Rate of phenotypic assortment in Tetrahymena thermophila.

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted.     

Regulatory Serotype Mutations in TETRAHYMENA PYRIFORMIS, Syngen 1

A method utilizing allelic exclusion has been developed to isolate mutants of Tetrahymena pyriformis, syngen 1, in which the normal pattern of expression of mutally exclusive surface antigens is altered. Cells homozygous for the recessive mutant allele R-1^r do not express the L, H and T serotypes when grown under conditions appropriate for their expression. Rather, a new immobilization antigen, r, is expressed. Cells homozygous for the recessive mutant allele R-3^r also express the r antigen instead of H serotypes, but are normal in their expression of T antigens. Genetic analyses show that R-1 and R-3 are not closely linked, that R-1 is linked to T by 9.3 units, and that R-3 may be loosely linked to the mt locus. Different linkage values were obtained, however, when different inbred laboratory strains were used, suggesting the possible existence of crossover modifying genes. The rates of assortment of R-1^R/R-1^r and R-3^R/R-3^r heterozygotes into pure sublines expressing either H or r serotypes are close to the values observed for the differentiation of heterozygotes at other loci. The data confirm the previous observation that genetic coupling relationships are not maintained in macronuclear phenotypes and are consistent with the hypothesis that the macronucleus contains 45 assorting subunits. The assortment of the double heterozygote R-1^R/R-1^r, R-3^R/R-3^r at R_f=0.0112 suggests that the units of assortment are not individual genetic loci or chromosome fragments, but that the units may be complete genomes.     

Macronuclear Genetics of Tetrahymena. II. Macronuclear Location of Somatic Mutations to Cycloheximide Resistance

Somatic cycloheximide-resistant mutants of syngen 1 of Tetrahymena pyriformis were isolated and genetically characterized. Two properties of the mutants were independently examined: (a) The transmission of the mutant phenotype during conjugation and (b) the kinetics of phenotypic assortment during vegetative propagation. The results of both studies strongly support the idea that these somatic mutations have a macronuclear location. The kinetics of assortment are consistent with the idea that the syngen 1 macronucleus contains about 45 assorting genetic units. The sib-selection method employed here, used in conjunction with the analysis of assortment kinetics and a previously described test for randomness of distribution, provides a probe of macronuclear genetics applicable to many ciliates, including those in which conjugation is not known to occur or is not under experimental control.     

New approaches to the problem of macronuclear assortment

Macronuclear assortment is a vegetative (somatic) process in the ciliate protozoan genus Tetrahymena which leads to the production of “homozygous” phenotypes from sexually-produced heterozygotes. The appearance of pure types follows a model of 45 randomly distributed and replicating subunits in the polyploid macronucleus. Models for the organization of macronuclear chromatin must explain the following: (1) haploid genomes are distributed at each fission; (2) not all loci assort; (3) different loci begin assortment at different times following conjugation. Therefore, either haploid subunits distributed at fission must frequently exchange parts (by somatic recombination, gene conversion, or progressive chromosome fragmentation), or they may actually disintegrate between fissions. Some of these possibilities can be distinguished by considering the kinetics of the appearance of pure types at two or more loci simultaneously. Conditions which give optimum discrimination between hypotheses of limited recombination and complete scrambling (by disintegration of haploid units between fissions) have been developed. A method for determining the number of assorting subunits in very young macronuclei is also given.     

Phenotypic Assortment in Tetrahymena Thermophila: Assortment Kinetics of Antibiotic-Resistance Markers, Tsa, Death, and the Highly Amplified Rdna Locus

Phenotypic assortment in Tetrahymena thermophila results from random distribution of alleles during amitotic division of the macronucleus. The rate of assortment is dependent on input ratio and the number of assorting units. The assortment of the antibiotic resistance markers Chx, Mpr and gal was determined and is consistent for each with the model of 45 assorting chromosomes. The gene tsA (previously ts-1) shows normal assortment, in contrast to previous reports. A mutation in the highly amplified ribosomal locus (rdnA2) assorts as if present at only 45 copies. Death of clones occurred at a rate consistent with assortment for a single gene.     

Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila

The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces. These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus. In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other. This phenomenon is called phenotypic assortment. We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece. The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups. Thus, coassortment allows the mapping of the somatic genome by purely genetic means. The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.     

Genetic Evidence Concerning the Structure of the Tetrahymena thermophila Macronucleus*†

Synopsis.
A satisfactory model of the Tetrahymena thermophila macronucleus must explain its genetic behavior in terms of its constituent molecules. Particular genetic phenomena requiring explanation are (a) phenotypic assortment , here interpreted as resulting from allelic disjunction rather than from differential gene expression; (b) unequal allelic input for some loci , interpreted as a consequence of unequal and selective replication of some alleles during early macronuclear development; (c) delayed assortment at some loci , interpreted as an effect of inequality of allelic input combined with a generalized elevation of DNA content during early clonal history; (d) linkage disruption , probably reflecting continuous somatic recombination rather than dissolution of chromosomes into small repliconic units; (e) assortment depression , brought about by the occasional association of homologous replicons (chromosomes) or else by a differential increase in some classes of replicons; (f) ploidy-related developmental differences in macronuclear primordia are interpreted on the basis of quantitative differences in DNA rather than in terms of an early perception of genic imbalance, (g) Ploidy independent macronuclear DNA content is consistent with several models of size regulation.     

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.     

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.     

Recombination and Assortment in the Macronucleus of TETRAHYMENA THERMOPHILA: A Theoretical Study by Computer Simulation

The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome. Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions. Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB. Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40%. At higher recombination frequencies, linked genes appear to assort independently. The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation. The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.     

Tetrahymena macronuclear genome mapping: colinearity Of macronuclear coassortment groups and the micronuclear map on chromosome 1l

The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment. While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces. We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible. To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus. Sixteen CAGs were identified, 7 of which contain at least two loci. We have concluded that CAGs represent a fundamental genetic feature of the MAC. The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs. These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.     

Evolution of Germline-Limited Sequences in Two Populations of the Ciliate Chilodonella uncinata

Ciliates are microbial eukaryotes that separate their nuclear functions into a germline micronucleus and a somatic macronucleus. During development of the macronucleus the genome undergoes a series of reorganization events that includes the precise excision of intervening DNA. Here, we determine the architecture of four loci in the micronuclear and macronuclear genomes of the ciliate Chilodonella uncinata and compare the levels of variation in micronuclear-limited sequences to macronuclear destined sequences at two of these loci. We find that within a population, germline-limited sequences are evolving at the same rate as other putatively neutral sites, but between populations germline-limited sequences are accumulating mutations at a much faster rate than other sites. We also find evidence of macronuclear recombination and incomplete elimination of intervening DNA, which result in increased diversity in the macronuclear genome. Our results support the assertion that the unusual genomic features of ciliates can result in rapid and unpredicted patterns of diversification.     

Macronuclear Genetics of Tetrahymena I. Random Distribution of Macronuclear Gene Copies in T. PYRIFORMIS, Syngen 1

The objective of this study was to test the idea that the macronuclear (somatic) genetic information is randomly distributed at each cell division in Tetrahymena pyriformis, syngen 1. We took advantage of a quick and reliable test for the detection of stable vegetative segregants in clones heterozygous for ts2, a heat-sensitive mutation. Clones that originated from cells inferred to contain very few (1-3) copies of the ts2⁺ allele in their macronuclei were selected for pedigree analysis. Experimental results were compared with the results of a computer simulation of the experiment. Our results are fully consistent with the predictions of the Allen-Nanney-Schensted model concerning the replication and distribution of functional gene copies. This model proposes that the macronucleus contains many functional copies of a given gene that are duplicated once during the cell cycle and are randomly distributed to the two-daughter nuclei. Our work has provided a more sensitive test of the assumption of randomness than was previously available. Our evidence for complete randomness suggests that 45, the effective number of macronuclear gene copies previously inferred from the segregation rate, indeed represents the actual number of copies. This conclusion, coupled with previously available evidence that the macronucleus is approximately 45-ploid, suggests that the segregating genetic units in the macronucleus are in effect haploid. This appears to remove the need to postulate inter-allelic repression to account for the phenomenon of phenotypic assortment. Our results, as well as those of others, also are inconsistent with any simple form of the master-slave hypothesis of Allen and Gibson.     

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed. © 1992 Wiley-Liss, Inc.     

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.     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.     

Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotic' chromatin distribution

The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.