Inactivation of a macronuclear intra-S-phase checkpoint in Tetrahymena thermophila with caffeine affects the integrity of the micronuclear genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.     

Suicide is not the inevitable outcome of "perpetual" selfing in tetrahymenines collected from natural habitats.

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms. Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing "immature" progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some "immature" progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette.     

Suicide is not the inevitable outcome of “perpetual” selfing in tetrahymenines collected from natural habitats

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms. Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing “immature” progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some “immature” progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette. © 1992 Wiley-Liss, Inc.     

A Study of Odd Mating-Type Determination Factor by Transfer of Macronuclear Karyoplasm in PARAMECIUM TETRAURELIA

The unique feature of the "B system" of mating-type determination found in Paramecium tetraurelia is the existence of a cytoplasmic difference between odd (O) and even (E) cells created and maintained by the action of their macronuclei. Thus far, the presence of a determining factor that controls the differentiation of the developing zygotic macronucleus for O mating type has not been verified. Results of crosses between cells of differing clonal age and complementary mating type suggest that, for one to two fissions after autogamy, O cells produce some factor that determines the gametic nucleus (micronucleus) as mating type O. Direct evidence for the production of O-determining factor by the young O macronucleus was obtained by transplanting young O macronuclear karyoplasm (a part of the macronucleus) into E cells: 32-35% of E exautogamous clones transformed to O; transformation of E exautogamous clones to O reached as high as 72% by transfer of young O macronuclear karyoplasm from a conjugant, 3-4 hr after mixing. This indicates that O determinants produced by the O macronucleus can also act during the sensitive period of development of the new macronucleus. These O-determining factors may be produced or activated at the sexual stage and then decrease in activity in subsequent fissions after new macronuclear reorganization.     

Molecular and Genetic Analyses of the B Type Surface Protein Gene from Paramecium Tetraurelia

The gene encoding the B type variable surface protein from Paramecium tetraurelia stock 51 has been cloned and sequenced. The 7,182 nucleotide open reading frame contains no introns and encodes a cysteine-rich protein that has a periodic structure including three nearly perfect tandem repeats in the central region. Interestingly, the B gene is located near a macronuclear telomere as was shown previously for two other paramecium surface protein genes. In this paper, we characterize four independent mutants with complete macronuclear deletions of the B gene. Previous analysis of different macronuclear deletion mutants of the A surface protein gene demonstrated two types of inheritance: typical Mendelian segregation (as illustrated by d12) and cytoplasmic inheritance (shown by d48). F(1) analysis of four B(-) mutants crossed with wild-type cells reveals heterozygous F(1) cell lines derived from both parental cytoplasms contain approximately the same copy number of the B gene, as expected for a recessive Mendelian mutation. Analysis of F(2) progeny from three of these four B(-) mutant crosses indicates that one of the three exhibits a Mendelian 1:1 segregation ratio of B(+) and B(-) cell lines. The other two show a preponderance of B(+) cells, but this is not correlated with the parental cytoplasmic type. In addition to having a large number of B(+) individuals, the d12.144, A(-), B(-) mutant produced some F(2) progeny that stably maintain less than normal macronuclear amounts of the A gene and/or the B gene.     

Sex allocation variation in Daphnia pulex

Daphnia (Crustacea: Cladocera) reproduce by cyclical parthenogenesis in which the sex of offspring is environmentally determined. Although numerous studies have demonstrated that factors such as crowding and short-day photoperiod stimulate male production, there is limited information on variation in allocation to male and female offspring for any species of Daphnia . The present study assessed the presence or absence of male production in 96 isofemale lines (clones) from each of eight populations of Daphnia pulex . An average of 37% (range 18–51%) of clones failed to produce males under crowded conditions in the laboratory. A subset of 14 of these non-male-producing clones also failed to produce males under short-day photoperiod (8L:16D). Three male-producing clones were within-clone mated as well as crossed to three non-male-producing clones to study the inheritance of the failure to produce males. The average frequency of non-male-producing F ₁ progeny was significantly higher (58%, N = 486) among the outcrossed progeny than the inbred progeny (5%, N = 86). In addition, when sixteen of the male-producing outcrossed progeny were within-clone mated, only 7% ( N = 106) of the resulting F ₂ progeny failed to produce males. These results are consistent with a genetic basis for the absence of male production. Average survival of the progeny from the nine outcrossed matings was more than twice (67%) that of the inbred progeny from the three within-clone matings (30%), suggesting that within-clone mating would result in significant inbreeding depression. We present a model that suggests that even low levels of inbreeding could allow non-male-producing females to be maintained in a population. The co-occurrence of non-male-producing females and females that produce both males and females in Daphnia pulex bears a similarity to the gynodioecious breeding system found in some plant species.     

Clonal Variation in Paramecium. I. Persistent Unstable Clones

Clones of Paramecium of identical serotype when cultured in test tubes may differ in their ability to give rise to subclones of this serotype. Characteristically, stable clones yield progeny indistinguishable from their parents, while from unstable clones diverse subclones with new serotypes can be isolated repeatedly. Stable lines are resistant to changes in culture medium and also are unaffected by most sera. In contrast, the numbers and kinds of serotypes displayed among subclones derived from unstable lines are often affected by these same agents. Stable and unstable clones are interconvertible when the medium from individual cultures is repeatedly and frequently replaced by fresh culture fluid. This effect is very likely a result of the removal of the initial exhausted medium with any cell products rather than the addition of fresh nutrient.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus II. Experimentally Induced Regeneration of Old Macronuclear Fragments1

Following conjugation in ciliates, the usual fate of the old pre-conjugant macronucleus is resorption. In some species, however, old macronuclei, or their fragments, have the ability to reform functional vegetative macronuclei when new macronuclear anlagen are defective. The present work on Euplotes shows that if anlagen are allowed to carry out their essential roles in early exconjugant development, including influence on cortical reorganization such that feeding can resume, they can then be permanently damaged by UV-microbeam irradiation and regeneration of old macronuclear fragments can occur. E. aediculatus exconjugants were anlage-irradiated at 40–60 hr of development and the irradiated cells cultured individually and fed. Squashes revealed enlargement and anteriorward migration of the persistent (posterior) macronuclear fragments. The first post-conjugant fission of such cells was delayed (times ranged 6–43 days) and did not seem to involve the damaged anlagen, which remained rudimentary, did not divide along with the cells, and were subsequently resorbed. It appeared that cell fission was supported by the fragments of the old macronuclei, which either divided or partitioned themselves between the two daughter cells. Mating tests performed on early clones derived from irradiated exconjugants revealed ample conjugation competence; intraclonal conjugation in such clones was also apparent. The absence of the immature period seen in normal exconjugants provides further evidence that the clones arose from cells with regenerated macronuclei.     

Disturbance of the determination of germinal and somatic nuclei by heat shock in Paramecium caudatum

During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.     

Traumatic Induction of Early Maturity in Tetrahymena

Exposure of conjugating pairs of Tetrahymena thermophila to high temperature (37°) during macronuclear development causes an abortion of many macronuclei, but it also often induces an early appearance of sexual maturity in clones completing macronuclear development. Lines become mature after about 15 cell divisions rather than after 50 or more cell divisions in untreated pairs. The phenotype resembles that associated with Em (early maturity) mutants but, because it is not transmitted to the progeny in the next generation, it must be considered a phenocopy. The hypothesis is developed that an early genotype-environment incompatibility, whether associated with an abnormal genotype or an unusual environment, activates a shunt mechanism permitting the organisms to undertake quickly an ordinarily forbidden sexual lottery.     

Epigenetic Regulation of Programmed Genomic Rearrangements in Paramecium aurelia1

ABSTRACT In ciliates, development of the polyploid somatic macronucleus after sexual events involves extensive and reproducible rearrangements of the germ-line genome, including chromosome fragmentation and precise excision of numerous internal sequence elements. In Paramecium aurelia, alternative macronuclear versions of the same germ-line genome can be maternally inherited across sexual generations, showing that rearrangement patterns are not strictly determined by the germ-line sequence. Homology-dependent maternal effects can be evidenced by transformation of the vegetative macronucleus with cloned macronuclear sequences: new fragmentation patterns or internal deletions are specifically induced during differentiation of a new macronucleus, in sexual progeny of transformed clones. Furthermore, transformation of the maternal macronucleus with germ-line sequences containing internal eliminated sequences (short single-copy elements) can result in a specific inhibition of the excision of the same elements in the zygotic macronucleus. These experiments show that the processing of many germ-line sequences in the developing macronucleus is sensitive to the structure and copy number of homologous sequences in the maternal macronucleus. The generality and sequence specificity of this trans-nuclear, epigenetic regulation of rearrangements suggest that it is mediated by pairing interactions between germ-line sequences and sequences imported from the maternal macronucleus.     

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.     

Intraspecific variability in response to stimuli for male and ephippia formation in Daphnia pulex

Natural clones and clones from laboratory crossings of Daphnia pulex have been tested in their response to stimuli for male and ephippia formation. The clones are from ponds in Illinois, USA, an area with both obligate and cyclical clones of D. pulex. The progeny from two types of crosses were studied: 1) crosses between an obligate clone producing males and a cyclical clone producing only females, and 2) self-fertilization within a cyclical clone producing both sexes. Both the natural and the artificial clones showed great variation in response to environmental stimuli for males. Ephippia response seems more equal among the clones, but two of the artificial clones could hardly be stimulated to produce ephippia in the induction experiments. In crowded cultures, however, they produced a few. Crossings between cyclicals and obligates yielded mostly cyclical progeny. Progeny from the selfed clone had low survival and fecundity.     

Abnormalities in Nuclear Behavior and Mating Type Determination in Cytoplasmically Bridged Exconjugants of Doublet Paramecium aurelia*

Exconjugant clones of Paramecium aurelia stock 51S, syngen 4, which fail to separate prior to the 1st fission have numerous cytologic and mating type determination anomalies. The doublets have abnormal distribution of macronuclear anlagen, fewer macronuclear fragments per cell, and abnormalities in numbers of micronuclei. Despite apparent cell fusion and mixture of cytoplasm, the singlets arising from each side of the doublet may be of opposite mating types, and mating type determination may remain unstable for 1 or more fissions in contrast to the usual pattern of mating type determination before the 1st postconjugation fission.     

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."     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.