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At least 19 recordsLinked to original sources
PubMed1984

Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA.

The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres. Despite these differences, terminal restriction fragments from O. fallax macronuclear DNA can support telomere formation in yeasts. Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed. One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA. After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O. fallax terminal sequence. In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes. Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA. These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA.

Animals↗
PubMed1998

A hairpin conformation for the 3' overhang of Oxytricha nova telomeric DNA.

The solution secondary structure of the Oxytricha nova telomeric 3' overhang, d(T4G4)2, has been investigated by Raman spectroscopy, hydrogen-deuterium exchange kinetics and gel electrophoresis. The electrophoretic mobility of d(T4G4)2 in non-denaturing gels indicates a highly compact conformation, consistent with a hairpin secondary structure. Raman markers show that the d(T4G4)2 hairpin contains equal numbers of C2'-endo/syn and C2'-endo/anti deoxyguanosine conformers, as well as G.G base-pairs of the Hoogsteen type. The hydrogen-deuterium exchange kinetics of d(T4G4)2, monitored by time-resolved Raman spectroscopy, reveal two kinetically distinct classes of guanine imino (N1H) protons. The more slowly exchanging fraction (kN1H(1)=4.6x10(-3) min-1), which represents 50% of N1H groups, is attributed to Hoogsteen-paired residues. The more rapidly exchanging fraction (kN1H(2)>/=0.3 min-1) is attributable to solvent-exposed residues. Raman dynamic probe of the kinetics of guanine C8H-->C8(2)H exchange in d(T4G4)2 reveals modest retardation vis-à-vis dGMP, which rules out quadruplex formation by the telomeric repeat and confirms an ordered secondary structure consistent with a Hoogsteen-paired hairpin. Similar Raman, hydrogen-isotope exchange and electrophoretic mobility experiments on the related telomeric model, dT6(T4G4)2, also reveal a hairpin stabilized by Hoogsteen G.G pairs. Presence of the 5' thymidine tail preceding the Oxytricha telomeric repeat has no apparent effect on the hairpin secondary structure. We propose a molecular model for the hairpin conformation of the Oxytricha nova telomeric repeat and consider its possible roles in mechanisms of telomeric DNA interaction in vitro and telomere function in vivo.

Animals↗
PubMed1999

Molecular mechanism of DNA recognition by the alpha subunit of the Oxytricha telomere binding protein.

Interactions between telomeric DNA and the alpha subunit of the heterodimeric telomere binding protein of Oxytricha nova have been probed by Raman spectroscopy, CD spectroscopy, and nondenaturing gel electrophoresis. Telomeric sequences investigated include the Oxytricha 3' overhang, d(T4G4)2, and the related sequence dT6(T4G4)2, which incorporates a 5'-thymidylate leader. Corresponding nontelomeric isomers, d(TG)8 and dT6(TG)8, have also been investigated. Both d(T4G4)2 and dT6(T4G4)2 form stable hairpins that contain Hoogsteen G.G base pairs [Laporte, L., and Thomas, G. J., Jr. (1998) J. Mol. Biol. 281, 261-270]. The alpha subunit binds specifically and stoichiometrically to the dT6(T4G4)2 hairpin and alters its secondary structure by inducing conformational changes in the 5'-thymidylate leader without extensive disruption of G.G base pairing. Conversely, binding of the alpha subunit to d(T4G4)2 eliminates G.G pairing and unfolds the hairpin. DNA unfolding is accompanied by conformational changes affecting both the backbone and dG residues, as evidenced by Raman and CD spectra. Interestingly, the alpha subunit also forms complexes with the nontelomeric isomers, d(TG)8 and dT6(TG)8, evidenced by altered electrophoretic mobility in nondenaturing gels; however, Raman and CD spectra of complexes of the alpha subunit with nontelomeric DNA suggest no significant changes in backbone or deoxynucleoside conformations. Similarly, the alpha subunit binds to but does not appreciably alter the secondary structure of duplex DNA. The present results show that while the alpha subunit has the capacity to bind to Watson-Crick and different non-Watson-Crick motifs, DNA refolding is specific to the Oxytricha telomeric hairpin and the retention of G.G pairing is specific to the telomeric sequence incorporating the 5' leading sequence. A model is proposed for alpha subunit binding to telomeric DNA, and the physiological role of the alpha subunit in telomere organization is discussed.

Animals↗
PubMed1995

Scrambling of the actin I gene in two Oxytricha species.

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.

Actins↗
PubMed2002

Secondary structure polymorphism in Oxytricha nova telomeric DNA.

Tandem repeats of the telomeric DNA sequence d(T4G4) of Oxytricha nova are capable of forming unusually stable secondary structures incorporating Hoogsteen hydrogen bonding interactions. The biological significance of such DNA structures is supported by evidence of specific recognition of telomere end-binding proteins in the crystal state. To further characterize structural polymorphism of Oxytricha telomeric DNAs, we have obtained and interpreted Raman, ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectra of the tandem repeats d(G4T4G4) (Oxy1.5), d(T4G4)2 (Oxy2) and dT6(T4G4)2 (T6Oxy2) and related non-telomeric isomers in aqueous salt solutions. Raman markers of Oxy1.5 identify both C2'-endo/anti and C2'-endo/syn conformations of the deoxyguanosine residues and Hoogsteen hydrogen bonded guanine quartets, consistent with the quadruplex fold determined previously by solution NMR spectroscopy. Raman, UVRR and CD signatures and Raman dynamic measurements, to monitor imino NH-->ND exchanges, show that the Oxy1.5 antiparallel quadruplex fold is distinct from the hairpin structures of Oxy2 and T6Oxy2, single-stranded structures of d(TG)8 and dT6(TG)8 and previously reported quadruplex structures of d(T4G4)4 (Oxy4) and dG12. Spectral markers of the telomeric and telomere-related DNA structures are tabulated and novel Raman and UVRR indicators of thymidine and deoxyguanosine conformations are identified. The results will be useful for probing structures of Oxytricha telomeric repeats in complexes with telomere end-binding proteins.

Animals↗
PubMedDate not supplied

Not-adaptive behavior of isotropically heated, inert populations of Oxytricha bifaria (Ciliophora, Stichotrichia).

The physiological effects on isotropically heated populations of Oxytricha bifaria cultured at 24 degrees C were investigated. At 34.6 degrees C ciliates became inert, and did not adaptively react to either cold or warm microgradients; they neither moved towards the favorable cold thermal source nor escaped from the unfavorable warm one. The inert oxytrichas were only able to perform the Side-Stepping Reaction (SSR) on the same spot. However, mobile ciliates at 31.6 degrees C reacted to the cold microgradient by immediately orienting themselves towards its source, without accelerating but reducing their SSR frequency. Moreover, in a warm microgradient such ciliates immediately increased their SSR frequency, then moved away from the thermal source. At 34.6 degrees C the behavior of ciliates was not-adaptive--not acting to guide the organisms to more favorable conditions--whereas at 31.6 degrees C it was still clearly adaptive. Therefore, the locomotory inertness of the oxytrichas at 34.6 degrees C was the result of thermal stress rather than their behavioral response to the environmental isotropy, in contrast to populations of the same species made inert at 9 degrees C.

Adaptation, Physiological↗
PubMed1991

Molecular evolution in hypotrichous ciliates: sequence of the small subunit ribosomal RNA genes from Onychodromus quadricornutus and Oxytricha granulifera (Oxytrichidae, Hypotrichida, Ciliophora).

The small subunit ribosomal RNA (16S-like rRNA) coding regions of the hypotrichous ciliates Onychodromus quadricornutus and Oxytricha granulifera were amplified using polymerase chain reaction techniques. Complete sequences were determined for the amplified genes and compared to those of other ciliated protozoa. In phylogenetic trees inferred using distance matrix methods oxytrichids are not seen as a cohesive phylogenetic group. Oxytricha nova is most closely related to Stylonychia pustulata in a lineage that also includes O. quadricornutus. This phylogeny contradicts phylogenetic schemes in which Onychodromus is considered to be a primitive hypotrichous ciliate and suggests that O. nova was misidentified as members of the genus Oxytricha.

Animals↗
PubMed1975

A very rapidly migrating f1 histone associated with gene-sized pieces of DNA in the macronucleus of Oxytricha sp.

Histones bound to gene-sized pieces of DNA in the macronucleus of Oxytricha sp. consist of five main fractions: f1, f2a1, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxytricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f1 fraction differs. Oxytricha f1 is unique among eukaryotes in that it migrates faster than f2a1 and is located nearest the cathode in polyacrylamide-urea gels.

Amino Acids↗
PubMed1988

Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules.

We have found abundant telomere-specific terminal transferase activity in crude macronuclear extracts from vegetatively growing cells of the hypotrichous ciliate Oxytricha nova. This activity adds two to seven tandem repeats of the sequence GGGGTTTT (the Oxytricha telomeric repeat) to the 3' end of oligonucleotide primers ending in repeats of G4T4 and always adds the repeats in the proper phase. The activity requires the presence of micromolar amounts of dGTP and dTTP as well as single-stranded oligomer primers ending 3' with repeats of the Oxytricha telomeric sequence. A nuclease activity is present in the extracts which is closely balanced with telomere terminal transferase activity. We propose a simple model for replication of the ends of linear DNA molecules based on the telomere terminal transferase.

Animals↗
PubMed1993

Developmental precise excision of Oxytricha trifallax telomere-bearing elements and formation of circles closed by a copy of the flanking target duplication.

The 4.1 kbp TBE1 elements of Oxytricha fallax and Oxytricha trifallax are deduced to transpose into a centrisymmetric target, CAnTG, and to duplicate the central AnT. Despite conserved C(A4C4)2 telomeric repeats at their tips, free TBE1s found during macronuclear development are not linear but 4.1 kbp circles closed on one copy of the AnT target duplication. The macronucleus-destined flanks are rejoined to regenerate the target, effecting efficient and precise somatic reversion of the germline transpositional mutation. A model is presented in which transposase catalyzes concerted precise rejoining of the flanks and cyclization of the excised element.

Animals↗
PubMed1995

Solution structure of the Oxytricha telomeric repeat d[G4(T4G4)3] G-tetraplex.

The solution structure of Oxytricha telomere sequence d[G4(T4G4)3] in 0.1 M Na+ containing solution has been determined using a combined NMR-molecular dynamics approach including relaxation matrix refinement. This four G4 repeat sequence folds intramolecularly into a right-handed G-tetraplex containing four stacked G-tetrads which are connected by two lateral T4 loops and a central diagonal T4 loop. The guanine glycosidic bonds adopt a syn-anti alternation along the full length of the d[G4(T4G4)3] sequence while the orientation around adjacent G-tetrads switches between syn.syn.anti.anti and anti.anti.syn.syn alignments. Four distinct grooves are formed by the parallel (two of medium width) and anti-parallel (one wide and one narrow width) alignment of adjacent G-G-G-G segments in the G-tetraplex. The T4 residues in the diagonal loop are well-defined while the T4 residues in both lateral loops are under-defined and sample multiple conformations. The solution structure of the Na(+)-stabilized Oxytricha d[G4(T4G4)3] G-tetraplex and an earlier solution structure reported from our laboratory on the Na(+)-stabilized human d[AG3(T2AG3)3] G-tetraplex exhibit a common folding topology defined by the same syn/anti distribution of guanine residues along individual strands and around individual G-tetrads, as well as a common central diagonal loop which defines the strand directionalities. The well-resolved proton NMR spectra associated with the d[G4(T4G4)3] G-tetraplex opens the opportunity for studies ranging from cation-dependent characterization of G-tetraplex conformation and hydration to ligand and protein recognition of the distinct grooves associated with this folding topology.

Animals↗
PubMed2001

Crystal structure of the N-terminal domain of Oxytricha nova telomere end-binding protein alpha subunit both uncomplexed and complexed with telomeric ssDNA.

Oxytricha nova telomere end-binding protein specifically recognizes and caps single strand (T(4)G(4))(n) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. Proteins homologous to the N-terminal domain of OnTEBP alpha subunit have now been identified in Oxytricha trifallax, Stylonychia mytilis, Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens, suggesting that this protein is widely distributed in eukaryotes. We describe here the crystal structures of the N-terminal single-stranded DNA (ssDNA)-binding domain of O. nova telomere end-binding protein alpha subunit both uncomplexed and complexed with single strand telomeric DNA. These structures show how the N-terminal domain of alpha alone, in the absence of the beta subunit and without alpha dimerization, can bind single-stranded telomeric DNA in a sequence-specific and 3'-end-specific manner. Furthermore, comparison of the uncomplexed and complexed forms of this protein shows that the ssDNA-binding site is largely pre-organized in the absence of ssDNA with modest, but interesting, rearrangements of amino acid side-chains that compose the ssDNA-binding site. The structures described here extend our understanding of structures of O. nova telomeric complexes by adding uncomplexed and complexed forms of monomeric alpha to previously described structures for (alpha 56/ssDNA)(2) dimer and alpha 56/beta 28/ssDNA ternary complexes. We believe that each of these four structures represent intermediates in an ordered assembly/disassembly pathway for O. nova telomeric complexes.

Animals↗
PubMed2004

Coding properties of Oxytricha trifallax (Sterkiella histriomuscorum) macronuclear chromosomes: analysis of a pilot genome project.

The macronuclear genomes of spirotrichous ciliates are almost entirely polyploid, single-gene chromosomes ("nanochromosomes"). We recently performed a pilot genome project for a member of this group, Oxytricha trifallax ( Sterkiella histriomuscorum), in which approximately 2000 nanochromosomes were cloned at random and end-sequenced. Here we describe the global properties of the coding regions predicted for these molecules, including nucleotide composition, codon usage, and intron properties. In identifying splice donor, acceptor and branch sites, we found that longer introns in Oxytricha have a stronger signal at the donor site than do smaller introns, as has been found for Caenorhabditis elegans and Drosophila, despite the overall small size of the introns. A systematic search for multi-gene chromosomes identified 11 candidate nanochromosomes. We compare the results from this large dataset with those obtained from earlier studies and with statistics recorded from ciliates and other eukaryotes.

Animals↗
PubMed1993

The beta subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA.

Telomeres, the ends of linear chromosomes, typically consist of tandem repeats of simple G-rich sequences. At high concentrations, single-stranded telomeric DNA can form dimers and tetramers involving G-quartets. We show that under physiological conditions, the beta subunit of the Oxytricha telomere-binding protein greatly accelerates G-quartet formation. The reaction occurs with oligonucleotides ending in the Oxytricha (T4G4T4G4) and Tetrahymena (T2G4T2G4) telomeric sequences; the sequence preceding these telomeric repeats can be nontelomeric, single-, or double-stranded. Protein deletion analysis indicates that the carboxy-terminal highly basic domain of the beta subunit, which is dispensible for telomeric complex formation, is sufficient for mediating G-quartet formation. The finding that a telomeric protein acts as a molecular chaperone for G-quartet formation provides a strong argument that such DNA structures exist in vivo at chromosome telomeres.

Animals↗
PubMed1994

Refined solution structure of the dimeric quadruplex formed from the Oxytricha telomeric oligonucleotide d(GGGGTTTTGGGG).

BACKGROUND: Telomeres, the structures at the ends of linear eukaryotic chromosomes, are essential for chromosome replication and stability. The telomeres of the unicellular ciliate Oxytricha contain a 3' single strand overhang composed of two repeats of the telomere repeat sequence d(TTTTGGGG). It has been proposed that oligonucleotides containing this repeat can form DNA quadruplexes via hydrogen bonding of the guanines into quartets. Such structures may be relevant to the biological function of the telomere, and in G-rich sequences elsewhere in the genome. RESULTS: We have previously determined from solution NMR data that the Oxy-1.5 Oxytricha repeat oligonucleotide d(GGGGTTTTGGGG) dimerizes to form an intermolecular quadruplex composed of four guanine quartets and with the thymines in loops across the diagonal at opposite ends of the quadruplex. We report here the refined solution structure of Oxy-1.5. This structure is compared with the previously published crystal structure of the same oligonucleotide. CONCLUSIONS: Oxy-1.5 forms a well-defined, symmetrical structure with ordered thymine loops. Both the solution and crystal structures of Oxy-1.5 are quadruplexes with alternating syn and anti glycosyl conformation of guanines along each strand of the helix and have thymine loops at opposite ends. However, the topology of the two structures is fundamentally different, leading to significant structural differences. A topological pathway for the formation and interconversion of the two structures is proposed.

Animals↗
PubMed1997

Solution conformations and interactions of alpha and beta subunits of the Oxytricha nova telomere binding protein: investigation by Raman spectroscopy.

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.

Animals↗
PubMed1998

Structural basis of DNA recognition and mechanism of quadruplex formation by the beta subunit of the Oxytricha telomere binding protein.

Interactions of the beta subunit of the Oxytricha nova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the beta subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes the formation of a parallel-stranded quadruplex only in dT6(T4G4)2, thus demonstrating the importance of the telomeric 5' tail for in vitro recognition and guanine quadruplex formation. While d(T4G4)2 is not a suitable substrate for quadruplex promotion by the beta subunit, the Raman spectra reveal other structural rearrangements of this DNA strand upon beta subunit binding, including changes in guanine glycosyl torsion angles from syn to anti and disruption of carbonyl hydrogen-bonding interactions. The conformation of d(T4G4)2 in the beta:d(T4G4)2 complex is suggested as a plausible intermediate along the pathway to formation of the parallel-stranded guanine quadruplex. Fluorescence band shifts indicate that at least one of the two tryptophans of the beta subunit is shielded from solvent as a consequence of DNA binding in both the beta:dT6(T4G4)2 and beta:d(T4G4)2 complexes. However, the Raman spectra of these complexes suggest no significant changes in the beta subunit secondary structure attendant with DNA binding. A model for beta subunit binding by Oxytricha telomeric DNA sequences and a mechanism for quadruplex formation are proposed. A key feature of this model is the use of a telomeric hairpin secondary structure as the recognition motif.

Animals↗
PubMed1994

DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases.

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.

Animals↗