Description of a new oxytrichid ciliate, Oxytricha buxai n. sp. and redescription of O. quadricirrata Blatterer and Foissner, 1988 based on morphology and 18S rDNA analyses.

The morphology of a new oxytrichid ciliate, Oxytricha buxai n. sp., isolated from a soil sample collected from the Buxa Tiger Reserve, West Bengal, India, was studied based on live observation and protargol impregnation. The new species is characterised by a body size of 85×35 µm in vivo, two macronuclear nodules with one or two micronuclei attached at variable positions, a few colourless cortical granules scattered throughout cortex, adoral zone of membranelles about 35% of body length with 26 membranelles on average, about 18 cirri in left and 16 cirri in right marginal row, right marginal row starts at the level of buccal vertex, usually 18 frontoventral transverse cirri, five dorsal kineties including one dorsomarginal row, three caudal cirri. Further, a redescription based on live and protargol-impregnated specimens of Oxytricha quadricirrata Blatterer and Foissner, 1988, isolated from a moss sample collected from the Kangra district, Himachal Pradesh, India, is provided. The Indian population of O. quadricirrata is similar in morphology to the type population. However, the dorsal side shows some variation, i.e., the presence of a second dorsomarginal row with one or two bristles and incomplete fragmentation of dorsal kinety 3 (vs single dorsomarginal row and complete fragmentation). The resting cyst is spherical and about 20 µm across, with a wrinkled surface. Morphogenesis is in typical Oxytricha pattern. Based on 18S rDNA, phylogenetic analyses show Oxytricha to be a polyphyletic genus. Further, O. quadricirrata clusters away from O. granulifera, thereby supporting the validity of the former.

Morphogenesis and molecular phylogeny of a freshwater ciliate, Oxytricha multilineata n. sp. (Ciliophora, Hypotrichia).

The morphology, morphogenesis, and molecular phylogeny of a new oxytrichid ciliate, Oxytricha multilineata n. sp. isolated from a tributary of the YuYao River, Ningbo, China, were investigated. Oxytricha multilineata n. sp. is characterized as follows: body size about 85-110 × 30-40 µm in vivo; 18 frontoventral-transverse cirri; paroral distinctly bipartite; seven dorsal kineties with cilia about 18 µm long; two macronuclear nodules and two micronuclei. The main events during morphogenesis follow a typical Oxytricha pattern with two exceptions: (1) only one anlage is formed initially within parental dorsal kineties 1-3, each of which then divides and is distributed into the two filial products, that is, the dorsal kineties anlagen are formed in primary mode; (2) dorsal kineties 3 and 4 are formed by incomplete fragmentation. Phylogenetic analyses based on SSU rDNA sequence data reveal that O. multilineata n. sp. clusters with Oxytricha lithofera, Heterourosomoida lanceolata and Quadristicha setigera, and species of the genus Oxytricha did not form a monophyletic group, however nodal support is not sufficiently high to recover a robust phylogeny of the Oxytrichidae.

Transcribed germline-limited coding sequences in Oxytricha trifallax.

The germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein-coding sequences whose gene expression is terminated by DNA elimination.

Dynamics and Barrier of Movements of Sodium and Potassium Ions Across the Oxytricha nova G-Quadruplex Core.

G-quadruplexes (GQs) are highly stable noncanonical forms of nucleic acids that are present in important genomic regions. The central core of the GQ is lined up by four closely spaced carbonyl groups from the G-quartets, and the resulting electrostatic repulsion is neutralized by the coordinating cations. In spite of several reports on GQ structure and cation-GQ interactions, the atomic- to molecular-level understanding of the ion dynamics and ion exchange in the GQ core is quite poor. Here, we attempt to elucidate the mechanism of Na+ and K+ binding to the GQ core and trace the exchange of these ions with the ions in bulk by means of all-atomic molecular dynamics (MD) simulations. One of the most studied GQs, Oxytricha nova telomeric G-quadruplex (OxyGQ), is taken as the representative GQ. Subsequently, umbrella sampling MD simulations were performed to elucidate the energetics of ion translocation from one end to the other end of the GQ central core. Our study highlights the importance of ion hydration for the uptake and correct positioning of the cations in the core. The free-energy landscape of ion transport has shown favorable in-plane binding of Na+ ions with GQ quartets, which matches very well with the crystal structure. The binding of K+ ions, on the other hand, was out-of-plane and its translocation required a larger barrier to cross.

Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences.

S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

Graph based analysis for gene segment organization In a scrambled genome.

DNA recombinant processes can involve gene segments that overlap or interleave with gene segments of another gene. Such gene segment appearances relative to each other are called here gene segment organization. We use graphs to represent the gene segment organization in a chromosome locus. Vertices of the graph represent contigs resulting after the recombination and the edges represent the gene segment organization prior to rearrangement. To each graph we associate a vector whose entries correspond to graph properties, and consider this vector as a point in a higher dimensional Euclidean space such that cluster formations and analysis can be performed with a hierarchical clustering method. The analysis is applied to a recently sequenced model organism Oxytricha trifallax, a species of ciliate with highly scrambled genome that undergoes massive rearrangement process after conjugation. The analysis shows some emerging star-like graph structures indicating that segments of a single gene can interleave, or even contain all of the segments from fifteen or more other genes in between its segments. We also observe that as many as six genes can have their segments mutually interleaving or overlapping.

Dominant protozoan species in rhizosphere soil over growth of Beta vulgaris L. in Northeast China.

This paper identified the dominant protozoan species in the four layers of rhizosphere soil during the six growth stages of Beta vulgaris L. and analyzed the correlations of the abundance and diversity of the dominant protozoan species with soil properties at different growth stages and soil depth. A total of 15 species of protozoa were identified; among them, Colpoda sp., Bodo sp., two kinds of Oxytricha sp., and Tachysoma sp. were the most dominant species of Beta vulgaris L. rhizosphere soil. The Colpoda sp. was eurytopic species in the Beta vulgaris L. rhizosphere soil and Tachysoma sp., Vorticella sp., Colpoda sp., Oxytricha sp.1, and Oxytricha sp. 2 were noted closely related to the acceleration function of circulation of N and P elements in soils. These dominant protozoan species were proposed to play a significant role of fertilization on N supply in rhizosphere soil during the initial growth of Beta vulgaris L.

Morphology and molecular phylogeny of Oxytricha seokmoensis sp. nov. (Hypotrichia: Oxytrichidae), with notes on its morphogenesis.

A new hypotrichous ciliate, Oxytricha seokmoensis sp. nov., was discovered in a soil from a forest in South Korea and described based on the observations of living and stained specimens. In addition, phylogenetic analyses were performed using the small subunit ribosomal RNA (18S rRNA) gene sequence. Morphologically, the new species is similar to the O. granulifera-complex in terms of ciliary structure and arrangement of cortical granules, but dorsal kineties 3 and 4 (not completely separated vs. separated) and macronuclear nodules in the cyst (separated vs. fused) differ. Oxytricha seokmoensis is most similar to O. pulvillus, but can be distinguished by the number of adoral membranelles (30-40 vs. 23-27), contractile vacuole (present vs. absent), number of left (27-37 vs. 17-25) and right (27-35 vs. 18-23) marginal cirri, and lepidosomes on the cyst surface (present vs. absent). In a phylogenetic tree, O. seokmoensis is distinctly separated from the O. granulifera clade, but is sister to the Paroxytricha clade. In addition, O. seokmoensis and P. longigranulosa have the smallest genetic difference (d = 0.015, 23 of 1579 nt difference). This close relationship is supported by incomplete dorsal kinety 3 fragmentation and separated macronuclear nodules in resting cysts.

Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA.

Extrachromosomal circular DNA (eccDNA) is both a driver of eukaryotic genome instability and a product of programmed genome rearrangements, but its extent had not been surveyed in Oxytricha, a ciliate with elaborate DNA elimination and translocation during development. Here, we captured rearrangement-specific circular DNA molecules across the genome to gain insight into its processes of programmed genome rearrangement. We recovered thousands of circularly excised Tc1/mariner-type transposable elements and high confidence non-repetitive germline-limited loci. We verified their bona fide circular topology using circular DNA deep-sequencing, 2D gel electrophoresis and inverse polymerase chain reaction. In contrast to the precise circular excision of transposable elements, we report widespread heterogeneity in the circular excision of non-repetitive germline-limited loci. We also demonstrate that circular DNAs are transcribed in Oxytricha, producing rearrangement-specific long non-coding RNAs. The programmed formation of thousands of eccDNA molecules makes Oxytricha a model system for studying nucleic acid topology. It also suggests involvement of eccDNA in programmed genome rearrangement.

Programmed Chromosome Deletion in the Ciliate Oxytricha trifallax.

The ciliate Oxytricha trifallax contains two nuclei: a germline micronucleus and a somatic macronucleus. These two nuclei diverge significantly in genomic structure. The micronucleus contains approximately 100 chromosomes of megabase scale, while the macronucleus contains 16,000 gene-sized, high ploidy "nanochromosomes." During its sexual cycle, a copy of the zygotic germline micronucleus develops into a somatic macronucleus via DNA excision and rearrangement. The rearrangement process is guided by multiple RNA-based pathways that program the epigenetic inheritance of sequences in the parental macronucleus of the subsequent generation. Here, we show that the introduction of synthetic DNA molecules homologous to a complete native nanochromosome during the rearrangement process results in either loss or heavy copy number reduction of the targeted nanochromosome in the macronucleus of the subsequent generation. This phenomenon was tested on a variety of nanochromosomes with different micronuclear structures, with deletions resulting in all cases. Deletion of the targeted nanochromosome results in the loss of expression of the targeted genes, including gene knockout phenotypes that were phenocopied using alternative knockdown approaches. Further investigation of the chromosome deletion showed that, although the full length nanochromosome was lost, remnants of the targeted chromosome remain. We were also able to detect the presence of telomeres on these remnants. The chromosome deletions and remnants are epigenetically inherited when backcrossed to wild type strains, suggesting that an undiscovered mechanism programs DNA elimination and cytoplasmically transfers to both daughter cells during conjugation. Programmed deletion of targeted chromosomes provides a novel approach to investigate genome rearrangement and expands the available strategies for gene knockout in Oxytricha trifallax.

Transformation with Artificial Chromosomes in Oxytricha trifallax and Their Applications.

Oxytricha trifallax, like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The macronucleus contains a somatic genome which is naturally fragmented into thousands of kilobase-sized chromosomes. Here, we develop a method to stably incorporate artificial chromosomes into the macronucleus. We report two cases of successful transformation and demonstrate the use of somatic transformation to investigate gene regulation and gene function in Oxytricha We show that the transformed artificial chromosomes are maintained through multiple asexual divisions. Furthermore, they support the transcriptional regulation of the native chromosome from which they were derived and are translated to produce functional proteins. To test if transformed chromosomes are amenable to practical applications, we generated a tagged version of a representative gene (AL1) and used it to co-precipitate associated proteins. This revealed an association with nucleic acid binding proteins, specifically RNA-binding proteins, and RNA immunoprecipitation of AL1 revealed its association with multiple RNAs. The use of artificial chromosomes in Oxytricha enables an array of genetic and molecular biological assays, as well as new avenues of inquiry into the epigenetic programming of macronuclear development and genome rearrangement.

Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

In situ microbiota distinguished primary anthropogenic stressor in freshwater sediments.

Conventional assessment and evaluation of sediment quality are based on laboratory-based ecotoxicological and chemical measurements with lack of concern for ecological relevance. Microbiotas in sediment are responsive to pollutants and can be used as alternative ecological indicators of sediment pollutants; however, the linkage between the microbial ecology and ecotoxicological endpoints in response to sediment contamination has been poorly evaluated. Here, in situ microbiotas from the Three Gorges Reservoir (TGR) area of the Yangtze River were characterized by DNA metabarcoding approaches, and then, changes of in situ microbiotas were compared with the ecotoxicological endpoint, aryl hydrocarbon receptor (AhR) mediated activity, and level of polycyclic aromatic hydrocarbons (PAHs) in sediments. PAHs and organic pollutant mixtures mediating AhR activity had different effects on the structures of microbiotas. Specifically, Shannon indices of protistan communities were negatively correlated with the levels of AhR mediated activity and PAHs. The sediment AhR activity was positively correlated with the relative abundance of prokaryotic Acetobacteraceae, but had a negative correlation with protistan Oxytrichidae. Furthermore, a quantitative classification model was built to predict the level of AhR activity based on the relative abundances of Acetobacteraceae and Oxytrichidae. These results suggested that in situ Protista communities could provide a useful tool for monitoring and assessing ecological stressors. The observed responses of microbial community provided supplementary evidence to support that the AhR-active pollutants, such as PAHs, were the primary stressors of the aquatic community in TGR area.

Russian Doll Genes and Complex Chromosome Rearrangements in Oxytricha trifallax.

Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax.

Small RNA-mediated regulation of DNA dosage in the ciliate Oxytricha.

Dicer-dependent small noncoding RNAs play important roles in gene regulation in a wide variety of organisms. Endogenous small interfering RNAs (siRNAs) are part of an ancient pathway of transposon control in plants and animals. The ciliate, Oxytricha trifallax, has approximately 16,000 gene-sized chromosomes in its somatic nucleus. Long noncoding RNAs establish high ploidy levels at the onset of sexual development, but the factors that regulate chromosome copy numbers during cell division and growth have been a mystery. We report a novel function of a class of Dicer (Dcl-1)- and RNA-dependent RNA polymerase (RdRP)-dependent endogenous small RNAs in regulating chromosome copy number and gene dosage in O. trifallax Asexually growing populations express an abundant class of 21-nt sRNAs that map to both coding and noncoding regions of most chromosomes. These sRNAs are bound to chromatin and their levels surprisingly do not correlate with mRNA levels. Instead, the levels of these small RNAs correlate with genomic DNA copy number. Reduced sRNA levels in dcl-1 or rdrp mutants lead to concomitant reduction in chromosome copy number. Furthermore, these cells show no signs of transposon activation, but instead display irregular nuclear architecture and signs of replication stress. In conclusion, Oxytricha Dcl-1 and RdRP-dependent small RNAs that derive from the somatic nucleus contribute to the maintenance of gene dosage, possibly via a role in DNA replication, offering a novel role for these small RNAs in eukaryotes.

A New Subspecies of Oxytricha granulifera (Hypotrichia: Oxytrichidae) from Mexico, with Notes on its Morphogenesis and Phylogenetic Position.

The genus Oxytricha Bory de Saint-Vincent in Lamouroux, Bory de Saint-Vincent and Deslongchamps, 1824 comprises about 38 species distributed worldwide and has been considered to be a nonmonophyletic group. Based on living observations, protargol preparations, and a small subunit ribosomal RNA (SSU rRNA) gene sequence, we describe a new subspecies Oxytricha granulifera chiapasensis n. subsp. This new taxon is morphologically characterized by undulating membranes basically in a Stylonychia-pattern, six dorsal kineties, size in vivo ca. 60-120 × 20-40 µm, 21-30 right and 21-31 left marginal cirri, 22-29 adoral membranelles, and spherical cortical granules arranged in longitudinal rows on the dorsal side. In terms of the SSU rRNA gene sequence, the new subspecies differs from populations of O. granulifera from GENBANK by 7-35 nucleotides. Phylogenetic analyses showed that Oxytricha granulifera gene sequences were nested into three groups, with the new subspecies included in one of them. Oxytricha granulifera chiapasensis n. subsp. is different from Oxytricha granulifera granulifera Foissner and Adam, 1983 and Oxytricha granulifera quadricirrata Blatterer and Foissner, 1988 based on: (i) undulating membranes in Stylonychia-pattern, (ii) formation of a sixth dorsal kinety during morphogenesis, (iii) the adoral membranelles number, and (iv) inhabiting freshwater habitats.

Molecules illuminate morphology: phylogenomics confirms convergent evolution among 'oligotrichous' ciliates.

'Oligotrichous' ciliates have been traditionally placed in a presumed monophyletic taxon called the Oligotrichia. However, gene sequences of the small subunit rRNA gene, and several other genes, suggest that the taxon is not monophyletic: although statistical support for this is not strong, the oligotrich Halteria grandinella is associated with the hypotrich ciliates and not with other oligotrich genera, such as Strombidium and Strombidinopsis. This has convinced some taxonomists to emphasize that morphological features strongly support the monophyly of the oligotrichs. To further test this hypothesis of monophyly, we have undertaken a phylogenomic analysis using the transcriptome of H. grandinella cells amplified by a single-cell technique. One hundred and twenty-six of 159 single-gene trees placed H. grandinella as sister to hypotrich species, and phylogenomic analyses based on a subset of 124 genes robustly rejected the monophyly of the Oligotrichia and placed the genus Halteria as sister to the hypotrich genera Stylonychia and Oxytricha. We use these phylogenomic analyses to assess the convergent nature of morphological features of oligotrichous ciliates. A particularly 'strong' morphological feature supporting monophyly of the oligotrichs is enantiotropic cell division, which our results suggest is nevertheless a convergent feature, arising through the need for dividing ciliates to undertake rotokinesis to complete cell division.

Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development.

The ciliate Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long noncoding (lnc) RNA "templates"-telomere-containing, RNA-cached copies of mature chromosomes-provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts. We find that during development, Oxytricha produces long noncoding RNA copies for over 10,000 of its 16,000 somatic chromosomes, consistent with a model in which Oxytricha transmits an RNA-cached copy of its somatic genome to the sexual progeny. Both the primary sequence and expression profile of a somatic chromosome influence the temporal distribution and abundance of individual template RNAs. This suggests that Oxytricha may undergo multiple rounds of DNA rearrangement during development. These observations implicate a complex set of thousands of long RNA molecules in the wiring and maintenance of a highly elaborate somatic genome architecture.

Preferential expression of scores of functionally and evolutionarily diverse DNA and RNA-binding proteins during Oxytricha trifallax macronuclear development.

During its sexual reproduction, the stichotrichous ciliate Oxytricha trifallax orchestrates a remarkable transformation of one of the newly formed germline micronuclear genomes. Hundreds of thousands of gene pieces are stitched together, excised from chromosomes, and replicated dozens of times to yield a functional somatic macronuclear genome composed of ~16,000 distinct DNA molecules that typically encode a single gene. Little is known about the proteins that carry out this process. We profiled mRNA expression as a function of macronuclear development and identified hundreds of mRNAs preferentially expressed at specific times during the program. We find that a disproportionate number of these mRNAs encode proteins that are involved in DNA and RNA functions. Many mRNAs preferentially expressed during macronuclear development have paralogs that are either expressed constitutively or are expressed at different times during macronuclear development, including many components of the RNA polymerase II machinery and homologous recombination complexes. Hundreds of macronuclear development-specific genes encode proteins that are well-conserved among multicellular eukaryotes, including many with links to germline functions or development. Our work implicates dozens of DNA and RNA-binding proteins with diverse evolutionary trajectories in macronuclear development in O. trifallax. It suggests functional connections between the process of macronuclear development in unicellular ciliates and germline specialization and differentiation in multicellular organisms, and argues that gene duplication is a key source of evolutionary innovation in this process.

Telomerase Mechanism of Telomere Synthesis.

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.