Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.     

Aminoglycoside binding to Oxytricha nova telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ～ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.     

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.     

Scrambled actin I gene in the micronucleus of Oxytricha nova.

In the hypotrichous ciliate Oxytricha nova the cloned precursor gene from the micronuclear genome that encodes actin I is composed of highly disordered blocks of deoxynucleotide sequences. We present and illustrate in detail a recombination model that explains how the actin I gene may be unscrambled during macronuclear development after cell mating. The model was described in a previous publication (Greslin et al.: Proc Natl Acad Sci USA 86:6264-6268, 1989). Here we show the data, described in the earlier publication, that support the model. The data show that scrambling is not an artifact of cloning. They rule against the presence of an unscrambled copy of the actin I gene in the micronucleus, which means that unscrambling must be a part of macronuclear development. Finally, the data prove that the actin I gene in O. trifallax is scrambled in a pattern that resembles the pattern in O. nova.     

Coding properties of macronuclear DNA molecules in Sterkiella nova (Oxytricha nova)

The DNA in the macronucleus of the stichotrichs like Sterkiella nova (formerly Oxytricha nova) occurs in short molecules ranging from approximately 200 bp to approximately 20,000 bp. It has been estimated that there are approximately 24,500 different sized DNA molecules in the macronucleus. Single genes have been assigned to approximately 130 different sized macronuclear molecules in various stichotrichs (12 in Sterkiella nova) and hypotrichs, suggesting that each of the -24,500 different sized molecules encodes a different gene. To test this proposition we sequenced 31 macronuclear molecules picked randomly from a plasmid library of macronuclear DNA and analyzed them for potential gene content. The open reading frames (ORFs) in three short molecules encode amino acid (aa) sequences that do not match sequences in GenBank. They may or may not encode genes. Twenty-eight of the 31 molecules contain ORFs encoding aa sequences with significant matches to sequences in GenBank. Six molecules contain more than one ORF with a significant match to GenBank. These results indicate that almost all, if not all of the -24,500 different molecules encode one or more genes, yielding an estimate of -26,800 genes in the macronucleus of S. nova.     

DNA primase and the replication of the telomeres in Oxytricha nova.

An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n. Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end. The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end. With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template. A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA. The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin. Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template. This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA. The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E. coli DNA polymerase I Klenow fragment. Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Evolution of internal eliminated segments and scrambling in the micronuclear gene encoding DNA polymerase alpha in two Oxytricha species.

To learn about the evolution of internal eliminated segments (IESs) and gene scrambling in hypotrichous ciliates we determined the structure of the micronuclear (germline) gene encoding DNA polymerasealpha(DNA polalpha) in Oxytricha trifallax and compared it to the previously published structure of the germline DNA polalphagene in Oxytricha nova . The DNA polalphagene of O.trifallax contains 51 macronuclear-destined segments (MDSs) separated by 50 IESs, compared to 45 MDSs and 44 IESs in the O.nova gene. This means that IESs and MDSs have been gained and/or lost during evolutionary divergence of the two species. Most of the MDSs are highly scrambled in a similar non-random pattern in the two species. We present a model to explain how IESs, non-scrambled MDSs and scrambled MDSs may be added and/or eliminated during evolution. Corresponding IESs in the two species differ totally in sequence, and junctions between MDSs and IESs are shifted by 1-18 bp in O.trifallax compared to the O.nova gene. In both species a short region of the gene is distantly separated from the main part of the gene. Comparison of the gene in the two species shows that IESs and scrambling are highly malleable over evolutionary time.     

The germline gene encoding DNA polymerase alpha in the hypotrichous ciliate Oxytricha nova is extremely scrambled.

We report the structure of the micronuclear (germline) gene encoding the large catalytic subunit of DNA polymerase alpha (DNA pol alpha) in the ciliate Oxytricha nova. It contains 44 internal eliminated segments (IESs) that divide the gene into 45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence, are missing from the 14 kb cloned gene. The missing MDSs have not been located but are at least several kilobases from the main body of the gene. The remarkably scrambled DNA pol alpha gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled, functional version of the gene during development of a new macronucleus. Unscrambling is hypothesized to occur by a homologous recombination mechanism guided by repeat sequences at MDS ends.     

Elimination of micronuclear specific DNA sequences early in anlagen development.

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.     

DNA G-quartets in a 1.86 A resolution structure of an Oxytricha nova telomeric protein-DNA complex.

The Oxytricha nova telomere end binding protein (OnTEBP) recognizes, binds and protects the single-stranded 3'-terminal DNA extension found at the ends of macronuclear chromosomes. The structure of this complex shows that the single strand GGGGTTTTGGGG DNA binds in a deep cleft between the two protein subunits of OnTEBP, adopting a non-helical and irregular conformation. In extending the resolution limit of this structure to 1.86 A, we were surprised to find a G-quartet linked dimer of the GGGGTTTTGGGG DNA also packing within the crystal lattice and interacting with the telomere end binding protein. The G-quartet DNA exhibits the same structure and topology as previously observed in solution by NMR with diagonally crossing d(TTTT) loops at either end of the four-stranded helix. Additionally, the crystal structure reveals clearly visible Na(+), and specific patterns of bound water molecules in the four non-equivalent grooves. Although the G-quartet:protein contact surfaces are modest and might simply represent crystal packing interactions, it is interesting to speculate that the two types of telomeric DNA-protein interactions observed here might both be important in telomere biology.     

Internal sequences are eliminated from genes during macronuclear development in the ciliated protozoan Oxytricha nova

During the life cycle of the hypotrichous ciliate Oxytricha nova, a macronucleus containing short, gene-sized DNA molecules is produced from a copy of the chromosomal micronuclear genome. In order to characterize the process of macronuclear development, we have isolated and determined the DNA sequence of a particular macronuclear gene and its micronuclear precursor. The results of this analysis indicate that macronuclear telomeric sequences (5'C4A4(3') repeats) are not present at the ends of the gene in its micronuclear chromosomal location and must be added during development. In addition, the micronuclear copy of the gene contains three short blocks of sequence that must be removed during development, implying the involvement of a nucleic acid-splicing process in generating mature macronuclear genes.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Thermodynamic and electrostatic properties of ternary Oxytricha nova TEBP-DNA complex

Telomeres constitute the nucleoprotein ends of eukaryotic chromosomes which are essential for their proper function. Telomere end binding protein (TEBP) from Oxytricha nova was among the first telomeric proteins, which were well characterized biologically. TEBP consists of two protein subunits (alpha, beta) and forms a ternary complex with single stranded telomeric DNA containing tandem repeats TTTTGGGG. This work presents the characterization of the thermodynamic and electrostatic properties of this complex by computational chemistry methods (continuum Poisson-Boltzmann and solvent accessible surface calculations). Our calculations give a new insight into molecular properties of studied system. Based on the thermodynamic analysis we provide a rationale for the experimental observation that alpha and ssDNA forms a binary complex and the beta subunit joins alpha:ssDNA complex only after the latter is formed. Calculations of distribution of the molecular electrostatic potential for protein subunits alone and for all possible binary complexes revealed the important role of the "guiding funnel" potential generated by alpha:ssDNA complex. This potential may help the beta subunit to dock to the already formed alpha:DNA intermediate in highly steric and electrostatic favorable manner. Our pK(a) calculations of TEBP are able to explain the experimental mobility shifts of the complex in electrophoretic non-denaturating gels.