Large scale synchronous mating and the study of macronuclear development in Euplotes crassus

After conjugation in hypotrichous ciliates, a new macronucleus is produced from a copy of the micronucleus. This transformation involves large-scale reorganization of DNA, with conversion of the chromosomal micronuclear genome into short, gene-sized DNA molecules in the macronucleus. To study directly the changes that occur during this process, we have developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus. Electron microscope studies show that the micronuclear chromosomes are polytenized during the first 20 h of macronuclear development. The polytene chromosomes lack the band-interband organization observed in other hypotrichs and in the Diptera. Polytenization is followed by transectioning of the chromosomes. We isolated DNA at various times of macronuclear development and found that the average molecular weight of the DNA decreases at the time of chromosome transectioning. In addition, we have shown that a small size group of macronuclear DNA molecules (450-550 base pairs) is excised from the chromosomal DNA approximately 10 h later in macronuclear development.     

Genome-wide characterization of Tetrahymena thermophila chromosome breakage sites. II. Physical and genetic mapping

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps.     

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.     

DNA intermediates and telomere addition during genome reorganization in Euplotes crassus

Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macronuclear development in the hypotrich Euplotes crassus. These molecules occur in integrated forms within polytene chromosomal DNA during macronuclear developmental. After transection of the polytene chromosomes, the three molecules occur in intermediate forms. One of the three molecules first appeared in a large intermediate that was subsequently replaced by a second intermediate, approximately 140 bp larger than the final molecule. The other two macronuclear molecules were detected only in intermediates approximately 140 bp larger than the mature form. These penultimate intermediates are larger by virtue of oversized telomeres, which are pared to yield the mature gene-sized molecules.     

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

A Chimeric Chromosome in the Ciliate Oxytricha Resulting from Duplication

In a process similar to exon splicing, ciliates use DNA splicing to produce a new somatic macronuclear genome from their germline micronuclear genome after sexual reproduction. This extra layer of DNA rearrangement permits novel mechanisms to create genetic complexity during both evolution and development. Here we describe a chimeric macronuclear chromosome in Oxytricha trifallax constructed from two smaller macronuclear chromosomes. To determine how the chimera was generated, we cloned and sequenced the corresponding germline loci. The chimera derives from a novel locus in the micronucleus that arose by partial duplication of the loci for the two smaller chromosomes. This suggests that an exon shuffling-like process, which we call MDS shuffling, enables ciliates to generate novel genetic material and gene products using different combinations of genomic DNA segments.     

Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development.

After mating, hypotrichous ciliated protozoa transform a set of their micronuclear chromosomes into thousands of short, linear DNA molecules that form the macronuclear genome. To examine micronuclear genome organization in the hypotrich Euplotes crassus, we have analyzed two cloned segments of micronuclear DNA as well as the macronuclear DNA molecules that are derived from them. E. crassus was found to display a number of features characteristic of other hypotrich genomes, including (i) clustering and close spacing of the precursors of macronuclear DNA molecules, (ii) the frequent occurrence of internal eliminated sequences within macronuclear precursors, (iii) overlapping macronuclear precursors, (iv) lack of telomeric repeats at the ends of macronuclear precursors, and (v) alternative processing of the micronuclear chromosome to yield multiple macronuclear DNA molecules. In addition, a moderately repetitive, transposonlike element that interrupts the precursors of two macronuclear DNA molecules has been identified and characterized. This transposonlike element, designated Tec1, is shown to be reproducibly removed from one of the macronuclear precursors during independent episodes of macronuclear development.     

Variation in macronuclear genome content of three ciliates with extensive chromosomal fragmentation: a preliminary analysis

The genome architecture of ciliates, including features such as nuclear dualism and large-scale genome rearrangements, impacts gene and genome evolution in these organisms. To better understand the structure of macronuclear chromosomes in ciliates with extensively processed chromosomes, a sample of complete macronuclear chromosomes was sequenced from three ciliate species: Metopus es (Class [Cl]: Armophorea), Nyctotherus ovalis (Cl: Armophorea), and Chilodonella uncinata (Cl: Phyllopharyngea). By cloning whole macronuclear chromosomes into a plasmid vector, we generated nine clones from each of M. es and C. uncinata, and 37 clones from N. ovalis. Analysis of these macronuclear chromosomes provides insight into the evolution of genome features such as chromosome content, gene structure, and genetic code. Phylogenetic patterns can be found in telomere structure and codon usage, which are both more similar in M. es and N. ovalis than C. uncinata. In addition, we provide evidence of lateral transfer of a bacterial endo-beta-mannanase gene onto a M. es chromosome and report the discovery of a 42-bp conserved sequence motif within N. ovalis untranslated regions.     

Chromosome Copy Number Variation and Control in the Ciliate Chilodonella uncinata

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.     

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.     

A high degree of macronuclear chromosome polymorphism is generated by variable DNA rearrangements in Paramecium primaurelia during macronuclear differentiation.

DNA rearrangements in Paramecium lead to the formation of macronuclear chromosomes, the sizes of which range from 50 and 800 kb (1 kb is 10(3) base-pairs). This process does not appear to be a simple size reduction of the micronuclear chromosomes by specific and reproducible DNA sequence elimination and chromosomal breakage followed by chromosomal amplification. On the contrary, this process generates a variety of different, but sequence-related, macronuclear chromosomes from a unique set of micronuclear chromosomes. This paper describes an attempt to understand the nature of the diversity of the macronuclear chromosomes and the mechanisms of their production. The structure of three macronuclear chromosomes, 480, 250 and 230 kb in size, have been determined utilizing chromosome-jumping and YAC-cloning techniques. The two smallest chromosomes correspond roughly to the two halves of the longest chromosome. The main contribution to the diversity arises from the chromosomal ends and is due to variable positions of the telomere addition sites and/or to variable rearrangements of DNA sequences. The 480 kb chromosome contains a region of variable length, which is likely to be due to a variable deletion, located at the position of telomerization seen in the two small chromosomes. A model of chromosomal breakage is proposed to rationalize this result where micronuclear DNA is first amplified, broken and degraded to various extent from the newly formed ends, which subsequently are either telomerized or religated. Potential implications of these processes for gene expression is discussed. Known phenotypes that have a macronuclear determinism could be explained by this type of process.     

COMPARTMENTALIZATION OF THE DEVELOPING MACRONUCLEUS FOLLOWING CONJUGATION IN STYLONYCHIA AND EUPLOTES

The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes and Stylonychia has been examined with the electron microscope. Banded polytene chromosomes can be seen in thin sections of the macronuclear anlagen during the early periods of exconjugant development. As the chromosomes reach their maximum state of polyteny, sheets of fibrous material appear between the chromosomes and transect the chromosomes in the interband regions. Individual bands of the polytene chromosomes thus appear to be isolated in separate compartments. Subsequently, during the stage when the bulk of the polytenic DNA is degraded (1), these compartments swell, resulting in a nucleus packed with thousands of separate spherical chambers. Individual chromosomes are no longer discernible. The anlagen retain this compartmentalized condition for several hours, at the end of which time aggregates of dense material form within many of the compartments. The partitioning layers disperse shortly before replication bands appear within the elongating anlagen, initiating the second period of DNA synthesis characteristic of macronuclear development in these hypotrichs. The evidence presented here suggests that the "chromatin granules" seen in the mature vegetative macronucleus represent the material of single bands of the polytene chromosomes seen during the earlier stages of macronuclear development. The possibility is also discussed that the degradation of DNA in the polytene chromosomes may be genetically selective, which would result in a somatic macronucleus with a different genetic constitution than that of the micronucleus from which it was derived.     

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.     

Nuclear Behavior of Spirostomum ambiguum During Conjugation with Special Reference to Macronuclear Development*

SYNOPSIS. During conjugation in Spirostomum ambiguum, the micronuclei divide thrice before synkaryon formation and 20 times thereafter. During the first meiotic division 18-24 bivalents, each about 0.5 μ or less appear on the spindle. They separate and pass to the poles. The details of the 2nd and 3rd prezygotic divisions and synkaryon formation by reciprocal exchange of gametic nuclei resemble those described for other ciliates in the literature. The synkaryon divides twice resulting in 4 nuclei; 2 of them become micronuclei and the remaining 2 macronuclear anlagen. The micronuclei enter into division, but this division is arrested in metaphase. The chromosomes in the macronuclear anlagen resemble those appearing in the Ist meiotic division in shape and size. In their maximum stage of development the macronuclear chromosomes are at least 3-4 times larger than those appearing in the arrested micronuclear metaphases in the same cell. There is no banding pattern of the chromosomes and therefore the possible extent of polyteny is difficult to evaluate. The chromosomes duplicate 3-4 times resulting in about 200–250 before they become indistinct as separate entities. Spirostomum is the only nonhypotrichous ciliate in which these cytologic features are described.     

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation and partial renaturation, a high percentage of total macronuclear DNA was found as single-stranded circles. Sequence analysis showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.     

Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.