Developmental regulation of gene expression in Tetrahymena

The onset of gene expression in Tetrahymena thermophila during macronuclear differentiation was investigated by assay of galactokinase in conjugating deoxygalactose-resistant heterokaryons. Our results distinguish three successive states of galactokinase gene expression for cells developing a new macronucleus: stage 0, refractory to induction; stage 1, inducible by refeeding; and stage 2, induced. The refractory period ends at 12 to 13 hr after the onset of conjugation; this corresponds to the time of pair separation, and occurs several hours after the new macronuclei have become morphologically distinguishable. Stage 1 cells behave indistinguishably from mature starved cells. Inhibitor studies suggest that galactokinase synthesis is induced coincidentally with the induction of bulk protein synthesis during conjugation: thus it behaves developmentally like a typical protein; and that galactokinase mRNA is probably transcribed within 1 hr prior to its translation. Thus, when conjugating cells are refed during the refractory period, some developmental condition prevents the swift induction of protein (and galactokinase) synthesis observed upon refeeding starved (nonmating) cells. The possible nature of this developmental phenomenon is discussed.     

The effects of actinomycin D,cycloheximide and puromycin on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae

The effects of 20-hydroxyecdysone (5 μg/g body weight), cycloheximide (5 μg/g body weight), puromycin (30 μg/g body weight) and actinomycin D (30 μg/g body weight) were investigated on the larval fat body cells of 48–70-hours-old last instar larvae of Pieris brassicae.20-Hydroxyecdysone was found to induce the formation of autophagic vacuoles 3 hr after its administration, but this was, however, prevented by simultaneous cycloheximide treatment in a parallel experiment. On the contrary, puromycin proved to induce autophagocytosis. These diverse effects of the two translational inhibitors on hormone-induced autophagocytosis may be explained by differences in their modes of action.Actinomycin D, when administered 21 hr prior to the hormone, exerted a preventive effect on induced autophagocytosis, but was ineffective when applied 3 hr before the injection of 20-hydroxyecdysone.It was concluded that the presence of RNA synthesized several hours prior to the hormone treatment was a prerequisite for induction of autophagocytosis by 20-hydroxyecdysone.     

Characterization of polyribosomes containing newly synthesized messenger RNA in preimplantation rabbit embryos

Conjugating cells of Stylonychia mytilus incorporate RNA precursors into RNA between 5–6 h after pairing. If RNA synthesis is inhibited during this period by Actinomycin D, the developmental processes are arrested immediately. After 6 h of pairing there is no detectable synthesis of RNA while protein synthesis continues for at least the next 33 h and during this time the cellular events leading to the macronuclear development are insensitive towards the action of actinomycin D. Treatment of live conjugating pairs (older than 6 h) with RNase solution digests the RNA of these cells and causes an immediate and irreversible arrest in development. On the other hand, a similar treatment of the vegetative cells, which show RNA synthesis, does not produce any irreversible effect. It is concluded that stable mRNA molecules are synthesized by the conjugants between 5–6 h following the onset of pairing.     

Mating type differentiation in Tetrahymena thermophila: Strong influence of delayed refeeding of conjugating pairs

Mating type differentiation in Tetrahymena thermophila is known to regularly involve stable hereditary alterations at a single chromosomal locus in the somatic (macro)nucleus. This differentiation is directionally affected by the temperature at which new macronuclei develop after fertilization. We now report large and predictable effects of delayed refeeding of conjugating pairs upon mating type differentiation, particularly among mat-2 homozygotes. The mating types whose frequency is affected the most are IV, VI, and VII, a set different from that most affected by temperature. We interpret our observations to reveal the existence of a second system which can participate in mating type differentiation, with different specificity from the system influenced by temperature under conditions of early refeeding of conjugating pairs. These observations enrich the phenomenology surrounding mating type differentiation in T thermophila and provide additional, easily controllable experimental conditions for the manipulation of mating type frequencies.     

Some Ultrastructural Changes Induced in Resistant and Susceptible Soybean Roots Following Infection by Rotylenchulus reniformis

A developmental electron microscopic study of the parasitism of Rolylenchulus reniforrnis in resistant ''Peking'' and susceptible ''Lee'' soybeans was made during a 21-day period under controlled conditions. Within 2 days of inoculation, the nematode had penetrated the cortical cells to the endodermis where it inserted its stylet, secreted and initiated syncytial formation and cell hypertrophy. Syncytia primarily involved pericycle tissues and, to a lesser extent, xylem parenchyma and endodermis. When identifiable, the cell into which the nematode stylet was inserted to initiate syncytial development was endodermal. Susceptible tissues exhibited two basic phases of development during this infection period: (i) an initial phase represented by partial cell wail lysis and separation; and (ii) an anabolic phase, characterized by organelle proliferation and development accompanied by secondary wall deposits, which provided nutrition for sessile female development. The resistant or hypersensitive reaction (HR) lacked the anabolic phase found in the susceptible reaction, and was characterized by an extension and usually accelerated type of Iysis found in the first phase of the syncytial development. The HR was usually very evident 4 days after inoculation, and could be identified by an almost complete lysis of the cell walls and cytoplasm. The possibility that the initial cell of the developing syncytium or "prosyncyte" may influence a susceptible or resistant reaction is discussed. Successive stages of cell wall dissolution and the deposition of secondary cell walls are described.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

Ultrastructure of salivary glands of Drosophila lebanonensis during normal development and after in vivo ecdysterone administration

Changes in the morphology of the salivary glands of Drosophila lebanonensis have been followed at both the light and electronmicroscopic level during a period of 30 hr before puparium formation and during puparium formation itself. Three striking differences were observed in comparison to other Drosophila species studied: (1) the secretion product of Drosophila lebanonensis has a different stainability to PAS reagent and uranyl acetate and no internal structures or “caps” can be observed; (2) the release of this secretion product is not restricted to a time period shortly before puparium formation but is a continuous process starting about 24 hr before puparium formation; and (3) the histolysis of these glands starts immediately after puparium formation, whereas in other Drosophila species this event starts 5 hr later.Puparium formation of Drosophila lebanonensis is controlled by the circadian oscillation. Injection of ecdysterone before the “gate” period results in changes in the cuticle as observed during normal development, but is not followed by the histolysis of the glands. Injection of ecdysterone after the “gate” is not followed by changes in the cuticle but histolysis is induced.     

Temporal relationships between leaving food, ecdysone release, mucoprotein extrusion and puparium formation in Drosophila virilis

The time sequence of various developmental processes at the end of larval life in Drosophila virilis larvae is reported. If reared at 25·3°C the larvae leave their food about 140 hr after oviposition; 6·6 hr later ecdysone release occurs, while 8·5 to 9 hr after leaving food the mucoprotein, synthesized and stored in the salivary gland cells, is extruded into the lumen of the gland. Puparium formation takes place 11·2 hr after leaving food. Changes in the puffing activity are correlated with these processes.     

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.     

Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila

Conjugating Tetrahymena were irradiated by ultraviolet-B (UV-B) at various stages of conjugation. When the conjugants were exposed to the UV-B at late meiotic prophase (the stage from pachytene to diplotene), abortive conjugation was induced at high frequencies. After completing meiosis, a significant number of the conjugants showed marked anomalies, i.e., failure of nuclear selection after meiosis, and abortion of the subsequent conjugation process such as a postmeiotic division to form gametic nuclei, nuclear exchange, synkaryon formation, and postzygotic development. The conjugating pairs retained the parental macronucleus and separated earlier as compared with a control. The resultant exconjugants degenerated meiotic products and became amicronucleates. These observations strongly suggest the presence of a UV-sensitive molecule that is expressed specifically at the meiotic prophase and that directs the subsequent development after meiosis. Dev. Genet. 23:151–157, 1998. © 1998 Wiley-Liss, Inc.     

Traumatic Induction of Early Maturity in Tetrahymena

Exposure of conjugating pairs of Tetrahymena thermophila to high temperature (37°) during macronuclear development causes an abortion of many macronuclei, but it also often induces an early appearance of sexual maturity in clones completing macronuclear development. Lines become mature after about 15 cell divisions rather than after 50 or more cell divisions in untreated pairs. The phenotype resembles that associated with Em (early maturity) mutants but, because it is not transmitted to the progeny in the next generation, it must be considered a phenocopy. The hypothesis is developed that an early genotype-environment incompatibility, whether associated with an abnormal genotype or an unusual environment, activates a shunt mechanism permitting the organisms to undertake quickly an ordinarily forbidden sexual lottery.     

Mating reaction in Saccharomyces cerevisiae

The process of mating reaction of Saccharomyces cerevisiae was studied by electron microscopy. Prior to the dissolution of the part of the cell walls separating the conjugating pair of cells, the thinning of the electron transparent layer of the cell wall occurs at the part toward which the nuclei are migrating. After the dissolution of the cell walls of the conjugating cells, the cell membranes become associated with each other, then to be broken and rejoined. The first diploid bud emerges from about the middle of the zygote. The morphological changes during the mating reaction are discussed in relation to the biochemical changes so far known.     

Early Changes in the Ultrastructure of Streptococcus faecalis After Amino Acid Starvation

Thin sections of Streptococcus faecalis (ATCC 9790) starved of one essential amino acid (threonine or valine) initially show rapid increases in (i) cell wall thickness, (ii) the apparent size of the central nucleoid region, and (iii) mesosomal membranes. The most rapid increases in all three variables occurred during the first 1 to 2 hr of starvation. After this initial period, the rates progressively decreased over the 20-hr observation period. During threonine starvation, the mesosomal membrane that accumulated in the first hour was subsequently degraded and reached a level similar to that found in exponential-phase cells after 20 hr. With valine starvation, mesosomal membrane continued to slowly accumulate over the entire 20-hr observation period. The mesosomes of the starved cells retained the same “stalked-bag” morphology of those in exponential-phase cells. These cytological observations agree with previously published biochemical data on membrane lipid and wall content after starvation.     

ECL cells of the rat stomach: development of lipofuscin in response to sustained gastrin stimulation

Ageing cells, especially post-mitotic cells, are known to accumulate pigments, i.e. highly electron-dense material, referred to as ceroid or lipofuscin. This material is formed as a consequence of autophagocytosis and peroxidation of the products undergoing degradation. The present study describes the development of lipofuscin in the ECL cells of the rat stomach. These cells produce and secrete histamine in response to gastrin. They are rich in secretory vesicles, which fuse to form vacuoles in hypergastrinaemic rats. Hypergastrinaemia was induced by continuous infusion of human Leu¹⁵-gastrin-17 for 6 days or by daily treatment with omeprazole for 10 weeks. Either treatment caused both vacuoles and lipofuscin bodies to appear in large numbers; the vacuoles disappeared promptly after interruption of the hypergastrinaemia, whereas the lipofuscin bodies remained. Antrectomy-evoked hypogastrinaemia was associated with a reduced number and volume density of lipofuscin bodies. Treatment with α-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, resulted in depletion of ECL-cell histamine and was found to prevent the omeprazole-evoked formation of vacuoles and lipofuscin. The numbers of both vacuoles and lipofuscin bodies were well-correlated with the serum gastrin concentration, suggesting that gastrin stimulates the development not only of vacuoles but also of lipofuscin, perhaps through enhanced autophagocytosis and/or oxidative stress. Thus, lipofuscin bodies may develop from vacuoles, and both vacuoles and lipofuscin bodies may reflect the efforts of overstimulated ECL cells to cope with the excessive formation of secretory products.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Probable somatic DNA rearrangements in mating type determination in Tetrahymena thermophila: A review and a model

Experimental data on mating type determination in T. thermophila, collected by Nanney, Allen, and their collaborators over a period of 25 years, are reinterpreted in the light of our current understanding of macronuclear genetics. A strong case is developed supporting the idea that mating type determination involves the developmental alteration of somatic DNA that occurs regularly in developing macronuclei in conjugating pairs. A. testable DNA deletion/splicing model is developed that although based on a few simple, plausible assumptions, explains the observations remarkably well. The model is in (at least) superficial analogy to the mechanism that must be involved to explain the somatic differentiation and alteration of DNA sequences that ultimately constitute an expressed vertebrate immunoglobulin gene. Because of the genetic, biochemical, and micromanipulative versatility of Tetrahymena, it may well turn out to be a uniquely suitable microbial eukaryotic experimental system for the study of developmental alterations of somatic DNA.     

Morphology and ultrastructure of the serosal cells (teratocytes) in Cardiochiles nigriceps Viereck (Hymenoptera : Braconidae) embryos

The morphogenetic changes of the serosal membrane during embryonic development of Cardiochiles nigriceps Viereck (Hymenoptera : Braconidae) were investigated. Eggs observed soon after oviposition into the natural host Heliothis virescens (F.) (Lepidoptera, Noctuidae) showed a transparent chorion and a uniform texture. After 5 hr, the embryo exhibited a distinct granular appearance and by 12 hr attained the germ band stage. A serosal membrane originated from the anterior pole of the embryo between 14 and 15 hr after the egg was laid, eventually forming with the cells both in the anterior and posterior pole a continuous envelope around the developing embryo.Ultrastructural observations revealed that the serosal cells in contact with the abdominal region of the embryo, beginning 24–25 hr after oviposition, formed a syncytium. However, the syncytial tissue did not extend to the cells around the head and thorax. The serosal cells at both embryo poles increased in size without losing their structural organization, and developed into teratocytes when the larva hatched. In contrast, the serosal cells surrounding the body of the embryo persisted longer on the head and thorax region of the newly hatched larva, while the syncytial tissue degraded more rapidly after hatching.In vitro rearing experiments showed that C. nigriceps embryos removed from parasitized host larvae just before and just after serosa formation, hatched only when the medium used was formulated with the addition of fetal bovine serum. Embryos did not develop or hatch when placed in a serum-free medium. Once the syncytium deriving from the serosal membrane became evident, embryos readily developed and hatched in serumfree media. The results of this study seem to suggest that the serosal embryonic membrane could have a nutritional role for the developing parasitoid embryo.     

Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A⁺) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.