Peter J. Bruns

Conjugation in Tetrahymena thermophila: A temporal analysis of cytological stages☆☆☆

Abstract The time course and synchrony of the stages of conjugation in Tetrahymena thermophila as defined by cytologically observable changes in the morphology and position of the nuclei were established. The time required for 50% of the pairs to enter or pass a particular stage, as well as the duration of each stage were determined. The relative synchrony of the pairs as they went through conjugation was followed by correlating the maximum percentage of the population found in a stage with the duration of that stage. The degree of synchrony between the pairs was found to be high under the conditions of this study, with very little decrease in synchrony seen during the initial 9 h of conjugation. Although some variability in the degree of synchrony was seen between different matings, there was little change detected in the duration of each cytological stage. Prolonged starvation of the cells prior to their mating resulted in a gradual loss of synchrony.

Long-term storage.

Publisher Summary This chapter presents a simple method for long-term storage of Tetrahymena thermophila , based on freezing starved cells. Starving cells before freezing greatly enhances the recovery of cells and eliminates the need for complicated freezing apparatuses or protocols. Tetrahymena thermophila and other ciliates require that growth be minimized or even eliminated during long-term storage: extended growth leads to strain degradation. Cultures of fresh exconjugants exhibit normal growth rates and can undergo normal conjugation, following a strain-specific period of immaturity. However, fertility begins to drop within a year or less of continuous growth in the laboratory; matings increasingly tend to undergo the abortive pathway called genomic exclusion. Cells still form pairs in these matings, but they fail to contribute any genome to the progeny. In addition, because they are not expressed and are therefore not selected against, micronuclear deletions accumulate with clonal age. The successful freezing of eukaryotic cells in liquid nitrogen is dependent on stringent control of a number of parameters, including: (1) the physiological state of the cells at the time of freezing; (2) the cryoprotectant and medium in which the cells are frozen; (3) the rate of cooling; (4) the temperature at which the cells are ultimately stored; and (5) the rate and temperature of thawing.

Chapter 13 Induction and Isolation of Mutants in Tetrabymena

Publisher Summary This chapter describes induction and isolation of mutants in Tetrahymena . Tetrahymena pyriformis is a unicellular animal well suited to studies of cell and molecular biology. It can be grown in pure culture to high concentrations, in practically unlimited volumes, with a remarkably short-vegetative doubling time (down to 2 hours under favorable conditions) and is thus extremely useful for the production and purification of cellular organelles and macromolecules. It is also well suited for genetic studies, having a diploid germ line with only five pairs of chromosomes, which are transmitted during conjugation according to the standard rules of the Mendelian genetics. The cells can be cloned efficiently, and conjugation is under precise experimental control. Clones can be readily frozen and indefinitely stored under liquid nitrogen. The chapter covers the methods used in the induction and isolation of Tetrahymena mutants, including (1) recent technical advances in handling the large numbers of cells required to isolate rare mutants and (2) recently developed strategies designed to increase the frequency of mutants. The chapter also describes elements of the Tetrahymena genetics, genetic analysis, and uses of the mutants. Design of conditions for isolating desired mutants is discussed in the chapter along with the limitations of the use of mutants.

Biolistic transformation of macro- and micronuclei.

Publisher Summary This chapter presents methods for the biolistic transformation of both macro- and micronuclei of Tetrahymena thermophila . DNA-coated gold particles are fired into starved vegetative cells and, during late phases of conjugation, into mating cells to introduce gene sequences into the macronucleus. Early stages of mating are bombarded to add genes to the micronucleus. Methods for cell preparation and a detailed protocol for shooting and recovering transformed cells are also presented. Development of successful transformation techniques in any ciliate is complicated by the separation of somatic and germinal functions into two distinctly different nuclei: the polyploid macronucleus, which solely directs the cells phenotype, and the transcriptionally inert diploid micronucleus, which contributes the genotype to subsequent sexual generations. Each of these nuclei is a unique target for DNA transformation, requires different strategies for the introduction of genes, and responds in different ways following successful transformation.

Costimulation in Tetrahymena pyriformis: A developmental interaction between specially prepared cells☆

Abstract Initiation and costimulation are two stages Tetrahymena pyriformis undergoes in the developmental pathway leading from vegetatively growing cells to mating pairs. Initiation is a specific period of starvation, requiring 2 hr at 30°C in 10 mM Tris. Costimulation requires cell-cell contact for about 1 hr at 30°C. An analysis of pair formation in mixtures of noninitiated and initiated mature cells demonstrates that cells of two mating types can costimulate only if they are both already initiated. Similarly, mixtures involving immature cells demonstrate that starved immature cells cannot stimulate initiated mature cells; a new technique is employed to obtain the immature cells. The work indicates that the events leading to pair formation are organized in a linear sequence.

Costimulation in Tetrahymena II. A nonspecific response to heterotypic cell-cell interactions☆

Abstract Costimulation, a developmental interaction requiring cell-to-cell contact, is the second stage in a linear sequence of events leading from vegetative growth to mating pairs in the ciliate Tetrahymena thermophila . This paper uses the kinetics of appearance of genetically marked progeny to measure accurately the duration of normal costimulation and then to examine the role of mating type in costimulation. Although diverse mating types are required for costimulation to occur, the costimulated cells response is not specific to the mating type of the cell contacting it; costimulation by one mating type appears to prepare a cell for mating with any complementary mating type.

Immobilization antigens of Tetrahymena pyriformis: I. Assay and extraction☆

Abstract A method using immunodiffusion has been established to assay the two mutually exclusive temperature dependent immobilization antigens, H and T, of Tetrahymena pyriformis . Specific antiserum was obtained by exploitation of allelic or temperature induced variations among inbred strains for absorption of antisera prepared against whole cells. The antigens were extracted both from isolated cilia and from whole cell bodies. Mild detergent extraction was found to be more efficient than mechanical disruption of the cells by freeze-thawing. The sedimentation behavior in sucrose density gradients of active H antigen was the same, whether freeze-thaw or detergent extracted; similarly, the sedimentation behavior of T was the same following the two extraction methods. Extraction with acetic acid, as reported by others, solubilized the same material as the detergent, but the acid denatured the antigen. An estimate of the molecular weight of the antigen of 29 000 for H and 23 000 for T was made.

The pre-pairing events in Tetrahymena thermophila: Analysis of blocks imposed by high concentrations of Tris-Cl☆

Abstract Initiation, a process that occurs at the very onset of the sexual cycle in the ciliate Tetrahymena thermophila, is blocked by concentrations of Tris-Cl in excess of 50 mM. Cells starved in 60 mM Tris-Cl proceed through the first 30 min of initiation before arresting. Cells allowed to initiate for 15–45 min in 10 mM Tris-Cl before exposure to 60 mM Tris-Cl accumulate at a second point about 50 min through the process. Two variants which behave differently than wild type in 60 mM Tris-Cl are described. A model for initiation is proposed and its implications are discussed.

A Simple, Efficient Technique for Freezing Tetrahymena thermophila

ABSTRACT. We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen. This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer. To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing. the frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.

Cell density as a selective parameter in Tetrahymena

Abstract Cells of various metabolic states of Tetrahymena pyriformis were separated by banding in linear density gradients of Ficoll. Whereas log phase cultures generated three distinct bands, stationary cultures produced only two, and starved cells demonstrated a unique, higher density. The value of this observation for the positive selection of nutritional auxotrophs and temperature sensitive mutants is discussed. In addition, a unique downward migration of a band of cells separated by the centrifugation is demonstrated and discussed.