Effects of some antibiotics on the stringent control of RNA synthesis in Escherichia coli.

Effects of neomycin, spectinomycin, tetracycline and chloramphenicol on the stringent control RNA synthesis and on ppGpp synthesis in the rel+-cells of Escherichia coli having a temperature-sensitive valyl-tRNA synthetase were examined. Without antibiotics, ppGpp began to accumulate and both RNA and protein syntheses were inhibited by transferring the exponentially growing cells from 30 degrees C (permissive temp.) to 40 degrees C (non-permissive temp.). Tetracycline or chloramphenicol, when added after the temperature shift, caused a resumption of RNA synthesis and decay of the accumulated ppGpp, while neomycin or spectinomycin had little effect both on RNA synthesis and the level of ppGpp. When the cells were treated with these antibiotics at permissive temperature, the shift of the temperature to 40 degrees C caused neither inhibition of RNA synthesis nor an accumulation of ppGpp. When neomycin or spectinomycin was added at the beginning of the temperature shift, RNA synthesis continued with an accumulation of ppGpp. Tetracycline or chloramphenicol had no such effect under the same conditions; RNA synthesis continued without an accumulation of ppGpp.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

A possible ribosomal-directed regulatory system in Euglena gracilis. Carbon dioxide fixation

It was shown that cycloheximide inhibits CO(2) fixation in Euglena cells in the dark, but no effect of chloramphenicol was found. The light-dependent CO(2) fixation was inhibited by chloramphenicol and by large amounts of cycloheximide, but was stimulated by small amounts of cycloheximide. The presence of the stimulatory concentration of cycloheximide abolished the inhibition effect of high concentrations of chloramphenicol. The results indicate that the light-dependent CO(2) fixation is controlled by a repression-derepression regulatory system, which seems to be independent of chlorophyll synthesis.     

Inactivation of protein synthesis stimulating activity in serum by cells

When Ehrlich ascites cells were cultivated in serum-free media their cellular protein synthetic rate declined to a new steady-state level and the cells stopped multiplying. On addition of serum the cellular protein synthetic rate increased to the level before serum starvation and cells resumed multiplication. The activity in serum stimulating protein synthesis was inactivated on incubation with cells. At cell concentrations of the usual culture conditions this inactivation took several hours; at very high cell concentrations it was complete in ten minutes. Serum-starved cells inactivated low serum (2%–6%) media in the same length of time. Studies of inactivation of high serum media demonstrated that cells had a limted capacity to inactivate. Cells grown in 10% serum were unable to inactivate. Inactivation was not due to accumulation in the medium of either low molecular or macromolecular cell products. Inactivation was strongly inhibited at 4° or by treatment of cells with fluoride or cycloheximide (long exposure): less inhibited by treatment with 2-deoxyglucose or glutaraldehyde; and slightly inhibited by treatment with cyanide or cycloheximide (short exposure). Inactivating ability was unaffected by trypsinization. These findings are best explained by the hypothesis that cells take up the serum activity by endocytosis.     

Effect of cordycepin (3''-deoxyadenosine) on virus-specific RNA species synthesized in Newcastle disease virus-infected cells.

Cordycepin (3'-deoxyadenosine) has no effect on the size or relative proportions of Newcastle disease virus-specific 18-22S mRNA species nor on the amount or size of the polyadenylic acid associated with them. Cordycepin does, however, cause an inhibition of incorporation of [3H]uridine into 50S virus-specific RNA relative to 18-22S RNA. This inhibition is probably not a direct effect of the drug on the synthesis of 50S viral RNA. Like cycloheximide, another drug which inhibits 50S RNA accumulation in paramyxovirus-infected cells, cordycepin inhibits protein synthesis as measured by amino acid incorporation. It is likely that the inhibition of 50S RNA accumulation is a secondary effect of protein synthesis inhibition. This is supported by the finding that concentrations of cordycepin and cycloheximide, which inhibit protein synthesis to the same extent, have the same effect on the ratio of 50 to 18-22S virus-specific RNA.     

Action of heliomycin on protein and mucleic acid synthesis in Staphylococcus aureus]

Heliomycin inhibited synthesis of RNA in Staph. aureua which was clearly shown in the study of the antibiotic effect on RNA synthesis in the lag phase of the culture development: heliomycin markedly lowered the maximum RNA level in the biomass observed in the culture at the beginning of the exponential growth. On further growth of the culture heliomycin induced a significant retardness of the process of the natural decrease in the RNA biomass level resulting in increased content of RNA in the cells growing in the presence of heliomycin as compared to the control culture. Retarded natural decrease in the RNA biomass level in the presence of heliomycin was observed also on the antibiotic addition just at the beginning of the exponential growth, during the period of maximum RNA accumulation in the cells. Heliomycin had no effect on synthesis and biomass levels of DNA. Heliomycin inhibited the protein synthesis and was close to chloramphenicol by the level of inhibition of the summation protein synthesis in the biomass. However, comparison of the effect of the above antibiotics on synthesis of beta-galactosidase, an individual enzyme protein showed that heliomycin was much less active as an inhibitor of protein synthesis in comparison to chloramphenicol.     

Irreversible Effects of Cycloheximide During the Early Period of Vaccinia Virus Replication

The presence of cycloheximide, an inhibitor of protein synthesis, during the period 30 to 60 min after vaccinia infection produced an irreversible block in virus replication. In contrast (i) cycloheximide given at earlier or later times, even for prolonged periods, did not prevent continuation of the infectious cycle after removal of the drug, and (ii) treatment with cycloheximide during the first 2 hr did not prevent virus growth when the early stages of replication proceeded more slowly due to infection with a low multiplicity of virus. These findings were interpreted as an indication that protein synthesis is required at a critical time in the virus growth cycle. Under the conditions in which brief cycloheximide treatment prevented virus growth, ribonucleic acid (RNA) synthesis continued at an undiminished rate for at least 2 hr after removal of the drug. Although this RNA appeared identical by polyacrylamide gel electrophoresis to "early" viral messenger RNA, it was not found associated with ribosomes or polyribosomes. Failure to observe viral protein synthesis was consistent with the latter finding. It appeared unlikely that the translational block resulted from inadequate removal of cycloheximide, since the effects of the drug were shown to be reversible at earlier or later times in infection or even at the same time when a lower multiplicity of virus was used. Interference with the normal synthesis of specific viral protein factors required for translation was postulated to explain the results.     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.     

Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation

Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by `magic spot'' nucleotides

During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.     

Protein Synthesis and Photosynthetic Recovery in the Resurrection Plant, Selaginella lepidophylla

The effects of inhibition of protein synthesis on whole-plant CO(2) exchange and on protein synthesis during hydration of the resurrection plant Selaginella lepidophylla (Hook and Grev.) were examined. Both chloramphenicol and cycloheximide inhibited the redevelopment of photosynthetic capacity which normally occurs within 24 hours of hydration in the light. The inhibitory effect of chloramphenicol was greater than that of cycloheximide. The onset of chloramphenicol inhibition of net photosynthesis occurred only after 12 hours of hydration. Cycloheximide stimulated net CO(2) influx early after rehydration and inhibited net CO(2) influx after 14 hours of hydration. Total protein synthesis, as measured by l-[(35)S]methionine incorporation, increased through 24 hours of hydration. Based upon the results obtained with the protein synthesis inhibitors, most protein synthesis within the first 12 hours of hydration was cytoplasm-directed, whereas the rate of organelle-directed protein synthesis remained low until 12 hours of hydration and increased rapidly thereafter. These data suggest that both organelle- and cytoplasm-directed protein synthesis are necessary for full photosynthetic recovery during rehydration of S. lepidophylla.     

Noncoordinate control of RNA synthesis in eucaryotic cells

Inhibition of protein synthesis in confluent monolayers of chick fibroblasts stimulates selectively the synthesis of 4S RNA, resulting in a net accumulation of 4S RNA in the inhibited cells. Under these conditions, inhibition of ribosomal RNA synthesis and processing occurs, as does a decrease in soluble uridine phosphate concentrations; increased pools of certain amino acids are also apparent. Recovery of cells from inhibition is accompanied by a rapidly increasing rate of protein synthesis that lasts for several hours. The small molecular weight RNA synthesized during inhibition of protein synthesis appears properly methylated, and in the presence of cycloheximide and actinomycin D shows a precursor-product conversion. Radiolabeled RNA synthesized during inhibition of protein synthesis is stable following the recovery of cells from inhibition. Stimulation of uridine incorporation into 4S RNA during arrest of protein synthesis is also demonstrated in high-density cultures of L- and Hep-2 cells, suggesting that this non-coordinate stimulation of 4S RNA may be a general property of eucaryotic cells.     

Nitrate Reductase Activity of Wheat Seedlings during Exposure to and Recovery from Water Stress and Salinity

Nitrate reductase activity was inhibited as a result of reduced soil moisture potentials or application of NaCI to nutrient solutions. The decrease in enzyme activity of wheat seedlings exposed to salinity, was found 24 hours after exposure to stress. The effect of stress on nitrate reductase was found in cell-free extracts as well as in riro in assays of intact leaf sections. A recovery in enzyme activity was found after irrigation or after removal of seedlings from salinity. While relative water content of the leaves was restored within 3 hours after removal of stress, full recovery of enzyme activity occurred only after 24 hours. Cycloheximide and chloramphenicol suppressed the activity of nitrate reductase in non-stressed seedlings, but had no effect on the activity of plants exposed to salinity. However, during removal of stress, cycloheximide prevented completely the recovery of nitrate reductase, while chloramphenicol did not interfere with the recovery of the inhibited enzyme activity. It is concluded that a fraction of nitrate reductase may be located in the cytoplasm and lost activity during stress, probably due to inhibited protein synthesis. Another fraction which may be associated with chloroplasts, was inhibited by stress due to conformational changes or partial denaturation.     

Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis

When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide. A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells. The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.     

Selective decrease in the rate of cleavage of an intracellular precursor to Rauscher leukemia virus p30 by treatment of infected cells with actinomycin D.

The cleavage of an intracellular 67,000- to 70,000-dalton precursor, termed Pr4 to Rauscher leukemia virus (RLV) p30 protein proceeded at a slower rate when virus-producing cells were treated with actinomycin D (AMD). Treatment with AMD also caused a slight accumulation of Pr4 in purified early virus particles produced by a cell line which usually produces virions that contain little Pr4. The cleavage of other intracellular viral precursor polypeptides was not affected by treatment with AMD. Treatment of infected cells with cycloheximide, on the other hand, allowed the cleavage of Pr4 to proceed at the usual rate for a short period of time before further cleavage was drastically slowed or prevented. The cleavage of several other viral precursor polypeptides was also inhibited by treatment with cycloheximide. Different lines of evidence suggest that the mechanism of action of AMD is not due to a possible indirect effect on protein synthesis. Thus, the rate of cleavage of Pr4 was not affected by the length of pretreatment with AMD between 1 to 8 h. In addition, the combined effect of AMD and cycloheximide, at their maximal inhibitory concentrations, was greater than the effect of either drug alone, indicating the involvement of two at least partially different mechanisms in the action of AMD and cycloheximide. Furthermore, AMD did not affect the pulse labeling of viral precursor polypeptides. These results suggest that the interaction with viral RNA, whose production is inhibited by AMD, accelerates the cleavage of Pr4 to p30 during virus assembly. A hypothetical model is presented to illustrate th possible advantages of having a step in virus assembly in which genomic RNA interacts with a precursor to capsid proteins before the cleavage of that precursor.     

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.     

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.     

Interleukin-1 beta stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production.

The cytokine interleukin-1 (IL-1 beta) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1 beta/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1 beta, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1 beta on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1 beta; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1 beta was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.     

Studies on frost hardiness in Chlorella ellipsoidea II. Effects of inhibitors of RNA and protein synthesis and surfactants on the process of hardening

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were brought into contact withinhibitors of RNA and protein synthesis, and surfactants duringor after hardening at 3°C. Cycloheximide, actinomycin D and 5-fluorouracil inhibited thedevelopment of frost hardiness in the algal cells. In contrastwith cycloheximide, chloramphenicol did not cause complete inhibitionof hardiness increase, even at a high concentration. These resultssuggest that protein synthesis on cytoplasmic ribosomes is indispensablefor the development of frost hardiness, but protein synthesison chloroplast ribosomes has little or no involvement in thehardening process. Triton X-100 and sodium dodecyl sulfate inhibited the developmentof frost hardiness, but sodium cholate and sodium deoxycholatedid not. Hardened cells were more susceptible to sodium dodecylsulfate, in contrast to sodium cholate, sodium deoxycholateand Triton X-100, than unhardened cells. From the results, considerablealterations in both lipids and proteins constituting cellularmembranes appear to be involved in the process of hardening. (Received December 3, 1975; )