James D. Berger

Gene expression and phenotypic change in Paramecium tetraurelia exconjugants.

A study of the patterns of phenotypic change in exconjugants using the recessive behavioural mutant pawn ( pwA ) and its wild-type allele shows that both cytoplasmic and nuclear factors contribute to phenomic lag. Following loss of the wild-type allele from the macronucleus, phenomic lag lasts for 6–11 cell cycles in various sublines of a single clone. Inherited cytoplasmic material is estimated to be responsible for phenomic lag of no more than 5–6 cell cycles. Longer persistence of the parental phenotype is due to continued gene activity in macronuclear fragments carrying the wild-type allele. Genes in fragments remain active and can result in maintenance of the parental phenotype as long as fragments are present (up to 11 cell cycles). Phenomic lag in the other direction, from pawn to wild type, varies from 0 to 2 cell cycles. The major cytoplasmic factor involved is the amount of wild-type material acquired from the mate during conjugation. Extensive cytoplasmic exchange often occurs during normal conjugation and can lead to change of phenotype as early as the first meiotic division. Phenotypic change due to gene expression in macronuclear anlagen brings about phenotypic change near the end of the first cell cycle in +/+ cells and about a cell cycle later in heterozygotes.

Growth rate of the marine planktonic ciliate Strombidinopsis cheshiri Snyder and Ohman as a function of food concentration and interclonal variability

A clonal culture of the ciliate Strombidinopsis cheshiri Snyder and Ohman was established from British Columbian coastal waters. Using the solitary 4 μm diatom Thalassiosim pseudonana (Hustedt) Hasle and Heimdal as food, a numerical response was obtained by keeping the ciliates in semi-continuous culture, measuring specific growth rates at different prey concentrations, and fitting the data to a rectangular hyperbolic function; this provided growth and mortality rates. From the experiments four pieces of information were obtained for S. cheshiri: 1) the threshold concentration (i.e., the prey concentration where ciliate growth = 0) was ~ 103· ml−1 (~6 ng C · ml−1); 2) the major increase in growth rate occurred between 103–104 prey · ml−1 (~ 6–60 ng C · ml−1); 3) the maximum growth rate was μ = 0.985 (~ 1.4 divisions · day −1); and 4) the ciliate was capable of selfing conjugation. The growth rate data were, however, variable due to sub-clonal differences (i.e., clones secondarily isolated from the original clone). Data obtained from examining cell isolates suggested that the sub-clonal differences were caused by clonal ageing and selfing conjugation. Consequently, abnormally low growth rates were assumed to be due to ciliates with a high mutational load; outliers were removed and not included in the final numerical response. This is the first observation of selfing conjugation in oligotrichs and one of only a few observations of clonal ageing for these ciliates. The results suggest that 1) this genus may be useful for future work on oligotrich mating and 2) older cultures of these ciliates should be used with caution when estimating ecological parameters.

Autogamy in Paramecium: cell cycle stage-specific commitment to meiosis

Autogamy is a process of meiosis and fertilization which takes place in unpaired Paramecium cells, and which is triggered by starvation. This study examines the consequences of nutritional down-shift at various points within the cell cycle on the occurrence of autogamy. It shows that cells become committed to autogamy in a two-step process. An initial point of commitment to autogamy occurs about 100 min prior to the median time of cell division (cell cycle duration, 330 min). Cells which have become committed to autogamy initiate meiosis following the next fission, others complete another vegetative cell cycle before undergoing meiosis. Treatments that perturb the cell cycle and displace the point of commitment of division also displace the point of initial commitment to autogamy to the same extent. The initial commitment to autogamy can be reversed by refeeding. The second, final, point of commitment to autogamy occurs about 30 min after the fission, immediately prior to initiation of meiosis, and coincides with the beginning of meiosis. If cells are refed at this point, or at later stages, autogamy continues. Autogamy is not well synchronized either in naturally starved cultures or in those subjected to abrupt nutritional down-shift. This is a consequence of the cell cycle stage dependence of entry into autogamy. Autogamy occurs synchronously in samples of dividers selected from asynchronous cultures 2 or more hours after nutritional down-shift. The timing of the events of conjugation and autogamy coincide when the pre-autogamous fission is aligned temporally with the initial contact of mating cells.

A cdc2‐Like Kinase Associated with Commitment to Division in Paramecium tetraurelia

ABSTRACT. Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2‐like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13suc1‐associated cdc2‐like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2‐like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.

Effects of gene dosage on protein synthesis rate in Paramecium tetraurelia: Implications for regulation of cell mass, DNA content and the cell cycle

Abstract Both cell mass and DNA content can vary considerably in Paramecium . This study examines protein and RNA synthesis rates in cells with various DNA contents. At lower DNA contents the protein synthesis rate is proportional to DNA content. At a characteristic ratio of gene dosage to cell mass, the protein synthesis rate levels off. This plateau rate of protein synthesis is strongly correlated with cell mass throughout the cell cycle. These results are consistent with two alternative interpretations. The results suggest that the protein synthesis rate is template-limited at low DNA contents and then levels off, either because the protein-synthesizing system is saturated with gene transcripts at high DNA contents, or because of a concentration-dependent feedback system which regulates the transcript concentration in the cytoplasm. A simple deterministic cell cycle simultation model shows that the saturation hypothesis is consistent with the observed effects of experimental increase in either DNA or protein content on the subsequent DNA and protein content and cell cycle length.

Timing of Life Cycle Morphogenesis in Synchronous Samples of Sterkiella histriomuscorum. II. The Sexual Pathway

Abstract Isolates of Sterkiella (Oxytrichidae, Stichotrichia, Ciliata) are commonly used to study macronuclear development. These organisms respond to changes in food abundance variably by encystment-excystment, conjugation, cannibalism or rescaling cell size. An isolate of Sterkiella histriomuscorum (previously Oxytricha fallax and O. trifallax) is used because two complementary mating types are available. We provide observations on conjugation in cultures of this isolate. Using synchronous samples of conjugants, the timing of stages of nuclear divisions during conjugation was determined. Following ex-conjugant cultures over time, the onset of clonal aging and senescence is described. Cells become sexually mature after a brief period of “adolescence”, during which time selfing is possible. Senescent cultures are less vigorous, unable to conjugate and encyst more readily. Excystment survival decreases with clonal age. These results can serve as reference for long-term cultures of this species and for analysing particular stages of developmental processes during conjugation.

The timing of initiation of DNA synthesis in Paramecium tetraurelia is established during the preceding cell cycle as cells become committed to cell division

The timing of initiation of DNA synthesis (IDS) in Paramecium is established before cell division at a point located at about 0.75 in the preceding cell cycle. This point occurs about 90 min prior to fission and coincides with the point at which cells become committed to cell division. The location of the point at which the timing of IDS is set was deduced from a series of nutrient-shift experiments. Changes in nutrient level lead to changes in the duration of the subsequent G1 interval when they occur more than 90 min prior to fission. Perturbation of the cell cycle so that the timing of commitment to cell division is altered, results in a parallel shift in the point at which the timing of IDS is established.

The Cell Cycle and Regulation of Cell Mass and Macronuclear DNA Content

The chief developmental pathways in unicellular eukaryotes, such as Paramecium, are the vegetative replication pathway, and the sexual pathway. Both phenomena have as their basis the cell cycle with its recurrent pattern of growth, replication and division. The regulation of events within the cell cycle requires that cell components be elaborated in such a way as to maintain balanced growth. Perturbations of cell mass or gene dosage must also be attenuated, and cellular components must be properly allocated to daughter cells at division. Macronuclear DNA content must also be regulated so that a constant gene concentration (gene dosage per unit cell mass) is maintained over time. In ciliates the maintenance of a constant gene concentration is a significant problem because of the variable gene dosage of the polygenomic macronucleus and its relatively imprecise amitotic macronuclear division process. The mechanisms regulating gene dosage and cell mass must also be integrated into the processes controlling cell cycle events. In addition, it is necessary that the alternative pathways (e.g. meiosis, or stationary phase arrest) be entered under the appropriate conditions and with proper phasing with respect to the underlying vegetative replication pattern.

Commitment to Division in Ciliate Cell Cycles

Near the end of the cell cycle, ciliates commit irreversibly to cell division. The point of commitment occurs at the time of oral polykinetid assembly and micronuclear anaphase. The commitment is a checkpoint which requisites a threshold cell mass/ DNA ratio and stomatogenesis. It is also a physiological transition point, involving cdk protein kinases similar to those of other eukaryotes. Both P34 kD and P36 kD kinases, similar to the S. pombe cdc2 kinases, have been described to have activity as monomers. Subsequent to commitment to division, dramatic cytoskeletal modifications occur for separation of organelles, cortex morphogenesis and cytokinesis. Numerous mutants affecting cytoskeletal function associated with the division process have been obtained in several species. Of these, only the ccl mutant in Paramecium affects cell cycle progression prior to commitment to division. The material reviewed is used to speculate about the mechanisms of regulation of pre‐fission morphogenesis and cell division related processes in ciliates.

Riding The Ciliate Cell Cycle—A Thirty-Five-Year Prospectivea

Abstract Studies of the ciliate cell cycle have moved from early examination of its biochemistry with heat-synchronized Tetrahymena through descriptive studies of Paramecium using small synchronous cell samples. These studies described what happens during the cell cycle and provided some initial insights into control, especially the idea that there was a point at which cells became committed to division. This early work was followed by an analytical phase in which the same small sample techniques, combined with gene mutations, were used to tease apart some major features of the regulation of cell growth kinetics, including regulation of macronuclear DNA content and regulation of cell size, the control of timing of initiation of macronuclear DNA synthesis, and the control of commitment to division in Paramecium. The availability of new molecular genetic approaches and new means of manipulating cells en masse made it possible to map out some of the basic features of the molecular biology of cell cycle regulation in ciliates. The challenge before us is to move beyond the ‘me-too-ism’ of validating the presence of basic molecular regulative machinery underlying the cell cycle in ciliates to a deeper analysis of the role of specific molecules in processes unique to ciliates or to analysis of the role of regulatory molecules in the control of cell process that can be uniquely well studied in ciliates.