forms of the elements. Various models have proposed a transposition-based mechanism for this excision. We have tested this hypothesis by determining the abundance of transcripts of Tec element open reading frames (ORFs) and the timing of their appearance. Transcripts are very low in abundance and are only detected by PCR amplification techniques. Thus, the low levels of transcripts argue against the participation of element-encoded functions in the Tec element elimination process. The element transcripts are only detected in RNA samples from mated cells, indicating that the micronucleus and/or developing macronucleus are transcriptionally active during the sexual phase of the life cycle. The transcription detected could allow a low level of germline-specific transposition for these elements.

The DNA in the transcriptionally active macronucleus of the hypotrichous ciliate Euplotes crassus exists as short, linear molecules with each molecule encoding a single genetic function. Previous work has indicated that coding regions occupy the majority of macronuclear DNA molecules. In the present study we have defined the transcription initiation sites and poly(A) addition sites for a number of different macronuclear genes in Euplotes crassus. Our results indicate that mature transcripts represent all but approximately 100-200 bases of the non-telomeric sequences in macronuclear DNA molecules. We have also examined the sequences in the vicinity of transcription start sites, poly(A) addition sites, and translation initiation sites for Euplotes species genes in an attempt to define the cis-acting elements that control these processes. Our results indicate that some of the common sequence elements known to control these processes in higher eukaryotes are likely not utilized by Euplotes genes. The data do indicate the presence of other conserved sequences both preceding and at the site of poly(A) addition, as well as at the site of translation initiation. These conserved sequences may serve an analogous role in these organisms. Finally, we have found that most macronuclear DNA molecules have transcription initiation sites within 30 bp of the telomere, suggesting that the telomere may play a role in promoting transcription.

The transposon-like elements TBE1, Tec1, and Tec2 of hypotrichous ciliated protozoa appear to encode a protein that belongs to the IS630-Tc1 family of transposases. The Anabaena IS895 transposase also is placed in this family. We note that most family members transpose into the dinucleotide target, TA, and that members with eukaryotic hosts have a tendency for somatic excision that is carried to an extreme by the ciliate elements. Alignments including the additional members, and also mariner elements, show that transposases of this family share strongly conserved residues in a large C-terminal portion, including a fully conserved dipeptide, Asp-Glu (DE), and a block consisting of a fully conserved Asp and highly conserved Glu, separated by 34 or 35 residues (D35E). This D35E motif likely is homologous to the previously characterized D35E motif of the family of retroviral-retrotransposon integrases and IS3-like transposases. Because it is known that the IS3-retroposon D35E region is a critical portion of a domain capable of various in vitro transposition-related reactions, the results suggest that the two families share homologous catalytic transposase domains and that members of both families may share a common transposition mechanism.

Approximately 60,000 transposon-like elements of the Tec1 and Tec2 families excise en masse from the micronuclear genome during formation of a macronucleus in Euplotes crassus. The circular product has been shown previously to contain the element inverted repeats joined head to head. To elucidate the mechanism of Tec excision, we have further characterized the circular products. DNA sequence analysis of cloned inverted repeat junctions and of population of supercoiled Tec circles shows that the inverted repeat junctions consist of both copies of the target site duplication surrounding 10 additional bases. The 10 bp differs for each junction. We demonstrate that the circles are highly sensitive to S1, mung bean and Bal 31 nucleases, and the site of sensitivity maps to the junction. Alkaline gel electrophoresis indicates that the junction does not contain a nick or gap; thus, a likely explanation for the nuclease sensitivity is the existence of a heteroduplex DNA structure at the junction. On the basis of these results, we present a model of Tec excision and discuss the relationship of Tec excision to IES elimination and chromosome fragmentation in E. crassus.

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.

During prolonged vegetative growth of a clonal line of Oxytricha nova, several macronuclear linear DNA molecules increased greatly in copy number over the rest of the approx. 24 000 kinds of molecules comprising the macronuclear genome. One of the amplified sequences was the linear DNA molecule encoding rRNA (rDNA). We have cloned and sequenced the other, smaller, amplified molecules and found that they comprise a gene family, with different allelic versions of one of the family members being amplified. Thus, increased replication is a general property of the molecules comprising this gene family. To date, no function has been assigned to these genes; thus, whether the amplification of these sequences has functional significance is unknown. The rDNA molecule and the two small amplified sequences increased 11-, 24- and 107-fold, respectively, during clonal growth of this line, eventually comprising up to 15% of the macronuclear DNA molecules. Seven other macronuclear DNA molecules did not vary substantially in copy number at different times during the clonal growth of this strain. Analysis of cell-to-cell differences in copy numbers in this clonally aged strain indicated more extensive variation than is evident when large populations from different times are compared.

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.