Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mating Types, Breeding System, Conjuàtion and Nuclear Phenomena in the Marine Ciliate Euplotes cristatus Kahl from the Gulf of Naples

SYNOPSIS. The marine ciliate Euplotes cristatus Kahl (Ciliophora, Hypotrichida), collected off Capri, Gulf of Naples, is described in detail. From populations, 6 different mating types, representing 1 variety or syngen, have been isolated. The breeding relations revealed a multiple mating type system characteristic of other members of the Hypotrichida that have been investigated. Presumably a 7th mating type was found which does not mate with any of the others. Although this may belong to another syngen, it could represent a mating type which has not yet reached sexual maturity or 1 which may be in a period of decline. Animals of different mating type do not mate immediately after being mixed but usually 3 or more hours later. An agglutination reaction involving many specimens is absent. Instead, 2 ciliates engage in a “pairing play” before joining firmly in conjugation. Well-fed or actively feeding and dividing ciliates do not mate; mating occurs only after the food becomes gradually depleted or when the food supply is sharply cut off. All mating types appear to be extremely stable. Neither selfing pairs (intraclonal conjugation) nor autogamy have been observed within any clonal culture during the several years under investigation. Cell-free filtrates from 1 mating type do not elicit mating or induce conjugation with specimens of a different mating type. The general pattern of nuclear events in conjugation and exconjugant reorganization is as follows: 1 preliminary division, 3 pregamic (prezygotic) divisions, fertilization, and generally 1 or occasionally 2 postzygotic divisions. The fate of micronuclear products may be determined by their size and location. Those which are larger and close to the cell membranes of the joined conjugants persist and/or divide. Those which are smaller are carried by cyclosis toward the center of each ciliate and degenerate. The degenerating macronucleus of each conjugant becomes segmented in a more or less uniform manner resulting in 4 subspherical masses. Two become localized in the anterior end of a conjugant and 2 in the posterior end. Those in the posterior end are always the first to degenerate completely and disappear. In nuclear reorganization of the exconjugant, fusion of the macronuclear anlage with parts of the old macronucleus does not occur.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

SYNTHESIS OF DEOXYRIBONUCLEIC ACID BY MICRO- AND MACRONUCLEI OF TETRAHYMENA PYRIFORMIS

Evidence as to the times of DNA synthesis in micronucleate Tetrahymena pyriformis (mating type II, variety 1) has been obtained by briefly exposing individuals of different ages to tritiated thymidine, returning them to non-radioactive medium, fixing at division, and preparing autoradiographs. A variable length of interphase, ranging from a few minutes to about 2 hours, has been found to precede the initiation of macronuclear DNA synthesis. Once begun, however, the period of synthesis appears to be similar in all cells, regardless of generation time, and has been estimated at 1 to 1½ hours. Under the conditions of these experiments, the time elapsing between the end of synthesis and subsequent division into daughter cells ranges from approximately 1½ to 2½ hours in generation times long enough to allow such variability. Division of the micronucleus occurs shortly before the cell begins to divide; its DNA synthesis starts immediately and continues after cell division for a total period estimated at about an hour.     

Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.     

Preconjugant cell interaction and cell cycle in the ciliate Euplotes crassus. Switching from the vegetative to the sexual stage.

Cells of Euplotes crassus can mate during the entire extent of G₁ as well in the very early macronuclear S phase. An arrest to cell cycling that occurs during mating is triggered by the ciliary agglutination that takes place between complementary cells, after a waiting period and before the cell bodies fuse into conjugal pairs. Cycling arrest is reversible. Sexually developing cells returned to nutrient conditions are able to reinitiate macronuclear DNA synthesis, although after a lag period. The synchronous reinitiation of synthesis is interpreted as a consequence of reprogramming events that occur as cells switch from the vegetative to the sexual stage of the life cycle.     

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.     

Direct observation of histone H2B-YFP fusion proteins and transport of their mRNA between conjugating Paramecia

Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P. caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B-PcVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B-PcVenus functioned normally. Seven hours after a transformant cell expressing histone H2B-PcVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B-PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear anlagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B-PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B-PcVenus was transferred from the transformed, to the untransformed cell during conjugation.     

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.     

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.     

Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development

BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.     

Adolescence and the Reversibility of Maturity in Euplotes crassus

ABSTRACT. The clonal life history of ciliated protists is characterized by a sequence of phenotypes; sexual immaturity, maturity, and senescence. The distinctiveness of immaturity and maturity has been investigated. Standard assays of the onset of maturity of progeny clones from a cross between stocks EC1 and EC2 of Euplotes crassus demonstrated significant differences among clones and among testers within clones. They also revealed that the first positive test(s) of a progeny subclone were typically followed by at least one negative test. Special protocols were devised to investigate if maturity was reversible at the cellular level. In these experiments, the first mating pair of a progeny subclone was split before the consummation of mating. From these two cells as well as from control progeny and tester cells, subclones were established and every leftover cell was tested for maturity after each transfer. Both standard and split-pair progeny subclones had immature and slow- to-mate cells. The number of fissions before progeny exhibited sexual behavior indistinguishable from the testers was more than twice that to the first mating reaction of a subclone. At the first sign of maturity, progeny lines are a heterogeneous population of cells able and not able to mate, but remarkably, clonal descendants of those able to mate may become unable to mate. The development of maturity is progressive, quantitative and non-monotonic rather than an instantaneous switch.     

A family of DNA sequences is reproducibly rearranged in the somatic nucleus of Tetrahymena.

A small family of DNA sequences is rearranged during the development of the somatic nucleus in Tetrahymena. The family is defined by 266 bp of highly conserved sequence which restriction mapping, hybridization and sequence analysis have shown is shared by a cloned micronuclear fragment and three sequences which constitute the macronuclear family. Genomic Southern hybridization experiments indicate there are five members of the family in micronuclear DNA. All of the family members are present in whole genome homozygotes and are therefore nonallelic. The three macronuclear sequences are all present in clonal cell lines and are reproducibly generated in every developing macronucleus. The rearrangement event begins 14 hours after conjugation is initiated and is nearly completed by 16 hours.     

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.     

Cloning and sequence analysis of the micronuclear and macronuclear gene encoding Rab protein of Euplotes octocarinatus

The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate. A Rab gene was isolated from the macronuclear plasmid mini-library of Euplotes octocarinatus. A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon UGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa. The protein shares 36% identity with Rab 1 protein of Plasmodium and yeast. Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, internal eliminated sequence (IES)1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5'-AAATTTT-3' direct repeats, and IES2 is flanked by 5'-TA-3' direct repeats.     

Integration Pattern of Human JC Virus Sequences in Two Clones of a Cell Line Established from a JC Virus-Induced Hamster Brain Tumor

The physical state of the JC virus (JCV) genome was studied in two clonal cell lines (clones 2 and 7) derived from a tissue culture cell line (HJC-15) established from a hamster brain tumor induced by JCV. Saturation-hybridization and reassociation kinetic analyses, using in vitro (32)P-labeled JCV DNA, indicated that clone 7 and 2 cells contain 9 to 10 and 4 to 5 copies per cell, respectively, of all or most of the viral genome. Both cell DNAs were analyzed by using the Southern blotting procedure with three restriction endonucleases: XhoI, which does not cleave JCV DNA; EcoRI, which cleaves once; and HindIII, which cleaves three times. With each DNA, a variety of JCV-specific DNA fragments were detected. The following conclusions are possible: (i) JCV DNA is integrated into cell DNA in both clonal lines; (ii) both clonal lines contain multiple copies of the viral genome integrated in a tandem head-to-tail orientation; (iii) neither clonal line contains detectable free-form I, II, or III JCV DNA; (iv) each clonal line contains multiple independent sites of JCV DNA integration; and (v) most or all of the sites of integration on the cellular or the viral genome, or both, are different in clone 7 DNA than in clone 2 DNA. Thus, although both clone 7 and clone 2 cells were established from the HJC-15 tumor cell line, they differ in the copy number and integration pattern of JCV DNA.     

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.     

Erythropoietic progenitors capable of colony formation in culture: response of normal and genetically anemic W-W-V mice to manipulations of the erythron

The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes. The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.