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At least 19 recordsLinked to original sources
PubMed1992

Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.

We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.

Amino Acid Sequence↗
PubMed1994

Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes.

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.

Animals↗
PubMed1999

Factors Affecting Predation by Cyclidium sp. and Euplotes sp. on PAH-Degrading and Nondegrading Bacteria.

Abstract If predators select for or against contaminant-degrading bacteria, it will affect bacterial survival and has important implications for bioremediation. Protozoa are important predators of bacteria. In order to determine whether protozoa preyed differentially on bacteria with different degradation abilities, two ciliates (Euplotes sp. and Cyclidium sp.) and three strains of PAH-degrading bacteria (Vibrio spp., degrading naphthalene, anthracene, or phenanthrene) were isolated from sediment from New York/New Jersey Harbor. By manipulating growth conditions, bacterial strains with different PAH-degradation abilities and different cell properties were produced. Stepwise regression models were used to analyze how clearance rates on suspended bacteria and grazing rates on bacteria attached to particles were affected by bacterial size, hydrophobicity, C:N ratio, protein content, and PAH-degradation ability. Clearance rates ranged from 0 to 49 nl ciliate-1 h-1 for Euplotes sp. and from 0 to 1.7 nl ciliate-1 h-1 for Cyclidium sp. Clearance rates of both ciliates were positively correlated with bacterial size, hydrophobicity, and protein content, and negatively correlated with C:N ratio. PAH degradation ability had no (for Euplotes sp.) or small (for Cyclidium sp.) effects on clearance rates. The models accounted for 63-75% of the variation in clearance rates on different bacteria. Only Euplotes sp. grazed on attached bacteria, at rates from 3 to 176 bacteria ciliate-1 h-1. A regression model with only C:N ratio and protein content explained 45% of the variation in grazing rates. These models indicate that multiple properties of bacteria affect their susceptibility to predation by ciliates, but PAH-degradation ability per se has little effect.

Journal Article↗
PubMed1991

Two introns in the pheromone 3-encoding gene of Euplotes octocarinatus.

The portion of the pheromone 3 (Phr3)-encoding gene (phr3) of Euplotes octocarinatus, corresponding to secreted Phr3 was isolated using the polymerase chain reaction and oligodeoxyribonucleotide primers flanking the Phr3-encoding cDNA. Unexpectedly, the sequence analysis revealed that this gene is interrupted by two introns with lengths of 63 bp and 72 bp. These introns belong to the class of nuclear pre-mRNA introns and contain typical 5'- and 3'-consensus sequences, as well as unique features. Our findings constitute the first example of introns in any Euplotes species and the first case of multiple introns in hypotrichous ciliates. In ciliates such as Euplotes, the macronucleus is the transcriptionally active nucleus. It develops from a micronucleus in a process which involves chromosome breakage and the elimination of up to 95% of the micronuclear genome. The existence of the introns in the macronuclear version of phr3 shows that, in spite of this extensive elimination, some noncoding sequences are nevertheless retained in the macronucleus of hypotrichous ciliates. It was recently discovered by Meyer et al. [Proc. Natl. Acad. Sci. USA 88 (1991) 3758-3761] that the Phr3-encoding cDNA contains three in frame TGA triplets coding for cysteine. The genomic sequence of phr3 confirms this finding.

Amino Acid Sequence↗
PubMed1998

The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule.

Deviations from the universal genetic code have evolved independently several times in ciliated protozoa. Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine. We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate. It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.

Animals↗
PubMedDate not supplied

Accumulation of telomerase RNA and telomere protein transcripts during telomere synthesis in Euplotes.

In the ciliate Euplotes crassus a complex series of developmental events lead to formation of a new macronucleus. Millions of telomeres are synthesized during this process. We have shown that transcript levels are tightly regulated throughout Euplotes conjugation and macronuclear development. Thus, expression of gene products needed for macronuclear development and telomere synthesis appears to be controlled at the level of RNA abundance. To learn more about the role played by telomerase and the Euplotes telomere protein during telomere synthesis, we have correlated changes in the abundance of telomerase RNA and telomere protein mRNA transcript with specific developmental events. Telomerase RNA levels increase steadily during the early stages of macronuclear development and reach a peak just after telomere addition. The telomere protein transcript rises and falls twice during conjugation and then rises again at the time of telomere addition. The increases in transcript levels during conjugation parallel micronuclear division suggesting that the telomere protein is synthesized at this time and hence may have a micronuclear function.

Actins↗
PubMed1998

The two gamma-tubulin-encoding genes of the ciliate Euplotes crassus differ in their sequences, codon usage, transcription initiation sites and poly(A) addition sites.

We have isolated and sequenced two gamma-tubulin (gamma-Tub)-encoding macronuclear genes of the ciliate Euplotes crassus (Ec), as well as their corresponding cDNAs. Our results reveal that the two genes (gamma-tub 1 and gamma-tub 2) have introns in homologous positions, but differ in their sequences, codon usage, transcription initiation sites and poly(A) addition sites. They both consist of three exons, two introns and two short non-coding sequences on both ends, and they both code for polypeptides of 462 amino acids (aa). The two genes share 76% identity at the nucleotide (nt) level, 86% at the deduced aa level and show 61-92% aa homology to the gamma-Tubs of other organisms. The gamma-tub 2 gene contains two in-frame UGA codons which, like UGA codons in other Euplotes genes, probably code for cysteines. No UGA triplet was found in the gamma-tub 1 gene. Further studies on the cDNA ends indicate that gamma-tub 1 uses at least three transcription initiation sites and two poly(A) addition sites. In contrast, only one transcription initiation site and one poly(A) addition site were identified in gamma-tub 2.

Amino Acid Sequence↗
PubMed1992

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.

Amino Acid Sequence↗
PubMed1996

Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.

Animals↗
PubMed1993

Telomere processing in Euplotes.

In Euplotes crassus millions of telomeres are synthesized during the sexual phase of the life cycle. Since these newly synthesized telomeres are longer than normal macronuclear telomeres, they must be trimmed to the mature size. We have examined the timing and mechanism of this trimming step. We have shown that a sudden decrease in telomere length takes place at a specific time during macronuclear development. The decrease in telomere length is not caused by incomplete replication of the most terminal DNA sequences; rather it is the result of an active processing event that occurs independently of DNA replication. The developmentally regulated telomere shortening that takes place in Euplotes is reminiscent of the sudden reductions in telomere length which have been observed in other eukaryotes.

Animals↗
PubMedDate not supplied

The alpha- and beta-tubulin genes of Euplotes octocarinatus.

The alpha- and the beta-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The alpha-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the beta-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the alpha-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most alpha-tubulin genes, but it supports for other hypotrichous ciliates. No evidence for the existence of more than one type of alpha- and one type of beta-tubulin genes could be obtained.

Amino Acid Sequence↗
PubMed1997

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.

Animals↗
PubMed1991

Structure of the macronuclear polyubiquitin gene in Euplotes.

The hypotrichous ciliate, Euplotes eurystomus, contains both a transcriptionally inactive micronucleus (MIC) and a transcriptionally active macronucleus (MAC) in the same cell. MAC DNA is small (0.5-20 kb), linear and highly amplified. Each DNA fragment consists of two telomeres, a single coding region, and the necessary control elements to regulate gene transcription and replication. The polyubiquitin gene consists of 898 bp, plus 28 bp of double-stranded and 14 bases of single-stranded DNA of the telomeric repeat G4T4 at each end. The coding region exists as three copies of the ubiquitin gene (690 bp) fused in a head-to-tail arrangement as in other organisms. The stop codon is TAA, as in other Euplotes genes, and is not the rare glutamine codon used in most other ciliates. The 3' nontranslated region contains two presumptive poly(A) addition sites; the 5' nontranslated region possesses two putative TATA boxes, several imperfect direct and inverted repeats, and a possible origin of replication. Nucleosome positioning studies reveal four tightly packed nucleosomes and a non-nucleosomal area containing the probable 5' control region as well as part of the coding region. The 5' area does not contain any DNAse I hypersensitive sites. Although the telomeres are protected from exonuclease digestion, they are not as well protected as Oxytricha telomeres against endonucleases and cleavage by methidium propyl Fe2+ EDTA.

Amino Acid Sequence↗
PubMed1990

Calcium-dependent sodium current in the marine ciliate Euplotes vannus.

Ca and Na inward currents were recorded upon depolarizations in Euplotes after the blockage of K outward currents with intracellular Cs ions. The Na current was analyzed under voltage clamp and had the following properties: it activated to a maximum within 150 msec and partly inactivated during sustained voltage steps. It had a positive equilibrium potential between 25 and 30 mV and could be carried by Na or Li ions but not by K, choline or Tris ions. The current revealed a prominent associated inward tail current which deactivated with a single-exponential time constant of 118 msec. Both the current and its tail were strongly reduced after reduction of the extracellular Na concentration. Externally applied K channel blocker tetraethylammonium chloride did not block the current. Either EGTA injection into the cell or nonlethal deciliation with ethanol eliminated the current and its tail. These results indicate the existence of a Na conductance within the membrane of Euplotes which is activated by the intracellular level of free Ca2+.

Animals↗
PubMed1985

Is the initiation of macronuclear DNA synthesis in Euplotes dependent on micronuclear functions?

To determine whether the micronucleus makes essential contributions during asexual reproduction, observations were made on cells of Euplotes octocarinatus from which the micronucleus had been removed with a micropipette. Most cells underwent one postenucleation division, then became arrested in macronuclear G1, slowed down in food uptake, developed macronuclear deformations, and finally died. Such cells could be rescued if a micronucleus was reimplanted before macronuclear deformations had developed. When provided with a new micronucleus, cells initiated macronuclear DNA synthesis about 12-16 h later. The data suggest that the micronucleus is involved in the control of the cell's transition from macronuclear G1 to S, and a model is proposed which postulates that in Euplotes macronuclear DNA synthesis is initiated when a micronucleus-encoded "initiator protein" has accumulated to a critical amount.

Animals↗
PubMed1987

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus.

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.

Animals↗
PubMed1989

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei.

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.

Animals↗
PubMed1989

Telomere terminal transferase activity from Euplotes crassus adds large numbers of TTTTGGGG repeats onto telomeric primers.

A telomere terminal transferase activity was identified in developing macronuclear extracts from Euplotes crassus. The activity was essentially unregulated in vitro: up to 50 tandem repeats of the Euplotes telomeric repeat sequence TTTTGGGG were added onto synthetic telomeric oligonucleotide primers. Both the structure of the telomere substrate and its 3'-terminal sequence were recognized. The activity was destroyed by low concentrations of RNase A.

Animals↗