DNA elimination and its relation to quantities in the macronucleus of Tetrahymena.

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriophage λ DNA fragments replicate in the Paramecium macronucleus: Absence of active copy number control

We show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley-Liss, Inc.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5’-TA-3’ dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60 000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

Differential DNA Amplification and Copy Number Control in the Hypotrichous Ciliate Euplotes crassus

ABSTRACT. During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.     

Variable copy number of macronuclear DNA molecules in Tetrahymena.

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Division and growth kinetics of the division mutant Conical of Tetrahymena : A contribution to regulation of generation time

The recessive mutant conical of Tetrahymena thermophila is characterized by unequal cytoplasmic division resulting in a large anterior (proter) and a small posterior (opisthe) daughter cell with similar macronuclear DNA contents. Opisthes have long and proters short generation times. This gives the opisthes more time to accumulate cell mass, thereby reducing differences in size between sister cells. Growth rates as determined by cytophotometry do not contribute to the reduction and eventual elimination of differences between sisters but rather should increase them, since small cells accumulate mass at a slower rate than large ones. By tracing consecutive generations it is shown that differences between sister cells in generation time as well as in cell size require more than one generation to be regressed to the mean of the whole population. These findings are incompatible with the probabilistic mode of regulation of generation time.     

Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

The Size of DNA Molecules and Chromatin Organization in the Macronucleus of the Ciliate Didinium nasutum (Ciliophora)

Pulsed‐field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short “gene‐sized” DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80–265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.     

Downward regulation of macronuclear DNA content in Paramecium tetraurelia : Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.     

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.     

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

The Full Schedule of Macronuclear DNA Synthesis is Not Required for Cell Division in Paramecium tetraurelia1 1

Macronuclear DNA synthesis normally continues until late in the cell cycle in Paramecium; however, blockage of macronuclear DNA synthesis after 0.72 in the cell cycle does not alter the occurrence or timing of the subsequent cell division. When DNA synthesis is blocked after cells have reached the transition point, macronuclear DNA content at the following division is reduced to about 75% of the normal level. The point at which macronuclear DNA synthesis is no longer required for division corresponds to the beginning of micronuclear mitosis and the early stages of oral morphogenesis.