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)

Gene concentration varies in the macronucleus of Tetrahymena.

The ratio of amounts of specific genes to total DNA (gene concentration) in different lines of Tetrahymena and at different conditions has been determined by hybridization with specific gene probes. The concentration of the DNA coding for rRNAs is specific for different strains. It also decreases with growth rate, the extent of reduction being different in various species. The rDNA concentration increases unproportionally when the size mutant D9 grows to double the volume upon transfer to the non permissive temperature. rDNA concentration, therefore, changes specifically with physiological status. Genes on other DNA fragments occur in other proportions than the rDNA. The concentration of the S25 ribosomal protein gene is reduced significantly upon entering the stationary phase, while the concentrations of the other genes tested appear unchanged. The results indicate that the amounts of specific DNA molecules in the macronucleus of Tetrahymena are controlled depending on the strains used and on the conditions.

The transient division block in the D9ts mutant of Tetrahymena is inducible at every cell cycle stage.

The temperature-sensitive mutant D9 of Tetrahymena thermophila doubles its size at restrictive temperature. It does so by complete cessation of cell division for a limited time. After resumption of proliferation, division rate and specific growth rate are the same as at the permissive temperature, thereby maintaining the new cell size. In this study a detailed analysis of the process of controlling the new cell size is presented, by probing the temperature sensitivity of cell cycle phases. It will be shown that high temperature affects the size-controlling system immediately upon shift in temperature. Temperature pulses are effective at every stage in the cycle and are executed at the time of expected division. After return to the permissive temperature, cells gradually recover from the temperature pulse as seen by a decrease in division delay. Preparation for the next division is unaffected by the temperature pulse. It occurs at the same time as in untreated controls. The results allow us to describe some features of the division initiating system.

Arrest of micronuclear DNA replication during genomic exclusion in Tetrahymena produces haploid strains.

Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion). These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content. However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated. When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid. Competence for diploidization is apparently limited to some period of time after nuclear transfer. Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena.

A characterization of Tetrahymena mRNA by in vitro translation: The effects of culture growth on the recruitment of poly (A)(+) and poly (A)(-) RNA.

Messenger RNA was extracted from exponentially growing and from stationary cultures of Tetrahymena thermophila. It was separated into polyadenylated and non-polyadenylated fractions which were used as templates in a rabbit reticulocyte protein synthesizing system. The translated proteins were analysed by one and by two dimensional electrophoresis. Our experiments were intended to answer the question to which extent the abundance and the specificity of mRNA facilitates or accompanies the passage of cells through one culture growth cycle. As illustrated by the identification of 113 proteins very few differences between translated messages accompany the transition to the stationary phase, the most obvious feature being a change in the intracellular location of translation activities. These data are discussed with special reference to the prevailing occupation of Tetrahymena which is biomass production.

Pattern of DNA increase in macronuclear anlagen of Tetrahymena.

By combining cytophotometry with autoradiography, five stages of macronuclear anlagen can be discriminated by their DNA content until the end of the first cell cycle after conjugation in Tetrahymena. DNA increases from 2C to about 32C. Each S-phase is followed by a non-synthetic period. Comparing the mean nuclear DNA content after and before each S-phase revealed that 16C anlagen contain significantly less DNA than twice the amount of 8C anlagen. This is unlike the situation in other S-phases during which the amount of DNA is precisely doubled. In the second cell cycle after conjugation some of the cells increase their macronuclear G2 DNA content beyond the 64C stage, while the majority show a mean G2 content of about 64C.

Analysis of a mutant expressing temperature-sensitive changes of cell size in Tetrahymena.

A temperature-sensitive mutant of Tetrahymena expresses an increase in cell volume by a factor of 2.5 upon shift to restrictive temperature. Cellular amounts of protein, RNA, and DNA increase at roughly the same proportions. The mutant cell size is attained by cessation of divisions immediately after temperature shift for a period of time which is about equal to one generation time. During this time cell growth and DNA replication continue at virtually unchanged rates. Maintained at the restrictive temperature the mutant cells divide at the same rate as the wild-type cells. Upon return to the permissive temperature, cell size is reduced by the combined effects of an accelerated division rate together with a decelerated growth rate.

Polysomal organization in growing and resting cultures and its relation to protein synthesis in Tetrahymena.

Cytosol from Tetrahymena cells growing at different rates was isolated and separated by centrifugation into polysomal and non-polysomal fractions. The RNAs of either fraction were separated chromatographically into poly(A)+ RNA and poly(A)-RNA. It was found that in resting cultures the total RNA per cell is only about half of that of rapidly growing cultures. All fractions of RNA were reduced proportionally. Thus, the percentage of polysomally bound total RNA (70% of cytosol RNA) and polysomally bound poly(A)+ RNA (72% of cytosol poly(A)+ RNA) is the same in growing and resting cultures. Differences, however, were found in the polysomal structure. Polysomes from resting cultures contained significantly fewer ribosomes. The amounts of RNA bound to polysomes were related to the rate of protein synthesis under different growth conditions. The decrease in cellular RNA corresponded well with the reduction in amino acid incorporation in resting cells. The rate of protein accumulation in resting cells, on the other hand, was considerably less, suggesting that polypeptides in resting cultures are less stable.

Mean macronuclear DNA contents are variable in the ciliate Tetrahymena.

Using cytophotometry the mean macronuclear DNA content of the ciliate Tetrahymena was found to be variable in two cell lines examined. While environmental changes (different culture fluids, temperature shifts) influence the mean DNA content of at least one of the two cell lines, spontaneous changes of the means are observed in both lines. The mean DNA contents of cultures maintained permanently in defined medium at various temperatures suggest the existence of a lower (7.5 pg) and an upper (13.6 pg) limit of the mean macronuclear DNA contents. Greatly differing means, either below or above those limits, were only occasionally found in cultures shifted to different temperatures for periods of less than 3 days. Eventually, the G2 mean macronuclear DNA contents of these cultures differed greatly from the doubled G1 mean value (maximum: mean G2 value equalling four times the G1 mean). Analysis of the maximum and minimum G1 DNA contents found in any culture indicates no absolute macronuclear DNA content triggering the regulation of macronuclear DNA content. There is no stringent correlation between the average cell volume and the average macronuclear DNA content. The data are discussed with reference to reported mechanisms of regulation of macronuclear DNA content.

External factors limiting the multiplication potential of Tetrahymena.

By variation of nutritional and other external conditions we have determined the factors that limit the multiplication rate and the culture growth in Tetrahymena thermophila. The enriched synthetic medium of Kidder & Dewey (1951), a culture temperature of 29 degrees C, and aeration by agitation were chosen as reference conditions. The final cell density is increased by and proportional to the amount of the complete set of nutrients. Testing single nutritional factors or groups of them revealed that only nitrogen sources yield higher cell densities. But none of them or any combination is as capable of increasing the cell density as the complete medium. Therefore, the medium has to be considered as well balanced. Ammonia, cell density, O2 supply, and pH have been excluded as factors limiting the capacity for multiplication. There are no known factors promoting or inhibiting culture growth.

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 macronuclear 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 of 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.

Increase in macromolecular amounts during the cell cycle of Tetrahymena: a contribution to cell cycle control.

Increases in RNA, protein and cell size were determined cytophotometrically during the cell division cycle of Tetrahymena. For these parameters different patterns were found. RNA accumulates slowly during G1 period and faster during macronuclear S. This agrees with the changing uridine incorporation rate which is at least partly related to the varying macronuclear DNA amount. Increases in protein content and cell size occur mainly during G1 and G2. This pattern was confirmed by determining the RNA : protein ratio in individual cells. It is minimal at the end of the G1 period. These findings and evidence from the literature suggest that initiation of DNA replication is under negative control by the relative RNA content of the cell.

Amount of DNA produced during extra S phases in Tetrahymena.

A protein mixture named LTx, was released from human tonsillar lymphocytes, when incubated at 37 degrees C in either "individual" or "mixed" cultures. LTx caused a decrease in the incorporation of 14-C- labelled amino acids of the target lymphocytes, while the release of 51- Cr from labelled target cells increased. Target cells were found to bind the "toxic" protein.