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.     

Polytene chromosomes and nucleic acid metabolism during macronuclear development in Euplotes

Polytene chromosomes in two species of Euplotes, E. woodruffi and E. eurystomus, have been described during the macronuclear development following conjugation. In these two species, the giant chromosomes appear briefly in the macronuclear anlagen and disappear completely later. DNA synthesis begins concomitantly with the appearance of the giant chromosomes and reaches a peak at the maximum stage of polyteny. Shortly thereafter DNA begins to break down and the breakdown products leave the macronuclear anlagen, reducing the DNA content in the anlagen to the amount present at the earlier stages of the polytene development of the chromosomes. A later phase of DNA synthesis occurs in the anlagen with the appearance of replication bands comparable to the bands which double the DNA in the somatic macronucleus. These replication bands initiate several rounds of DNA synthesis which finally lead to the development of the vegetative macronucleus. RNA synthesis occurs uniformly on the giant chromosomes and no special RNA producing puffs or other regions are noticed on them.Research supported by American Cancer Society grant E 434 to David M. Prescott and by the Deutsche Forschungsgemeinschaft to Dieter Ammermann.     

Ciliary protein conservation during development in the ciliated protozoan, Oxytricha

The ciliated protozoan Oxytricha fallax possesses multiple highly localized clusters of basal bodies and cilia, all of which are broken down and rebuilt during prefission morphogenesis-with one major exception. The adoral zone of membranelles (AZM) of the ciliate oral apparatus contains approximately 1,500-2,000 basal bodies and cilia, and it is the only compound ciliary structure that is passed morphologically intact to one daughter cell at each cell division. By labeling all proteins in cells, and then picking the one daughter cell possessing the original labeled AZM, we could then evaluate whether or not the ciliary proteins of the AZM were diluted (i.e., either by degradation to constituent amino acids or by subunit exchange) during cell division. Autoradiographic analysis demonstrated that the label was highly conserved in the AZM (i.e., we saw no evidence of turnover), and electrophoretic data illustrate that at least one of the proteins of the AZM is tubulin. We, therefore, conclude that for at least some of the ciliary and basal body proteins of Oxytricha fallax, AZM morphological conservation is essentially equivalent to molecular conservation.     

Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its micronucleus, which contains chromosome-sized DNA, into a macronucleus containing linear, gene-sized DNA molecules. A region of the micronuclear genome has been defined that gives rise to two distinct macronuclear DNA molecules during development. Through analysis of recombinant macronuclear and micronuclear clones, the generation of the two macronuclear DNA molecules was shown to be the result of alternative use of chromosome fragmentation sites. In addition, evidence was obtained that adjacent micronuclear precursors of macronuclear DNA molecules can overlap by a few base pairs. The significance of these findings in relation to developmental chromosome fragmentation is discussed.     

DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of oxytricha

We have measured the reassociation kinetics of DNA from the micronucleus and from the macronucleus of the hypotrichous cillate Oxytricha. The micronuclear DNA reassociates with at least a two-component reaction, indicating the presence of both repeated and non-repeated sequences. The kinetic complexity of micronuclear non-repeated DNA is in the range of 2 to 15 × 10¹¹ daltons; the haploid DNA content of the micronucleus is 4 × 10¹¹ daltons (0.66 pg), measured microspectrophotometrically. The DNA of the macronucleus reassociates as a single second-order reaction, with a kinetic complexity of 3.6 × 10¹⁰ daltons. A comparison of the kinetic complexities of micronuclear and macronuclear DNAs suggest a 5 to 30 fold reduction in DNA sequence complexity during the formation of a macronucleus from a micronucleus. Macronuclear DNA is in pleces with an average molecular weight of 2.1 × 10⁶ daltons. Since the kinetic complexity of macronuclear DNA is 3.6 × 10¹⁰ daltons, the macronucleus must contain about 17,000 different kinds of DNA pieces.Each macronucleus contains 3.5 × 10¹³ daltons (58 pg) of DNA, indicating that each sequence must be present about 1000 times per macronucleus or 2000 times per cell.     

Copy number variations of 11 macronuclear chromosomes and their gene expression in Oxytricha trifallax

Genome-wide methylation studies frequently lack adequate controls to estimate proportions of background reads in the resulting datasets. To generate appropriate control pools, we developed technique termed nMETR (non-methylated tag recovery) based on digestion of genomic DNA with methylation-sensitive restriction enzyme, ligation of adapter oligonucleotide and PCR amplification of non-methylated sites associated with genomic repetitive elements. The protocol takes only two working days to generate amplicons for deep sequencing. We applied nMETR for human DNA using BspFNI enzyme and retrotransposon Alu-specific primers. 454-sequencing enabled identification of 1113 nMETR tag sites, of them ~65% were parts of CpG islands. Representation of reads inversely correlated with methylation levels, thus confirming nMETR fidelity. We created software that eliminates background reads and enables to map and annotate individual tags on human genome. nMETR tags may serve as the controls for large-scale epigenetic studies and for identifying unmethylated transposable elements located close to genomic CpG islands.     

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.     

Distribution of a centrosomal antigen during morphogenesis in the ciliated protozoan Euplotes

Ciliates assemble basal bodies in great number at many stages of the life-cycle. In order to understand their assembly mechanisms, we screened a library of monoclonal antibodies directed against pericentriolar material. One of these antibodies, CTR210, was used previously to follow steps of this assembly process: in Paraurostyla, new basal bodies appear along a scaffold of linear structures recognized by this antibody. The very unusual behavior of this antigen deserved confirmation in other species. In the present study, we show by immunofluorescence that, in another phylogenetically very distant species, Euplotes, basal bodies are assembled in the same pathway during division. In addition, this antibody recognizes a filamentous ring located at the division furrow and linking many basal body assemblages. By cell fractionation and cytoskeletal extraction, we obtained fractions enriched in basal bodies and associated material. Such fractions still display a high complexity in protein composition. These fractions were used to characterize the main target of the antibody as a doublet of 45 kDa. These results confirm previous results in terms of functionality of the protein recognized by the antibody, but raise new questions in terms of the assignment of the recognized protein to the HSP70 family as hypothesized previously.     

Secretory organelle biogenesis: Trichocyst formation in a ciliated protozoan

Summary— By classical electron microscopy and immunoelectron microscopy, the biogenesis of trichocyst secretory granules has been followed in the ciliated protozoan Pseudomicrothorax dubius. The very early pre-trichocysts form by fusion of bristle-coated, electron-dense vesicles (dense vesicles) with electron-translucent vesicles (clear vesicles), both of which originate in a well-developed trans-Golgi network (TGN). The pre-trichocyst grows by further fusion with dense and clear vesicles as well as with other pre-trichocysts until it reaches its maximum diameter of about 2 μm. Dense and clear vesicle formation from the TGN has been followed, and the fusion sequence of dense vesicles with the pre-trichocyst has been documented. The contents of the dense vesicles are the precursors of the trichocyst tip, which is composed of four arm-like rods, whereas the shaft precursors are supplied by the clear vesicles. The first evidence of trichocyst shaft formation is the appearance of a paracrystalline, dense core condensation center in the pre-trichocyst. Following shaft formation, the trichocyst tip forms by fusion and condensation of the dense arm precursors along each of the four sides of the shaft. Docking of the fully formed trichocyst in the cell cortex is described. Pre-trichocyst biogenesis in cells grown with and without Se is compared.     

The development of the macronucleus in the ciliated protozoan Stylonychia mytilus

Ciliated protozoa are characterized by generative micronuclei and vegetative polyploid macronuclei. Micronuclei of Stylonychia mytilus contain 1 600 times as much DNA per haploid genome as E. coli. Most of this DNA is shown to be repetitive. The development of the macronucleus involves, as demonstrated by cytology, only 1/3 of the chromosomes which in a first replication phase are polytenized in probably 5 replication steps and appear as giant chromosomes. At this developmental stage considerable amounts of repetitive DNA are still present in the chromosomes. During the subsequent disintegration phase more than 90% of the DNA are eliminated from the macronucleus anlage. The remainder is further replicated five times and composes the final macronucleus. Since this DNA reassociates with a reaction rate almost identical to an ideal second order reaction its kinetic complexity can be determined by comparison with the kinetic complexity of E. coli DNA. Macronuclear DNA reassociates with a kinetic complexity of 26 times the kinetic complexity of E. coli DNA (corrected for GC content) which indicates that macronuclear DNA sequences exist at a ploidy level of 4 096 C. We assume that macronuclear DNA may be present only once per haploid genome. In this case it represents only 1.6% of the DNA in micronuclei or 10% of the DNA in the giant chromosome stage.     

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.     

Telomere formation on macronuclear chromosomes of Oxytricha trifallax and O. fallax : alternatively processed regions have multiple telomere addition sites

Background  

Ciliates employ massive chromatid breakage and de novo telomere formation during generation of the somatic macronucleus. Positions flanking the 81-MAC locus are reproducibly cut. But those flanking the Common Region are proposed to often escape cutting, generating three nested macronuclear chromosomes, two retaining "arms" still appended to the Common Region. Arm-distal positions must differ (in cis ) from the Common Region flanks.     

Three different macronuclear DNAs in Oxytricha fallax share a common sequence block.

The three members of a cross-hybridizing family of macronuclear DNAs (4,890, 2,780, and 1,640 base pairs) from the protozoan Oxytricha fallax have in common a conserved sequence block 1,300 to 1,550 base pairs long. Adjacent to the common block in the two larger DNAs are sequences which are unique to them, whereas the smallest DNA contains few if any additional sequences. The family reappears when the macronucleus is replaced after conjugation and can be detected in another O. fallax subspecies. In a random collection of cloned macronuclear DNAs, 6 of 15 hybridize to macronuclear DNA families. This high frequency suggests that families sharing common sequence blocks have an important role in macronuclear function.     

Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex

Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented. A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends. Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule. We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can be sufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA.