Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.     

The Full Schedule of Macronuclear DNA Synthesis is Not Required for Cell Division in Paramecium tetraurelia1 1

Macronuclear DNA synthesis normally continues until late in the cell cycle in Paramecium; however, blockage of macronuclear DNA synthesis after 0.72 in the cell cycle does not alter the occurrence or timing of the subsequent cell division. When DNA synthesis is blocked after cells have reached the transition point, macronuclear DNA content at the following division is reduced to about 75% of the normal level. The point at which macronuclear DNA synthesis is no longer required for division corresponds to the beginning of micronuclear mitosis and the early stages of oral morphogenesis.     

Selective Autolysis of Nuclei as a Source of DNA Precursors in Paramecium aurelia Exconjugants*

The fate of labeled DNA in macronuclear fragments of starving Paramecium aurelia exconjugants was studied by quantitative autoradiography. Labeled material originally contained in DNA of macronuclear fragments is incorporated into macronuclear anlagen. During the starvation period the mean number of macronuclear fragments per cell decreased exponentially while there was an approximately exponential increase in the volume of macronuclear anlagen. Fragments appeared to be selectively and individually autolyzed. Labeled material originally contained in fragments was largely if not completely conserved through 108 hr of starvation during which more than 90% of the fragments were lost. Soluble labeled material was detectable after autolysis of fragments began, but finally almost all labeled material was incorporated into macronuclear anlagen.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.     

The Time of DNA Synthesis During the Interdivision Growth Cycle of Tetrahymena pyriformis Fed on Living Bacteria

SYNOPSIS. It has been possible to obtain selective labeling of the macronucleus of Tetrahymena pyriformis fed on living Escherichia coli. The bacteria themselves, a thymidine requiring mutant, were labeled by exposure to tritiated thymidine in a lettuce infusion medium supplemented with trypticase broth. Various patterns of labeling were seen in synchronized Tetrahymena when the radioactive bacteria were given at particular times during the growth cycle. These patterns have been interpreted as indicating the duration of the G₁, S, and G₂ periods; they also suggest that a soluble pool of thymine exists in this animal from one S period to the next.     

Selective inhibition of DNA synthesis in macronuclear fragments in Paramecium aurelia exconjugants and its reversal during macronuclear regeneration

In Paramecium exconjugants very rapid DNA synthesis takes place in the developing macronuclear anlagen, while DNA synthesis is suppressed in macronuclear fragments. The rate of DNA synthesis in fragments (as a percentage of the rate in anlagen or macronuclei in the same cells) decreases by about 40% during each successive cell cycle over at least the first five cell cycles after conjugation, even though macronuclear anlagen are fully mature by the end of the second cell cycle. — Suppression of DNA synthesis in macronuclear fragments is reversible. If macronuclear anlagen are removed at fission, a very high rate of DNA synthesis resumes in macronuclear fragments after a two-hour lag. The total rate of synthesis in the ensemble of macronuclear fragments in cells without anlagen is greater than that in anlagen in control cells. Thus, suppression of DNA synthesis in macronuclear fragments is not the result of any stable differentiation or irreversible change in the fragments but is the result of, and dependent on, the presence of macronuclear anlagen. — The results of injection of cytoplasm from vegetative cells into normal exeonjugants suggest that normal macronuclei produce an inhibitor which selectively suppresses DNA synthesis in macronuclear fragments. In control cells the relative rate of DNA synthesis in fragments ranged from 40 to 70% of that in anlagen in the same cells, while in injected cells the relative rate of incorporation of DNA precursors was suppressed to as little as 7%. The mean level of incorporation into fragments in injected cells was significantly lower than that in controls, suggesting that the injected cytoplasm contained an inhibitor.Contribution 822, Zoology Department, Indiana University. Supported in part by contract COO-235-66 of the USAEC and by grant No. Gm 15410-05 of the USPHS to T. M. Sonneborn.This paper is a portion of a dissertation submitted in partial fulfillment of the equirements for the degree of Doctor of Philosophy.     

Regulation of macronuclear DNA content in Paramecium tetraurelia

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

DNA replication and the nuclear membrane

To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.     

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation and partial renaturation, a high percentage of total macronuclear DNA was found as single-stranded circles. Sequence analysis showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.     

Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena

Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei.     

Tritiated thymidine effects on DNA, RNA, and protein synthetic rates in synchronized L-cells

Exponentially growing L -cells were synchronized by the double thymidine-block method and exposed to high specific activities of tritiated thymidine. DNA, RNA, and protein synthetic rates were measured through one cell cycle with 1-hour pulses of the appropriate C¹⁴-labelled precursors. Equivalent doses of tritiated water were substituted for tritiated thymidine in some experiments. Total amounts of DNA and histones per nucleus were determined photometrically in Feulgen and fast-green stained cells. It was observed that incorporated tritiated thymidine has an effect distinct from that of tritiated water and that it enhances the incorporation of the precursors at specific stages of the cell cycle, to a degree roughly proportional to the dose. Photometric data indicated an increase in DNA net synthesis and a metabolic instability of histones in the H³-thymidine-treated cells, resulting in higher DNA:histone ratios.     

Electron microscope autoradiography of DNA synthesis in the replication band of two hypotrichous ciliates

The synthesis of DNA in two hypotrichous ciliates, Styx sp. and an amicronucleated strain of Oxytricha sp., was studied by high voltage (1000 kV) electron microscopy. High voltage EM permits use of thick sections (0.25-0.40 micron), including serial sections; thick sections produce strong autoradiographic images with relatively short exposure times. The autoradiographs show that DNA synthesis occurs in a narrow part of the rear zone of a replication band in the macronucleus. Macronuclear DNA synthesis occupies a substantial part of the interdivision interval, and micronuclear DNA synthesis in Styx sp. takes place in early prophase at a time when macronuclear DNA synthesis is in its terminal phase.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.     

Electron Microscope Autoradiography of DNA Synthesis in the Replication Band of Two Hypotrichous Ciliates1

The synthesis of DNA in two hypotrichous ciliates, Styx sp. and an amicronucleated strain of Oxytricha sp., was studied by high voltage (1000 kV) electron microscopy. High voltage EM permits use of thick sections (0.25-0.40 μm), including serial sections; thick sections produce strong autoradiographic images with relatively short exposure times. The autoradiographs show that DNA synthesis occurs in a narrow part of the rear zone of a replication band in the macronucleus. Macronuclear DNA synthesis occupies a substantial part of the interdivision interval, and micronuclear DNA synthesis in Styx sp. takes place in early prophase at a time when macronuclear DNA synthesis is in its terminal phase.     

Synthesis of ribosomal DNA in conjugating Tetrahymena.

In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.     

DNA of ciliated protozoa

DNA was isolated from macronuclei and micronuclei of the ciliated protozoan, Stylonychia mytilus under conditions that minimize the possibility of DNA degradation. Macronuclear DNA has an S value of 10 to 11 in sucrose gradients. Macronuclear DNA has an average molecular weight of 1.15×10⁶ daltons and a range of molecular weights of 1.0×10⁶ to 1.95×10⁶ daltons. The average length of macronuclear DNA, measured by electron microscopy, is 0.80 microns and the range is 0.2 to 2.2 microns. Almost all micronuclear DNA pieces are too long to be measured by electron microscopy. The shortest piece of micronuclear DNA found was 15.0 microns in length.