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PubMed1957

An electron microscope study of the cytology of the protozoan Euplotes patella.

1. Structurally the "sensory bristles" in Euplotes patella are typical cilia, but no ciliary rootlets connect their bases. 2. The "neuromotor fibrils" are composed of filaments 21 mmicro in diameter. At the point of junction of the filaments with the peripheral ciliary fibrils a granular structure 65 to 90 mmicro in diameter is seen which has dense central and peripheral zones separated by a less dense layer. Information on the interconnection of organelles is expanded. 3. A system of subpellicular fibrils is described. The external fibrillar system described by others could not be found. 4. The motorium is shown to be a mass of intertwining rootlet filaments. 5. The micronucleus is shown to have a spongy, dense material in a less dense material, all of which is surrounded by a double-layered membrane. 6. The double-layered macronuclear membrane contains annuli whose outside diameter is 70 mmicro; the macronuclear bodies are sometimes closely applied to the membrane. In the macronuclear reorganization bands, the solution plane is a fine network, while the reconstruction plane is devoid of structure at the level of resolution observed. 7. The mitochondria are composed of tubules, only occasionally oriented, usually embedded in a surrounding material of lower density. 8. Microbodies whose diameters are 250 to 350 mmicro are frequently observed in close association with mitochondrial surfaces. 9. The food vacuoles, contractile vacuoles, and ciliary vacuoles are bounded by single-layered membranes. In the food vacuoles, the bacteria are surrounded by membranes individually or in small groups. 10. Cytoplasmic rods localized in the oral region, and cytoplasmic granules dispersed at random, are described. No typical ergastoplasm, endoplasmic reticulum, or Golgi material was observed.

Animals↗
PubMed1959

Macronuclear duplication in the ciliated protozoan Euplotes.

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.

Animals↗
PubMed1999

Differentiation of chromatin during DNA elimination in Euplotes crassus.

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 approximately 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.

Animals↗
PubMed2000

The autocrine mitogenic loop of the ciliate Euplotes raikovi: the pheromone membrane-bound forms are the cell binding sites and potential signaling receptors of soluble pheromones.

Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding.

Amino Acid Sequence↗
PubMed1996

Differential replication and DNA elimination in the polytene chromosomes of Euplotes crassus.

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.

Animals↗
PubMed2003

Identification of a novel "chromosome scaffold" protein that associates with Tec elements undergoing en masse elimination in Euplotes crassus.

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.

Amino Acid Sequence↗
PubMed2001

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon.

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.

Amino Acid Sequence↗
PubMed2000

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting.

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Amino Acid Motifs↗
PubMed1992

Gene dosage as a possible major determinant for equal expression levels of genes encoding RNA polymerase subunits in the hypotrichous ciliate Euplotes octocarinatus.

Ciliated protozoa harbor two different types of nuclei in each cell. The diploid micronucleus is the transcriptionally inactive generative nucleus, while the macronuclous contains a highly amplified transcriptionally active genome of lower complexity. The macronuclear genes encoding the two largest subunits of both RNA polymerases I and II of Euplotes octocarinatus were identified by a novel method of two step PCR walking, employing primer pairs derived from telomeric sequences of the organism and known conserved RNA polymerase polypeptide sequences, respectively. The relative gene dosage was determined. The genes are present in equal copy numbers for the respective matching subunits. Northern hybridizations showed comparable amounts of transcripts, as well, within the matching pairs. Mapping of the 5'-termini of the transcripts of the gene sized chromosomes showed that the upstream nontranscribed regions are very short and contain characteristic sequence motifs which could be the determinants of equal promoter strengths for subunits of a common RNA polymerase.

Amino Acid Sequence↗
PubMed1992

TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.

Amino Acid Sequence↗
PubMed1994

Developmentally regulated, low abundance Tec element transcripts in Euplotes crassus--implications for DNA elimination and transposition.

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.

Animals↗
PubMed1996

A nascent micronuclear pseudogene in the ciliate Euplotes crassus.

The macronuclear genome of Euplotes crassus contains two different genes, EFA1 and EFA2, encoding EF-1alpha proteins. They are derived from micronuclear precursors in the course of a sexual process termed conjugation. We have found that two apparent micronuclear precursors exist for EFA1. They differ in their potential coding sequences and in the internal sequences interrupting the genes, which are normally removed during the processing from micronuclear into macronuclear genes. One of these genes is not processed into a macronuclear gene and has accumulated C-->T transitions in a limited section of the coding region. The gene obviously constitutes a recent duplication which has lost its ability to be processed into a macronuclear gene and has therefore become a micronuclear pseudogene. The true EFA1 precursor harbours a novel type of internal sequence in addition to a classical AT-rich IES. As usual, only one micronuclear EFA2 precursor gene was found. Its coding sequence is interrupted by a 79 bp TeIIES.

Amino Acid Sequence↗
PubMed1997

The telomere binding protein of Euplotes crassus prevents non-specific transcription initiation but has no role in positioning transcription initiation complexes.

Model substrates mimicking the telomeric as well as the 5'-untranslated regions in front of a 5'-terminal part of a macronuclear gene of Euplotes crassus were transcribed in vitro using cellular extracts. The obtained transcripts were characterized by primer extension and shown to start at the natural initiation points. The situation changed in the absence of telomere binding protein or with substrates lacking functional telomeric sequences. In these cases non-specific transcription was observed. Since it had been previously found that transcription starts are frequently located at an apparently fixed distance from the telomere, a model substrate was constructed which carried a duplication of the non-transcribed region. This resulted in only one transcription start site, the position of which was conserved relative to the start of the open reading frame but moved away from the telomere by the distance of the duplication.

Animals↗
PubMed1997

dGTP-dependent processivity and possible template switching of euplotes telomerase.

We have measured the processivity of telomeric DNA extension by Euplotes aediculatus telomerase at various concentrations of the nucleotide substrates dGTP and dTTP. The maximum processivity (approximately 3 repeats) was observed at approximately 100 microM of each dNTP. Processivity decreased as the dNTP concentrations were reduced and, surprisingly, as the concentration of dGTP was increased. Also, the characteristic banding pattern generated by telomerase extension of DNA primers shifted in response to changes in dGTP concentration. One pattern with 8 nt periodicity was predominant at dGTP concentrations </=16 microM, while at >/= 250 microM an 8 nt repeat pattern out-of-phase with the first was observed; at intermediate concentrations the two patterns coexisted. We propose that two different segments of the RNA subunit can serve as the template for repeat synthesis; nt 42-49 at low dGTP concentrations and nt 36-43 at high dGTP concentrations. An alternative model for the low dGTP pattern involves an internal pause site but no pause at the end of the template and is, therefore, considered less likely. Because the effects of dGTP on processivity and banding pattern appear to be distinct from nucleotide binding in the polymerase active site, we propose a second dGTP binding site involved in template selection and processivity.

Animals↗
PubMed1998

Reiterative dG addition by Euplotes crassus telomerase during extension of non-telomeric DNA.

Telomerase from the ciliate Euplotes crassus incorporates G4T4telomeric repeats onto both telomeric and non-telomeric single-stranded DNA 3'-ends via reverse transcription of a templating domain in its RNA subunit. Here we describe an unusual mode of template copying that is characteristic of DNA synthesis onto non-telomeric 3'-ends in vitro . When dTTP was eliminated from telomerase reactions, telomeric primers or DNA products generated from the telomerase endonuclease were extended by precise copying of the RNA template. In contrast, telomerase catalyzed the addition of up to 13 dG residues onto primers with non-telomeric 3'-ends under the same reaction conditions. Introducing mismatches in the 3'-terminus of telomeric primers that reduced primer complementarity to the RNA template induced reiterative dG incorporation, indicating that the reaction is influenced by Watson-Crick base pair formation between the primer and the RNA template. Unexpectedly, the reiterative dG addition mode was confined to telomerase derived from developing cells that undergo new telomere formation. This reaction was not observed in vegetatively growing cells. We postulate that indiscriminate dG addition by telomerase occurs by reiterative copying of C residues in the telomerase RNA templating domain and reflects lateral instability of the primer-template interaction during de novo telomere formation.

Animals↗
PubMed1998

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

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.

Animals↗
PubMed1999

Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.

5' Untranslated Regions↗
PubMed2002

De novo telomere addition to spacer sequences prior to their developmental degradation in Euplotes crassus.

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3' overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.

Animals↗