Sex determination in hydra: control by a subpopulation of interstitial cells in Hydra oligactis males

The stability of sexual phenotype was examined in a single clone of Hydra oligactis males maintained at two culture temperatures, 18 and 22 degrees C. At these temperatures animals of this species do not reproduce sexually, but reproduce asexually by budding, and males and females are morphologically indistinguishable. When the temperature is lowered to 10 degrees C gametogenesis is induced and sexual phenotype can be assayed. Males cultured for several years at 18 degrees C expressed a stable sexual phenotype when induced to undergo gametogenesis; males remained male. Those cultured at 22 degrees C for 1 year, however, expressed a low frequency of sex reversal from male to female; males ceased sperm differentiation and began producing eggs. Male sex reversal in cultures maintained at the higher temperature was correlated with the loss of a specific subpopulation of interstitial cells, those that bind the monoclonal antibody, AC2, which labels cells specific to the spermatogenic pathway in H. oligactis males. When interstitial cells restricted to this pathway were reintroduced into sex-reversed males (phenotypic females), the male phenotype was reestablished and animals reverted to sperm production. To further investigate the role of AC2+ cells in the masculinization of females, normal males (containing AC2+ cells) and sex-reversed males (lacking AC2+ cells) were grafted to females. In grafts between normal males and females, egg production ceased and sperm differentiation ensued, whereas those between sex-reversed males and females continued to produce eggs. Thus, the presence of AC2+ interstitial cells is strictly correlated with male sexual phenotypes and it is only in their absence that the female phenotype is expressed.     

Genetic analysis of developmental mechanisms in hydra. XVIII. Mechanism for elimination of the interstitial cell lineage in the mutant strain Sf-1

The interstitial cell lineage in mutant strain sf-1 of hydra is temperature sensitive and is lost rapidly from tissue when the animal is cultured at a restrictive temperature of 23 degrees C or higher. The mechanism responsible for this cell elimination process was investigated. Sf-1 polyps were treated at a restrictive temperature of 27 degrees C for varying lengths of time, their tissues were macerated, and the resultant dissociated cells were examined for evidence of phagocytosis after Feulgen staining. It was found that large phagocytic vacuoles were present in the cytoplasm of some epithelial cells. These vacuoles contained partially degraded cells, whose nuclei had highly-condensed and intensely Feulgen-positive chromatin granules. This indicated that, as in colchicine-treated (Campbell, 1976) or starved (Bosch and David, 1984) wild-type hydra, the epithelial cells in strain sf-1 engulfed and disintegrated other cells in the phagocytic vacuoles. The incidence of phagocytosis was higher in sf-1 tissue maintained at elevated temperature than in sf-1 tissue maintained at normal temperature. However, the observed incidence was relatively low (maximally 0.14 phagocytosed cells per epithelial cell) and appeared to be too low to account for the very rapid interstitial cell loss occurring in this strain. We concluded that elimination of the interstitial cell lineage at a restrictive temperature in strain sf-1 takes place in part by phagocytosis and in part by other yet-unidentified mechanisms (cf., Marcum et al., 1980).     

The action of caffeine on X-irradiated HeLa cells. IX. Hypothermic effects

Hypothermic enhancement of the lethal effect of 3.5 Gy of 220-kV X rays in the absence of caffeine as well as in its presence (4 mM) was examined at temperatures between 10 and 34 degrees C in monolayer cultures in the G1 phase of the cell cycle. Correction has been made for the toxicity of low temperatures, and of caffeine at low temperatures, by concomitantly measuring cell killing in unirradiated cells. In the absence of caffeine, incubation of irradiated cells for up to 34 h at temperatures in the range 15 to 30 degrees C (or possibly 34 degrees C) enhances killing compared to that observed at 38 degrees C; the amount of enhancement is about the same throughout this range, but is nil at 10 degrees C. The enhanced killing induced by caffeine at 38 degrees C decreases as the temperature is lowered to 15 degrees C; there is no enhancement at 10 degrees C. Less killing is manifested in the range 15 to 25 degrees C in the presence of caffeine than in its absence. Recovery (loss of sensitivity to caffeine) and fixation of potentially lethal damage were studied in late-S/G2-phase cells at reduced temperatures by delaying treatment with caffeine for increasing times after irradiation. As the temperature is progressively lowered to 20 degrees C, less recovery is manifested after 5 h of incubation; no recovery is detected in the range 10 to 20 degrees C. Despite extensive recovery at 34 degrees C, no fixation is observed at that (or any lower) temperature in G2-phase cells: the cells are able to recover essentially fully when returned to 38 degrees C. In addition, responses of unirradiated control series to incubation at low temperatures appear to differ from those reported by others for longer treatment times of different cell systems.     

Effect of restrictive temperature on cell wall synthesis in a temperature-sensitive mutant of Bacillus stearothermophilus.

A temperature-sensitive mutant of Bacillus stearothermophilus, TS-13, was unable to grow above 58 degrees C, compared to 72 degrees C for the wild type. Actively growing TS-13 cells lysed within 2 h when exposed to a restrictive temperature of 65 degrees C. Peptidoglycan synthesis stopped within 10 to 15 min postshift before a shut down of other macromolecular syntheses. Composition of preexisting peptidoglycan was not altered, nor was new peptidoglycan of aberrant composition formed. No significant difference in autolysin activity was observed between the mutant and the wild type at 65 degrees C. Protoplasts of TS-13 cells were able to synthesize cell wall material at 52 degress C, but not at 65 degrees C. This wall material remained closely associated with the cell membrane at the outer surface of the protoplasts, forming small, globular, membrane-bound structures which could be visualized by electron microscopy. These structures reacted with fluorescent antibody prepared against purified cell walls. Production of this membrane-associated wall material could be blocked by bacitracin, which inhibited cell wall synthesis at the level of transport through the membrane. The data were in agreement with previous studies showing that at the restrictive temperature this mutant is unable to alter its membrane fatty acid and phospholipid composition with temperature such that it is not able to maintain a membrane lipid composition which permits normal membrane function at the restrictive temperature.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

Heat-induced disturbances of intracellular movement and the consistency of the aqueous cytoplasm in HeLa S-3 cells: a laser-Doppler and proton NMR study.

Intracellular particle movements, of both saltatory and streaming types, in HeLa S-3 cells were simultaneously interrupted after 1 h exposure of cells to 43 degrees C, within 10 min at 44 degrees C and within 5 min at 45 degrees C. Intracellular movement inhibited after 15 min at 44 degrees C and 10 min at 45 degrees C was not reversible in cells rescued at 37 degrees C. Brownian motion was not observed in heat-treated cells while they were maintained at elevated temperatures, but became pronounced in blebbing which occurred shortly after they were returned to 37 degrees C. Returning these cells to 45 degrees C intensified the Brownian activity inside blebs, and rapidly induced cell lysis. The same heat-treated cells were simultaneously studied by laser-Doppler microscopy, which confirmed: a) that flow (cytoplasmic streaming) is completely arrested at 44 degrees C within 10 min, b) flow recovered in 10-15 min in cells rescued after 10-15 min at 44 degrees C, c) submicroscopic particles down to the size of water molecules had faster self-diffusion coefficients at 44 degrees C than at 37 degrees C. Proton nmr studies on cells exposed from 4 to 45 degrees C gave corrected relaxation times T1 and T2 which rose with temperature in a predictable manner. Inhibition of cellular movement at elevated temperatures was not specifically attributable to the depletion of intracellular ATP levels.     

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. II. Relationship of myoblast fusion with calcium and temperature.

The effects of calcium and temperature on fusion of quail embryonic myoblasts were examined using cells transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). The transformed quail myoblasts (QM-RSV) fused to form myotubes at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature. On incubation at 41 degrees C, a period of more than 10 hr was needed for the myoblasts to become fusion-competent, but calcium was not needed for development of fusion-competence. Once the cells had become competent, fusion proceeded even at 35.5 degrees C. These results suggest that the src gene product expressed at 35.5 degrees C may control the fusion of cells in the competent stage by inactivating a component(s) that is associated with fusion-competence. However, fusion of even myoblasts in the competent stage was blocked in calcium-deficient medium, suggesting that calcium is essential for the fusion, probably at a step immediately before membrane union. Unlike fusion, other biochemical processes of differentiation proceeded even in calcium-deficient medium, indicating a distinction of fusion from these other processes during myoblast differentiation.     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors

To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33 degrees C) and restrictive (39.5 degrees C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5 degrees C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33 degrees C respond only to isoproterenol (ISO, 10(-6) M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5 degrees C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10(-7) M dDarginine vasopressin (dDAVP/basal: 18.2, apparent Ka: 5 X 10(-9) M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (approximately 50 fmol/10(6) cells) which are undetectable when SV40 genome is activated (33 degrees C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5 degrees C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.     

Morphological and functional differentiation in epithelial cultures obtained from human skin explants

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-phase cells were found to be totally unlabelled. The grain count distributions revealed similar cell cycle subpopulations as have been shown to occur in vivo. The relationship of these subpopulations to the differentiation process is discussed.     

Molecular basis for the maximum growth temperature of an obligately psychrophilic yeast, Leucosporidium stokesii.

Cells of the obligately psychrophilic yeast Leucosporidium stokesii were subjected to permissive (15 and 20 degrees C) and restrictive (23 and 25 degrees C) temperatures to determine the event(s) responsible for the low maximum growth temperature of this organism. An investigation of subcellular morphology by nuclear staining revealed that buds formed at 20 degrees C were anucleate but showed nuclear migration within the parent cell. Cells incubated initially at 23 degrees C and then shifted down to a permissive growth temperature of 15 degrees C in the presence of a deoxyribonucleic acid (DNA) synthesis inhibitor, hydroxyurea, confirmed the observation that the anucleate condition of atypical buds was the result of temperature-sensitive DNA synthesis. Concomitantly, the incorporation of labeled adenine into DNA was inhibited at 23 and 25 degrees C. The synthesis of ribonucleic acid, however, was enhanced at 23 degrees C but impaired at 25 degres C. Similarly, protein synthesis was unaffected at either restrictive temperature.     

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate.     

Germ cells in Hydra oligactis males. II. Evidence for a subpopulation of interstitial stem cells whose differentiation is limited to sperm production

Animals containing germline-restricted interstitial cells were obtained by treating males from a clone of Hydra oligactis with hydroxyurea (HU) to lower the interstitial population to 1 or 2 cells per animal. A 3-day HU treatment produced animals whose interstitial cells did not form somatic cells, but did produce sperm. The isolation of these cells in HU-treated animals has lead us to propose that the interstitial cell population may contain subpopulations which possess different growth dynamics and developmental potentials. Through asexual propagation, we have cloned several animals containing only sperm precursor interstitial cells and have examined the growth and differentiation behavior of these cells in offspring propagated over a 2-year period. Evidence has been obtained which demonstrates (1) the extensive self-renewal capacity of the sperm precursor interstitial cells, and (2) the restricted differentiation capacity of these interstitial stem cells. Factors which affect cells entering and traversing the spermatogenic pathway are also presented.     

Effect of freezing temperature,at which straws were plunged into liquid nitrogen,on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.     

The role of lymphotoxin in target cell destruction induced by mitogen-activated human lymphocytes in vitro. II. The correlation of temperature and trypsin-sensitive phases of lymphotoxin-induced and lymphocyte-mediated cytotoxicity.

The in vitro destruction of phytohemagglutinin (PHA) coated Beta L cells by non-immune human lymphocytes was resolved into two distinct phases--lymphocyte dependent and lymphocyte independent. The initial or lymphocyte-dependent phase occurred within the first 2 hr and proceeded equally well at 34 and 37 degrees C. The amount of lymphotoxin (LT) secreted by PHA-activated human lymphocytes in vitro to PHA stimulation was the same at 34 and 37 degrees C. Antiserum and complement inactivation of the aggressor lymphocytes at various intervals revealed that target cell lysis was lymphocyte independent. However, the latter phase was temperature dependent, i.e., proceeding at the permissive temperature of 37 degrees C, but inhibited at the restrictive temperature of 34 degrees C. Further experiments revealed that LT-induced destruction had the same temperature sensitivity as target cell cytolysis occurring during the lymphocyte-independent step. Trypsin treatment of target cells during an early period of the lymphocyte-independent phase protected the target cell from subsequent death, indicating the aggressor lymphocyte has deposited a cytotoxic effector material on its surface. These results suggest the lymphocyte-dependent stage involves the processes required for the induction of LT synthesis and secretion. The actual cytolysis occurring during the lymphocyte-independent stage may be caused by LT or LT-like material(s) deposited on the target cell surface by the mitogen-activated human lymphocyte.     

Effects of the Mitotic Cell-Cycle Mutation cdc4 on Yeast Meiosis

The mitotic cell-cycle mutation cdc4 has been reported to block the initiation of nuclear DNA replication and the separation of spindle plaques after their replication. Meiosis in cdc4/cdc4 diploids is normal at the permissive temperature (25 degrees) and is arrested at the first division (one-nucleus stage) at the restrictive temperature (34 degrees or 36 degrees). Arrested cells at 34 degrees show a high degree of commitment to recombination (at least 50% of the controls) but no haploidization, while cells arrested at 36 degrees are not committed to recombination. Meiotic cells arrested at 34 degrees show a delayed and reduced synthesis of DNA (at most 40% of the control), at least half of which is probably mitochondrial. It is suggested that recombination commitment does not depend on the completion of nuclear premeiotic DNA replication in sporulation medium.--Transfer of cdc4/cdc4 cells to the restrictive temperature at the onset of sporulation produces a uniform phenotype of arrest at a 1-nucleus morphology. On the other hand, shifts of the meiotic cells to the restrictive temperature at later times produce two additional phenotypes of arrest, thus suggesting that the function of cdc4 is required at several points in meiosis (at least at three different times).     

Relaxation effect of chloramphenicol on the stringent control in Escherichia coli.

In 10B601 (rel+) strain possessing a temperature-sensitive valyl-tRNA synthetase, chloramphenicol prevented the formation of guanosine-3'-diphosphate-5'-diphosphate (ppGpp) as well as the stringent control of stable RNA synthesis, under the conditions where the incorporation of valine into protein was still detectable i.e. at the lower restrictive temperatures. On the other hand, the effect of chloramphenicol was not observed at higher restrictive temperatures above 42 degrees C where the incorporation of valine was completely absent. Pretreatment of 10B601 cells with chloramphenicol before transfer to a high restrictive temperature (43.5 degrees C) did retard the onset of accumulation of ppGpp after the shift-up. Duration of the lag period was dependent on the concentration of chloramphenicol added. In parallel with the inability of the cells to accumulate ppGpp, stable RNA synthesis was permitted to continue at that high temperature. These results suggest that chloramphenicol traps aminoacyl-tRNA at the A-sites of ribosomes by damming-up the small flow of aminoacyl-tRNA under the restrictive supply of amino acids. Unchanged tRNA which has been located at the A-site is replaced by the charged one, thus resulting in the suppression of ppGpp formation and in the restoration of stable RNA synthesis.