Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum by the heat-sensitive system which triggers mitosis and S phase

Temperature shifts from 22 to 32 °C perturb one of the systems responsible for mitosis triggering in the plasmodia of Physarum (Myxomycetes). In order to determine if the same regulatory mechanism could also be involved in some other cell cycle events, the effects of temperature shifts on the peak of thymidine kinase (EC 2.7.1.21, ATP : thymidine 5′-phosphotransferase) synthesis have been studied. At 22 °C, the increase in thymidine kinase (tdk) activity begins shortly before mitosis and is thus always associated with the end of the G2 phase, the mitosis and the beginning of the S phase. The consequences of temperature shifts depend upon their position in the cell cycle. In all cases, a peak of tdk occurs concomitantly with the 32 °C mitosis. But, when the temperature shift is applied 90-15 min before the control metaphase at 22 °C, another peak of tdk is observed at 32 °C in absence of mitosis, but at the same time as the control mitosis at 22 °C. These results indicate that the increase in the synthesis of tdk is controlled by the heat-sensitive regulatory system which plays a role in the onset of mitosis and S phase. We further suggest that the increase in the synthesis of tdk and the triggering of mitosis are both controlled by the amount of a heat-sensitive effector. But the former takes place when the amount of the effector reaches a critical value lower than the value necessary to trigger mitosis.     

Regulation of mitosis onset and thymidine kinase synthesis during the cell cycle of Physarum polycephalum: action of aphidicolin

In Physarum polycephalum (Myxomycetes) aphidicolin has been found to delay metaphase onset when applied to synchronous plasmodia 3 h before control metaphase. In contrast to the action of temperature shifts, aphidicolin treatment did not delay the initiation of the increase of thymidine kinase synthesis (EC 2.7.1.21, ATP-thymidine 5' phosphotransferase) and the decrease of the synthesis of thymidine kinase occurred normally after completion of mitosis in presence of aphidicolin. The amount of thymidine kinase synthesized was larger for aphidicolin treated plasmodia than in the control due to both a longer period of increased synthesis and a higher maximum rate of synthesis. These results were interpreted by postulating the presence of two regulatory pathways. The first one acting on the increase of the synthesis of thymidine kinase and on mitosis onset was sensitive to temperature shifts from 22 to 32 degrees C. The second one acting on mitosis onset only was sensitive to aphidicolin.     

A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature

Summary A heat resistant mutant of E. coli dnaAts46 was isolated, which grows normally only at temperatures above 39°. After a temperature shift from 42° to 32° the mutant overproduces DNA relative to protein. This is due to overinitiation of rounds of chromosome replication at low temperature, as indicated by hybridization and other experiments. The mutation is cotransduced by Pl with ilv and could not be separated from dnaAts46 by transduction.     

In vivo- undin vitro-verhalten temperatursensitiver mutanten des Tabakmosaikvirus

Summary It has been found that certain mutants of TMV with known amino acid exchanges produce abnormally low quantities of infectious virus particlesin vivo at 32°C, although the particles themselves are stable at even higher temperatures. A correlation between temperature sensitivity of the mutantsin vivo and the inability of their A-Proteins to reaggregate to rodsin vitro at 30°C is demonstrated. It is concluded that the amino acid exchanges in coat protein lead to temperature sensitivity of the mutants described.     

Activation parameters and molecular changes induced by substrate hydrolysis of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of three different soluble forms of the enzyme

Summary The Arrhenius plots for the active and low activity soluble forms of the ATPase purified from the membranes ofMicrococcus lysodeikticus grown at 30°C presented discontinuities at 30 and 33°C, respectively. Their activation parameters differed, being highest for the low activity form of the enzyme.Both forms underwent changes in their molecular properties as a consequence of being enzymically active, i.e., upon incubation with substrates at an adequate temperature. These changes consisted of a decrease in the relative mobilities of some of their subunits in dodecyl sulphate polyacrylamide gel electrophoresis, and the temperature at which they occurred depended on the energy of activation of the particular form of the ATPase used. The low activity form required an incubation temperature of 50°C, whereas for an active form 37°C was sufficient.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Extreme temperatures and thermal tolerances for seedlings of desert succulents

Summary Extreme temperatures near the soil surface, which can reach 70°C at the main study site in the northwestern Sonoran Desert, markedly affect seedling survival. Computer simulations indicated that for the rather spherical barrel cactus Ferocactus acanthodes (Lem.) Britt. & Rose the maximum surface temperature decreased 8°C and the minimum temperature increased 3°C as the seedling height was increased from 1 mm up to 50 mm. Simulated changes in shortwave and longwave irradiation alone showed that shading could decrease the maximum temperature by about 5°C for the common desert agave, Agave deserti Engelm., and raise the minimum 1°C. Actual field measurements on seedlings of both species, where shading would affect local air temperatures and wind speeds in addition to irradiation, indicated that shading decreased the average maximum surface temperature by 11°C in the summer and raised the minimum temperature by 3°C in winter.Seedlings grown at day/iight air temperatures of 30°C/20°C tolerated low temperatures of about -7°C and high temperatures of about 56°C, as measured by the temperature where stain uptake by chlorenchyma cells was reduced 50%. Seedling tolerance to high temperatures increased slightly with age, and F. acanthodes was more tolerant than A. deserti. Even taking the acclimation of high temperature tolerance into account (2.7°C increase per 10°C increase in temperature), seedlings of A. deserti would not be expected to withstand the high temperatures at exposed sites, consistent with previous observations that these seedlings occur only in protected microhabitats. Based primarily on greater high temperature acclimation (4.3°C per 10°C), seedlings of F. acanthodes have a greater high temperature tolerance and can just barely survive in exposed sites. Wide ranges in photoperiod had little effect on the thermal sensitivities of either species. When drought increased the chlorenchyma osmotic pressure from about 0.5 MPa to 1.3 MPa, seedlings of both species became about 2°C less tolerant of high temperatures, which would be nonadaptive in a desert environment, and 2°C more tolerant of low temperatures, which also occurs for other species.In conclusion, seedlings of A. deserti and F. acanthodes could tolerate tissue temperatures over 60°C when acclimated to high temperatures and below -8°C when acclimated to low temperatures. However, the extreme environment adjacent to desert soil requires sheltered microhabitats to protect the plants from high temperature damage and also to protect them from low temperature damage at their upper elevational limits.     

The annual reproductive cycle of the freshwater mussel Dreissena polymorpha Pallas in lakes

Summary The annual development of the gonads of Dreissena polymorpha was studied at three sampling sites in two lakes over 3 and 1 1/2 years, respectively. A resting stage occurred after the last spawning in summer/autumn. Oogenesis (accompanied by multiplying segmentation of the oogonia and early growth processes of its oocytes) restarted in specimens at least 1 year old at low temperatures (below 10° C) during winter and early spring. At one location (Fühlinger See) the onset of the spawning season was correlated with an increase of water temperatures above 12° C. At 2 m depth, two main spawning periods in May and August were normally recognized, the first at temperatures of 12–16° C, the second at 16–21° C. It was clearly demonstrated for the first time in Dreissena polymorpha that the oocytes became mature in successive cohorts within one gonad. A female mussel may spawn several times during the reproductive season. At 9 m depth, the onset of spawning also started at about 12° C; this occurred in late summer, with two spawning periods within 1 month at a temperature range of 12–16° C. At another location (Heider Bergsee) the size of the gonads and the oocytes was reduced during April of both years studied, when food supply was low simultaneously with rapidly rising water temperatures in this shallow lake. There was no spawning period during spring. The major spawning period was delayed until July (temperatures 19–22°C). This shows (1) the synchronizing influence of low winter temperatures on the annual reproductive cycle and (2) a temperature threshold of at least 12° C for the start of the spawning processes. The results are discussed with regard to the geographical limits of further spread of Dreissena polymorpha.     

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe

Summary A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25°C but become blocked at DNA replication or mitosis when incubated at 36°C. Thirtynine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambigously assigned to either DNA replication or mitosis but were definitely required for one or the other.Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36°C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons.A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.     

Body surface temperatures of jerboas (Allactaga) in uniform thermal environments

Summary Body surface temperatures of threeAllactaga elater and oneA. hotsoni were measured by infrared radiography at ambient temperatures of 1° to 42°C. In each test the radiant temperature of environmental surfaces was the same as air temperature.At ambient temperatures of 40–42°C, the temperature of the entire body surface was close to ambient temperature. As ambient temperature was lowered toward 1°C, forehead and back temperatures became increasingly greater than ambient temperature (Fig. 3), indicating an increasing thermal flux across these parts of the body. Forehead and back temperatures were linear functions of ambient temperature below thermoneutrality and behaved as expected according to a model of thermal exchange developed here. The surface temperature of the extraordinarily large pinnae remained close to ambient temperature down to 10°C (Fig. 3), indicating that deep pinna temperature likely falls with decreasing ambient temperature and that the pinnae, despite their size, are not major sites of heat loss at low ambient temperatures.     

Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.     

The Effect of Temperature on the Lipid Composition of the Green Alga Botryococcus

The lipid composition of the green alga Botryococcus was studied at three different cultivation temperatures: suboptimal (18°C), optimal (25°C), and supraoptimal (32°C). Cultivation at the supraoptimal temperature was found to considerably inhibit the synthesis of nearly all intracellular lipids, except for triacylglycerides, and to influence their fatty acid composition. In particular, the content of trienoic fatty acids was significantly lower at the supraoptimal than at the optimal cultivation temperature. At the same time, the fatty acid composition of the extracellular lipids of the alga virtually did not depend on cultivation temperature.     

Diel activity and thermoregulatory behavior of a fully aquatic frog: Xenopus laevis

Ten adult Xenopus laevis were tested individually for 48-hr periods, following an initial 24-hr introductory period, in electronic shuttleboxes which allowed them to control water temperatures without operant conditioning. Locomotor activity was recorded via photocell-monitored light beams. The frogs were nocturnal, being nearly twice as active at night as during the day. The mean preferred temperature was 22.4°C, with no significant difference between night (22.5°C) and day (22.3°C), although the modal preferendum shifted from 24°C by day to 22°C at night, with a corresponding change in skewness. The range of voluntarily occupied temperatures was 14–32°C by day and 14–29°C at night. The median thermal preferendum was 22°C both day and night.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Temperature shiftup suppresses synthesis of extracellular proteins inBacillus megaterium

The effects of temperature shifts on the synthesis of an extracellular proteinase and extracellular proteins inBacillus megaterium were studied. A shiftdown (42° to 28°C) brought about an immediate increase of proteinase synthesis by 70%. A shiftup (28° to 42°C) caused a temporary suppression of enzyme formation, which was, at least partially, owing to the inhibition of its mRNA synthesis. The shiftup also brought about a temporary decrease of excretion of all extracellular proteins.     

A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

The sexual agglutination substance is secreted through the yeast secretory pathway in Saccharomyces cerevisiae

Summary A Saccharomyces cerevisiae a strain carrying the secretory mutation sec1, sec7 or sec18 showed no sexual agglutination ability when treated with pheromone at the restrictive temperature 36° C, although the a agglutination substance had accumulated in the cytoplasm. These cells became sexually agglutinable, with a concomitant decrease in the agglutination substance in the cytoplasm, when the temperature was shifted from 36° C down to the permissive temperature 24° C after the addition of, cycloheximide. The a agglutination substance was barely detectable in sec53 cells (a) treated with pheromone at 36° C, indicating that the active a agglutination substance was formed after the export of its precursor into the endoplasmic reticulum. These results indicate that the a agglutination substance is exported through the yeast secretory pathway and that pheromone acts at the level of synthesis of the precursor molecule of the substance. An strain carrying sec1, sec7 or sec18 behaved just like an a strain carrying the sec gene in the induction of agglutination ability by the opposite mating type sex pheromone.     

Duration of larval and spawning periods inPagrus auratus (Sparidae) determined from otolith daily increments

Synopsis Counts of pre-metamorphic and post-metamorphic daily increments in the sagittae of settled juvenilePagrus auratus were used to determine duration of the larval period and to back-calculate spawning dates. The duration of the larval period was 18–32 days, and was longer for snapper spawned early in the spawning season, when water temperatures were low, than for snapper spawned later in the season when temperatures were high. Sagitta size at metamorphosis was unrelated to duration of the larval period or temperature, and mean increment width during the larval period increased with temperature. These results suggest that metamorphosis is size- rather than age-dependent. Back-calculated spawning dates ranged from September to March, and peaked in November-January. Maximum spawning season duration was five months. Spawning onset was earlier when spring water temperature was higher than normal, and first spawning occurred at 14.8–15.6 °C over three seasons, indicating that spawning onset is temperature-dependent.     

The physiology of hypothermia in the black-capped Chickadee,Parus atricapillus

Summary The shivering, body temperature, and metabolic response to stable and decreasing ambient temperature were measured in winter acclimatized Black-capped Chickadees,Parus atricapillus. Shivering activity, measured by duration and amplitude of bursts, increased curvilinearly from thermoneutral temperatures of 27°C down to 0°C. This parabolic shivering response may be a major component of the curvilinear response of metabolism to decreasing ambient temperature.Birds exposed to 0°C exhibited metabolism 32–45% lower than predicted for a 12-g homeotherm and body temperatures 10°C below the pre-experimental nocturnal body temperature. This hypothermia was not the result of a breakdown in thermoregulation, but was a controlled effort serving to reduce overnight energy expenditure. It is suggested that (1) hypothermia was achieved by decreased shivering by pectoral muscles during exposure to decreasing ambient temperatures, (2) the rate of body temperature decline was moderated by intermittent and reduced bursts during the cooling period, and (3) body temperature was maintained at a particular level during exposure to a stable low ambient temperature by intense bursts lasting one to three minutes.The physiology of hypothermia in chickadees is similar to torpor; however, chickadees did not arouse to a normal diurnal body temperature in the laboratory, and their hypothermia was not induced by inanition or prolonged exposure to cold, as reported for other species capable of torpor.     

Mutants of Agrobacterium tumefaciens with temperature sensitivity in respect to their tumor inducing ability

Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.