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At least 19 recordsLinked to original sources
PubMedDate not supplied

Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle.

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.

Animals↗
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↗
PubMed2005

Sequencing of random Euplotes crassus macronuclear genes supports a high frequency of +1 translational frameshifting.

Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more +1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes, and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus. Three new candidate +1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C(2)H(2)-type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.

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↗
PubMed2002

A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea).

The cosmopolitan genus Euplotes is remarkable among ciliates for its species richness. To understand the still tangled taxonomy and phylogenetic relationships within the genus, small subunit rRNA sequences of 11 morphologically defined species colonizing different habitats were determined. Euplotes 18S rRNA is unique among ciliates for its anomalous length and high evolutionary rate. Phylogenetic reconstruction pointed to a high divergence between this genus and the other Spirotrichea, together with a high variability within the genus. Some of the relationships within the Euplotes group were also resolved. Data from the literature, based on morphological features, habitat, and symbiotic relationships, have been compared with our results and are critically discussed. In many cases, the molecular phylogenetic analysis disagreed with species relatedness established on morphological and ecological grounds. The occurrence of a radiation phenomenon in the evolution of the genus is postulated.

Animals↗
PubMed2004

Evolution of programmed ribosomal frameshifting in the TERT genes of Euplotes.

A number of recent studies indicate that programmed + 1 ribosomal frameshifting is frequently required for the expression of genes in species of the genus Euplotes. In E. crassus, three genes encoding the telomerase reverse transcriptase (TERT) subunit have been previously found to possess one or two + 1 frameshift sites. To examine the origin of frameshift sites within the Euplotes group, we have isolated segments of the TERT gene from five Euplotes species. Coupled with phylogenetic analysis, the results indicate that one frameshift site in the TERT gene arose late in the evolution of the group. In addition, a novel frameshift site was identified in the TERT gene of E. minuta, a species where frameshifting has not been previously reported. Coupled with other studies, the results indicate that frameshift sites have arisen during the diversification of the euplotids. The results also are discussed in regard to the mutations necessary to generate frameshift sites, and the specialization of TERT protein function that has apparently occurred in E. crassus.

Amino Acid Sequence↗
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↗
PubMed2001

The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1.

Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.

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↗
PubMedDate not supplied

Molecular cloning of the gene encoding an acidic ribosomal protein of the P2 family from the ciliate Euplotes raikovi.

We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear "gene-sized" molecules with Euplotes telomeres at the ends. The longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. To our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.

Amino Acid Sequence↗
PubMedDate not supplied

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia).

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.

Animals↗
PubMed2003

Origin usage during euplotes ribosomal DNA amplification.

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.

Animals↗
PubMed2006

Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes.

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

Animals↗
PubMed2002

Encystment-inducing factors in the ciliate Euplotes elegans.

Although starvation is considered one of the most important induces of ciliate encystment, its nature has been unclear. Euplotes is a well-known ciliate genus, but the relationship in Euplotes between encystment and food has not been reported. The encystment of Euplotes elegans is facilitated when it is transferred to Chalkley's solution without bacteria as food. A higher ciliate density also facilitates encystment. Thus, starvation and ciliate density needed to be examined. Ciliates were inoculated into 3 treatments: Chalkley's solution with formaldehyde-fixed bacteria as nutritive particles (FFB group), with polystyrene latex particles as non-nutritive particles (PLP group), and without particles (control group). Cysts appeared fastest and ciliate numbers increased in the FFB group. Although the encystment kinetics of the PLP group was similar to that of the control group, the encystment rate of the PLP group was lower than that of the control group in the earliest phase. This suggests that the ciliates were temporarily deceived into feeding on PLP, because they had food vacuoles containing PLP during the earliest phase of incubation. A cell-free old culture solution from a stationary phase, which probably contained excreted substances from high-density ciliates, also facilitated encystment.

Animals↗
PubMed2006

[Cloning and sequence analysis of a novel member of the rab gene family from Euplotes octocarinatus].

Rab proteins belong to a subfamily of small GTP-binding proteins of the Ras superfamily, which play an important role in intracellular vesicular traffic. In this study, a rab gene was obtained from Euplotes octocarinatus by polymerase chain reaction (PCR) and RT-PCR. The rab gene from macronucleic DNA was 884 bp in length, including non-coding regions and telomeric sequences at both ends. The rab gene from micronuclear DNA (723 bp), lacking of internal eliminated sequences, was identical to rab gene from macronuclear DNA. RT-PCR showed that the opening reading frame of the rab gene was 663 bp long. The rab gene from macronuclear DNA contained an intron of 60 bp at the position from 153 bp to 212 bp of macronuclear DNA. The rab gene had two in-frame TGAs encoding for cysteine in Euplotes octocarinatus. The rab gene used TAG as stop codon, which was the first report in Euplotes octocarinatus. The result of BLAST in NCBI demonstrates that the Rab shares a homology of 49-52% at the amino acid level with Rab1 proteins from a number of other eukaryote, which suggesting that the Rab is a Rab1 homolog. The rab gene was therefore designated Eo-rab-1N (GenBank accession number: DQ105562). The evolution of Eo-rab-1N was analyzed using phylogenetic tree of amino acids sequences of Rab1 obtained from GenBank.

AT Rich Sequence↗
PubMed2005

Ribosomal cold-adaptation: characterization of the genes encoding the acidic ribosomal P0 and P2 proteins from the Antarctic ciliate Euplotes focardii.

Molecular adaptation at low temperature requires specificities represented mainly by modifications in the gene sequence and consequently in the protein primary structure. To characterize the molecular mechanisms responsible for ribosome cold-adaptation, we compared the ribosomal P0 and P2 genes from the Antarctic ciliate Euplotes focardii with homologous genes from mesophilic organisms, including the ciliates Tetrahymena thermophila and non cold-adapted Euplotes species. This analysis revealed the presence of non synonymous mutations unique to E. focardii. In the P0 protein the mutations produced amino acid substitutions that increased the molecular flexibility that may facilitate a conformational adjustment associated with the interaction with the GTPase center of the large subunit rRNA, and increased the hydrophobicity of the region involved in the interaction with P1/P2 heterodimer, probably to keep associated the ribosomal stalk in the cold. In the P2 protein the mutations produced amino acid substitutions that increased the N-terminus flexibility, which may facilitate interactions with P1 protein in the formation of the heterodimer, and reduced the mobility of the C-terminus, to stabilize the stalk during ribosomal activity. Finally, P proteins appeared to be valid markers for investigating the phylogenetic origin of early eukaryotes.

Adaptation, Physiological↗