ABSTRACT
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.
Subject(s)
DNA, Protozoan/genetics , Evolution, Molecular , Gene Expression Profiling , Macronucleus/metabolism , Oxytricha/metabolism , Protozoan Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Macronucleus/genetics , Oxytricha/genetics , Oxytricha/growth & development , Phylogeny , Protozoan Proteins/genetics , RNA-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
DNA Replication , DNA, Single-Stranded/metabolism , RNA/metabolism , Ribonucleoproteins/metabolism , Telomerase/metabolism , Telomere/enzymology , Animals , Catalytic Domain , DNA, Single-Stranded/genetics , Gene Expression Regulation , Humans , Microsatellite Repeats , Nucleic Acid Conformation , Oxytricha/genetics , Oxytricha/metabolism , RNA/genetics , Ribonucleoproteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Telomerase/genetics , Telomere/chemistry , Tetrahymena thermophila/genetics , Tetrahymena thermophila/metabolismABSTRACT
Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell.
Subject(s)
Ciliophora/genetics , Dyneins/chemistry , Dyneins/genetics , Evolution, Molecular , Amino Acid Sequence , Ciliophora/metabolism , Oxytricha/genetics , Oxytricha/metabolism , Paramecium/genetics , Paramecium/metabolism , Phylogeny , Tetrahymena/genetics , Tetrahymena/metabolismABSTRACT
Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.
Subject(s)
Gene Rearrangement , Genome, Protozoan , Oxytricha/growth & development , Oxytricha/genetics , Cell Nucleus/metabolism , Chromosomes/metabolism , Molecular Sequence Data , Oxytricha/cytology , Oxytricha/metabolismABSTRACT
Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells - many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes.
Subject(s)
Chromosomes , Gene Expression Regulation/physiology , Genes, Protozoan/physiology , Macronucleus , Oxytricha , Polyploidy , Chromosomes/genetics , Chromosomes/metabolism , Macronucleus/genetics , Macronucleus/metabolism , Oxytricha/genetics , Oxytricha/metabolismABSTRACT
Several independent lines of evidence suggest that the modern genetic system was preceded by the 'RNA world' in which RNA genes encoded RNA catalysts. Current gaps in our conceptual framework of early genetic systems make it difficult to imagine how a stable RNA genome may have functioned and how the transition to a DNA genome could have taken place. Here we use the single-celled ciliate, Oxytricha, as an analog to some of the genetic and genomic traits that may have been present in organisms before and during the establishment of a DNA genome. Oxytricha and its close relatives have a unique genome architecture involving two differentiated nuclei, one of which encodes the genome on small, linear nanochromosomes. While its unique genomic characteristics are relatively modern, some physiological processes related to the genomes and nuclei of Oxytricha may exemplify primitive states of the developing genetic system.
Subject(s)
Biological Evolution , Genome, Protozoan , Oxytricha/genetics , Cell Division , DNA Replication , Genomic Instability , Oxytricha/cytology , Oxytricha/metabolismABSTRACT
We took advantage of the unusual genomic organization of the ciliate Oxytricha trifallax to screen for eukaryotic non-coding RNA (ncRNA) genes. Ciliates have two types of nuclei: a germ line micronucleus that is usually transcriptionally inactive, and a somatic macronucleus that contains a reduced, fragmented and rearranged genome that expresses all genes required for growth and asexual reproduction. In some ciliates including Oxytricha, the macronuclear genome is particularly extreme, consisting of thousands of tiny 'nanochromosomes', each of which usually contains only a single gene. Because the organism itself identifies and isolates most of its genes on single-gene nanochromosomes, nanochromosome structure could facilitate the discovery of unusual genes or gene classes, such as ncRNA genes. Using a draft Oxytricha genome assembly and a custom-written protein-coding genefinding program, we identified a subset of nanochromosomes that lack any detectable protein-coding gene, thereby strongly enriching for nanochromosomes that carry ncRNA genes. We found only a small proportion of non-coding nanochromosomes, suggesting that Oxytricha has few independent ncRNA genes besides homologs of already known RNAs. Other than new members of known ncRNA classes including C/D and H/ACA snoRNAs, our screen identified one new family of small RNA genes, named the Arisong RNAs, which share some of the features of small nuclear RNAs.
Subject(s)
Chromosomes/chemistry , Genes, Protozoan , Oxytricha/genetics , RNA, Untranslated/genetics , Base Sequence , Conserved Sequence , Genome, Protozoan , Genomics/methods , Introns , Oxytricha/metabolism , RNA, Small Nuclear/genetics , RNA, Small Nucleolar/genetics , RNA, Untranslated/chemistry , RNA, Untranslated/metabolismABSTRACT
Hg(2+) is able to inhibit the peroxidase-like DNAzyme function of a T-containing G-quadruplex DNA via Hg(2+)-mediated T-T base pairs, which enables the visual detection of Hg(2+) in the TMB-H(2)O(2) reaction system with high selectivity and sensitivity.
Subject(s)
DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , G-Quadruplexes , Mercury/analysis , Mercury/chemistry , Vision, Ocular/physiology , Animals , Humans , Oxytricha/chemistry , Oxytricha/metabolism , Sensitivity and Specificity , Spectrophotometry , Telomere/chemistry , Telomere/metabolismABSTRACT
The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.
Subject(s)
DNA, Protozoan/metabolism , DNA, Single-Stranded/metabolism , Oxytricha/metabolism , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Animals , DNA, Protozoan/chemistry , DNA, Single-Stranded/chemistry , Electrolytes/pharmacology , Entropy , Lithium Chloride/pharmacology , Models, Molecular , Mutation/genetics , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Telomere/genetics , ThermodynamicsABSTRACT
During the course of a pilot genome project for the ciliate Oxytricha trifallax, we discovered a fusion gene never before described in any taxa. This gene, FSF1, encodes a putative fusion protein comprising an entire formaldehyde dehydrogenase (FALDH) homolog at one end and an S-formylglutathione hydrolase (SFGH) homolog at the other, two proteins that catalyze serial steps in the formaldehyde detoxification pathway. We confirmed the presence of the Oxytricha fusion gene in vivo and detected transcripts of the full-length fusion gene. A survey of other large-scale sequencing projects revealed a similar fusion protein in a distantly related ciliate, Tetrahymena thermophila, and a possible fusion of these two genes in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, but in the reverse order, with the SFGH domain encoded upstream of the FALDH domain. Orthologs of these fusion proteins may be widespread within the ciliates and diatoms.
Subject(s)
Diatoms/genetics , Formaldehyde/metabolism , Genes, Protozoan , Oxytricha/genetics , Amino Acid Sequence , Animals , Diatoms/metabolism , Inactivation, Metabolic , Molecular Sequence Data , Oxytricha/metabolism , Tetrahymena/geneticsABSTRACT
We have isolated an cDNA after applying a DDRT-PCR analysis on mRNA from mature resting cysts of the ciliate Oxytricha (Sterkiella) nova. From this cDNA fragment the complete macronuclear minichromosome was obtained by using the Mac-End-PCR method. After cloning and sequencing, this cDNA shown certain similarity to HMG-like proteins. The analysis of the inferred amino acid sequence shown that this putative HMG-like protein has one HMG-box interrupted by a intron. The analysis of others characteristics (including a 3D model) confirms that it is a HMGB family protein. It is the first time that a macronuclear gene encoding a putative HMG-box protein is isolated from resting cysts of a stichotrich ciliate. The possible implications of this stored mRNA in the ciliate cryptobiotic stage are discussed.
Subject(s)
DNA, Complementary/genetics , DNA, Complementary/isolation & purification , HMGB Proteins/genetics , Oxytricha/metabolism , RNA, Messenger, Stored/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/metabolism , HMGB Proteins/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Oxytricha/cytology , Oxytricha/genetics , RNA, Messenger, Stored/isolation & purification , RNA, Messenger, Stored/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
DNA guanine (G) quadruplexes are stabilized by an interesting variation of the hydrogen-bonding schemes encountered in nucleic acid duplexes and triplexes. In an attempt to use this mode of molecular recognition, we target a dimeric G-quadruplex formed by the Oxytricha nova telomeric sequence d(G(4)T(4)G(4)) with a peptide nucleic acid (PNA) probe having a homologous rather than complementary sequence. UV-vis and CD spectroscopy reveal that a stable hybrid possessing G-quartets is formed between the PNA and DNA. The four-stranded character of the hybrid and the relative orientation of the strands is determined by fluorescence resonance energy transfer (FRET) experiments. FRET results indicate that (i) the two PNA strands are parallel to each other, (ii) the two DNA strands are parallel to each other, and (iii) the 5'-termini of the DNA strands align with the N-termini of the PNA strands. The resulting PNA(2)-DNA(2) quadruplex shows a preference of Na(+) over Li(+) and displays thermodynamic behavior consistent with alternating PNA and DNA strands in the hybrid. The formation of this novel supramolecular structure demonstrates a new high-affinity DNA recognition mechanism and expands the scope of molecular recognition by PNA.
Subject(s)
DNA/chemistry , Peptide Nucleic Acids/chemistry , Animals , Circular Dichroism , DNA/metabolism , G-Quadruplexes , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oxytricha/genetics , Oxytricha/metabolism , Peptide Nucleic Acids/metabolism , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Telomere/genetics , ThermodynamicsABSTRACT
The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.
Subject(s)
DNA, Fungal/metabolism , DNA, Single-Stranded/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Animals , Base Sequence , Binding Sites , Cyclin B/chemistry , Cyclin B/metabolism , DNA, Fungal/chemistry , DNA, Single-Stranded/chemistry , Electrophoretic Mobility Shift Assay , Kinetics , Macromolecular Substances , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Oxytricha/chemistry , Oxytricha/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/isolation & purification , Shelterin Complex , Sodium Chloride/chemistry , Telomere/chemistry , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/isolation & purification , ThermodynamicsABSTRACT
Telomeres are protein-DNA complexes that cap chromosome ends and protect them from being recognized and processed as DNA breaks. Loss of capping function results in genetic instability and loss of cellular viability. The emerging view is that maintenance of an appropriate telomere structure is essential for function. Structural information on telomeric proteins that bind to double and single-stranded telomeric DNA shows that, despite a lack of extensive amino-acid sequence conservation, telomeric DNA recognition occurs via conserved DNA-binding domains. Furthermore, telomeric proteins have multidomain structures and hence are conformationally flexible. A possibility is that telomeric proteins take up different conformations when bound to different partners, providing a simple mechanism for modulating telomere architecture.
Subject(s)
DNA/metabolism , Homeodomain Proteins/metabolism , Oncogene Proteins v-myb/metabolism , Telomere/chemistry , Telomere/metabolism , Animals , DNA-Binding Proteins/metabolism , Oxytricha/chemistry , Oxytricha/metabolismABSTRACT
In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs. which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Developmental , Oxytricha/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , DNA, Protozoan/genetics , DNA, Protozoan/isolation & purification , DNA, Protozoan/metabolism , Molecular Sequence Data , Oxytricha/growth & development , Oxytricha/metabolism , RNA, Messenger/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits alpha and beta on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the alpha subunit. Moreover, the alpha-DNA cross-linked complex was a substrate for telomerase. At higher alpha concentrations, two alpha subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this alpha . DNA . alpha complex may contribute to regulation of telomere length. The alpha . beta . DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of alpha and beta inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.
Subject(s)
DNA, Protozoan/metabolism , DNA-Binding Proteins/metabolism , Oxytricha/metabolism , Protozoan Proteins/metabolism , Telomerase/metabolism , Telomere/metabolism , Animals , DNA Nucleotidylexotransferase/metabolism , DNA, Single-Stranded/metabolism , DNA-Directed DNA Polymerase/metabolism , Electrophoresis, Agar Gel , Macromolecular Substances , Models, Genetic , Oxytricha/genetics , Protein Binding , Protozoan Proteins/antagonists & inhibitors , Repetitive Sequences, Nucleic Acid , Species Specificity , Telomerase/antagonists & inhibitorsABSTRACT
Solution conformations of the alpha and beta subunits of the Oxytricha nova telomere binding protein have been investigated by Raman spectroscopy. Raman spectra have also been obtained for a deletion mutant of the beta subunit, betaC232, which retains the N-terminal domain that is active in ternary complex (alpha:beta:DNA) formation but lacks the C-terminal domain that is active in catalyzing guanine quadruplex formation. The Raman spectra show that alpha, beta, and betaC232 are rich in beta-strand secondary structure ( approximately 40-50%) and turns. The Raman signature of the C-terminal 153 amino acids of beta, generated by subtracting the spectrum of betaC232 (residues 1-232) from that of the full subunit, indicates that the domain active in guanine quadruplex formation contains less beta-strand secondary structure and more irregular structure than the domain active in alpha:beta:DNA formation. Raman markers also provide information about the environments and orientations of several key side chains, including tryptophan residues in N- and C-terminal domains of the beta subunit. Both alpha and beta denature between 30 and 40 degrees C, as evidenced by large changes in Raman bands diagnostic of main chain conformation and side chain environments. The Raman spectrum of an equimolar alpha/beta mixture exhibits no evidence of specific interaction between the subunits; further, the denaturation profile of this mixture is indistinguishable from the sum of denaturation profiles of the constituent subunits, consistent with the absence of appreciable interaction between alpha and beta throughout the range 0-50 degrees C. The present results provide insights into the solution conformations of the Oxytricha telomere binding protein subunits and serve as the basis for future study of subunit interactions with telomeric DNA.
Subject(s)
DNA-Binding Proteins/chemistry , Oxytricha/metabolism , Protein Structure, Secondary , Protozoan Proteins/chemistry , Animals , Drug Stability , Heating , Macromolecular Substances , Mutation , Protein Conformation , Solutions , Spectrum Analysis, Raman , ThermodynamicsABSTRACT
This work presents the further purification of a Cd-linking protein in Oxytricha granulifera by reverse-phase chromatography. This protein contains 25% cysteine and no aromatic amino acid. It may be considered as a chelatin with some similarity to metallothioneins. During the purification of another Cd-linking compound, we were able to demonstrate that the H protein precursor of glycine cleavage is present in Oxytricha. This is the first finding of the presence of this system in Protozoa.
Subject(s)
Cadmium/metabolism , Glycine/metabolism , Metallothionein/isolation & purification , Metallothionein/metabolism , Oxytricha/metabolism , Amino Acid Sequence , Amino Acids/analysis , Animals , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Metallothionein/chemistry , Microscopy, Electron , Molecular Sequence Data , Molecular Weight , Sequence Homology, Amino AcidABSTRACT
In the macronucleus of the ciliate Oxytricha nova, telomeres end with single-stranded (T4G4)2 DNA bound to a heterodimeric telomere protein (alpha beta). Both the alpha and beta subunits (alpha-TP and beta-TP) were phosphorylated in asynchronously growing Oxytricha; beta-TP was phosphorylated to a much higher degree. In vitro, mouse cyclin-dependent kinases (Cdks) phosphorylated beta-TP in a lysine-rich domain that is not required for specific DNA binding but is implicated in higher order structure formation of telomeres. Therefore, phosphorylation of beta-TP could modulate a function of the telomere protein that is separate from specific DNA binding. Phosphoamino acid analysis revealed that the mouse Cdks modify predominantly threonine residues in beta-TP, consistent with the observation that beta-TP contains two consensus Cdk recognition sequences containing threonine residues. In Xenopus egg extracts that undergo cell cycling, beta-TP was phosphorylated in M phase and dephosphorylated in interphase. This work provides the first direct evidence of phosphorylation at telomeres in any organism, as well as indirect evidence for cell cycle regulation of telomere phosphorylation. The Cdc2/cyclin A and Cdc2/cyclin B kinases are required for major mitotic events. An attractive model is that phosphorylation of beta-TP by these kinases is required for the breakdown of telomere associations with each other and/or with nuclear structures prior to nuclear division.
Subject(s)
Oxytricha/metabolism , Telomere/metabolism , Amino Acid Sequence , Animals , Cell Cycle , DNA, Single-Stranded/metabolism , Molecular Sequence Data , Oxytricha/cytology , Phosphorylation , Proteins/chemistryABSTRACT
The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs.