Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide.     

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.     

Control of Deoxyribonucleic Acid Synthesis in a Bacillus subtilis Mutant Temperature Sensitive for Initiation of Chromosome Replication

We used a Bacillus subtilis mutant described previously, which is temperature sensitive for initiation of replication. The inhibition of deoxyribonucleic acid synthesis occurring at 45 C was shown to be reversed when the temperature is lowered even in the absence of protein synthesis. If the bacteria are returned to 30 C, after a prior period at 45 C, they are able to initiate the first round of replication in the presence of chloramphenicol, but the initiation of the second round still requires protein synthesis. This paper shows that the proteins necessary to initiate the second round of replication can be present in bacteria long before this round is initiated. In addition, the appearance of these proteins seems to be influenced by the length of the previous 45 C period. Although similar reinitiation kinetics are observed at 30 C after prior 45 C periods of 30 or 65 min, the ability to initiate the second round without further protein synthesis appears much earlier after a longer exposure at 45 C. To explain these results, a hypothesis is presented which assumes that two different proteins are both necessary for initiation. Only one of these proteins could be accumulated at 45 C during the inhibition of deoxyribonucleic acid synthesis. A peculiarity of initiation material in mutant Ts 37 is that it may be active at 45 C if it has been exposed previously at 30 C.     

Reinitiation of chromosome replication in a thermosensitive DNA mutant of Escherichia coli. II. Synchronization of chromosome replication after temperature shifts

A short incubation at the non-permissive temperature, 10 to 15 minutes at 40 °C, suffices to induce chromosome reinitiation in CRT 266, a thermosensitive DNA mutant of Escherichia coli. In order to acquire the potentiality to reinitiate chromosome replication, protein synthesis is necessary, both during the 40 °C incubation and also during the first 15 minutes after returning to 30 °C.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.     

Effects of sodium chloride on prevention of thermodormancy, ethylene and protein synthesis and respiration in Grand Rapids lettuce seeds

Grand Rapids lettuce ( Lactuca saliva L.) seeds entered into a state of secondary dormancy (thermodormancy) when they were imbibed at 40°C for 72 h. The effect of 40°C in inducing thermodormancy was largely reduced by imbibing seeds at 40°C in solutions of polyethylene glycol (PEG), mannitol and NaCl. Despite similar water potentials of solutions, NaCl pretreatment was more effective. Subsequent germination in the dark at 25°C of saline, high-temperature-pretreated seeds required only gibberellic acid (GA₃), as was the case with nonthermodormant seeds. Thermodormancy reduced total respiratory capacity (V_T) and increased the ratio of alternate pathway (V_alt) to cytochrome pathway (V_cyt) respiration. This was prevented by saline pretreatment. Ethylene production and protein synthesis were depressed in thermodormant seeds, and this was partly alleviated by saline pretreatment. The patterns of protein synthesis in saline- and nonsaline-freated seeds at 40°C were similar, differing only in that the saline treated seeds produced in addition a 78 kDa polypeptide. The pattern of protein synthesis at 40°C differed significantly from that at 25°C.     

Gene expression during leaf development in Lolium temulentum: Patterns of protein synthesis in response to heat-shock and cold-shock

At an optimal growth temperature of 20°C, expending 4th leaves of Lolium temulentum L. synthesised a broad spectrum of polypeptides which altered with the maturity of the leaf tissue. Elevation of the temperature to 35°C, or above, induced synthesis of heat-shock proteins (hsp), and all parts of the 4th leaf were capable of this response. The threshold temperature for induction of hsp synthesis was little affected by the growth temperature (5 or 20°C). In contrast, a sudden 15–18°C decrease in temperature did not result in a marked alteration of protein synthesis patterns. It is concluded that in this species adaptation to rapid temperature reduction is not mediated by stress protein synthesis.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Translational efficiency of heat-induced messages in Drosophila melanogaster cells

When Drosophila cells are exposed to elevated temperatures, pre-existing polysomes are depleted and normal cellular protein synthesis is greatly reduced. Polysomes rapidly reform on newly synthesized messenger RNA as the so-called heat shock proteins become the major products of protein synthesis in the cell. These circumstances afford the opportunity to calculate rates of initiation and elongation of protein synthesis directly from measurements of the quantity of actively translated messenger RNA and the quantity of protein produced over a given period. Ribosomes were found to initiate on heat-induced messages in Drosophila with a frequency of between 9 and 14 initiations per minute at 37 °C. This rate is close to that reported for other eukaryotic systems at similar temperatures. Thus, although heat treatment causes a profound change in the patterns of protein synthesis, it does not deleteriously affect the capacity of cells to synthesize protein.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

Cell division in Escherichia coli: evidence for regulation of septation by effector molecules

Evidence regarding the regulation of cell division has been obtained from the study of septation in a mutant of Escherichia coli. The mutant, MX74T2 ts52, gradually stops dividing when transferred from 30 to 41°C in rich medium, but forms long filaments and continues to synthesize DNA and protein. These filaments serve as test objects for the investigation of the regulation of septation. A synchronous cell division of the filaments is induced after 15 minutes, even at 41°C, by the addition of chloramphenicol (100 μg/ml.), rifampicin (200 μg/ml.), or by transfer to minimal medium. Blocking of protein formation with puromycin (500 μg/ml.) or amino-acid analogues does not permit septation. Thus, septation appears to be coupled to inhibition of peptide bond formation rather than protein synthesis. A model for the control of cell division is proposed in which a small effector molecule that is related to peptide bond formation is needed for septation.     

PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.     

Photoinhibition and recovery of photosynthesis in intact barley leaves at 5 and 20°C

Photoinhibition of photosynthesis and its recovery were studied in intact barley ( Hordeum vuigare L. cv. Gunilla) leaves grown in a controlled environment by exposing them to two temperatures, 5 and 20°C, and a range of photon flux densities in excess of that during growth. Additionally, photoinhibtion was examined in the presence of chloramphenicol (CAP, an inhibitor of chloroplast protein synthesis) and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Susceptibility to photoinhibition was much higher at 5 than at 20°C. Furthermore, at 20°C. CAP exacerbated photoinhibition strongly, whereas CAP had little additional effect (10%) at 5°C. These results support the model that net photoinhibition is the difference between the inactivation and repair of photosystem II (PSII); i.e. the degradation and synthesis of the reaction centre protein, Dl. Furthermore, the steady-state extent of photoinhibition was strongly dependent on temperature and the results indicated this was manifested through the effects of temperature on the repair process of PSII. We propose that the continuous repair of PS II at 20°C conferred at least some protection from photoinhibition. At 5°C the repair process was largely inhibited, with increased photoinhibition as a consequence. However, we suggest where repair is inhibited by low temperature, some protection is alternatively conferred by the photoinhibited reaction centres. Providing they are not degraded, such centres could still dissipate excitation energy non-radiatively, thereby conferring protection of remaining photochemically active centres under steady-state conditions.
A fraction of PS II centres were capable of resisting photoinhibition when the repair process was inhibited by CAP. This is discussed in relation to PS II heterogeneity. Furthermore, the repair process was not apparently activated within 3 h when barley leaves were transferred to photoinhibitory light conditions at 20°C.     

Potato cold hardiness development and abscisic acid. II. De novo synthesis of proteins is required for the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

During cold acclimation of potato plantlets ( Solanum commersonii Dun, PI 458317), there are two transitory increases in free ABA content corresponding to a three-fold increase on the 2nd day and a five-fold increase on the 6th day (Ryu and Li 1993). During this period, plantlets increased in cold hardiness from −5°C (killing temperature, control grown at 22/18°C, day/night) to −10°C by the 7th day of exposure to 4/2°C (day/night). This increase in free ABA was not found when cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis, was added to the culture medium 6 h before exposure to low temperatures. Plantlets treated with CHI did not acclimate to cold, maintaining a hardiness level (−5°C) similar to that of the 22/18°C-grown plantlets. When the CHI-treated plantlets were exposed to low temperatures for 3 days, transferred to CHI-free culture medium and grown at low temperatures, the plantlets showed a transitory increase in free ABA 2 days later. This increase was followed by the development of cold hardiness (−8°C). Application of CHI to the culture medium after 3 days of cold acclimation, when the first ABA peak and a partial development of cold hardiness (−8°C) had occurred, blocked the second transitory increase in free ABA and resulted in no further development of cold hardiness. These results suggest that de novo synthesis of proteins is required for these transitory increases in free ABA during cold acclimation of potato plantlets.     

Induction and synthesis of tubulin during the cell cycle and life cycle of Chlamydomonas reinhardi

Two types of tubulin induction are observed in Chlamydomonas reinhardi. One is elicited by flagellar detachment and the other occurs as a normal event of the vegetative cell cycle. In the former case, a strong and extensive induction of tubulin synthesis occurs following deflagellation of cells in all phases of the life cycle [vegetative, gametic, and (early) zygotic]. Synthesis is initiated in all three cell types within 15 min after deflagellation. In gametic and zygotic cells, tubulin synthesis so induced accounts for 15 to 20% of the total protein synthesis during the 1-hr peak period of tubulin production. The ability to support both tubulin synthesis and flagellar regeneration is lost in zygotes at 1.5 hr after the initiation of zygotic development. This alteration represents one of several dramatic shifts in the programming of protein synthesis that occur during the first 4 hr of zygotic differentiation in C. reinhardi. The second (i.e., cell cycle-dependent) type of induction is observed in synchronously growing vegetative cells at ～1.5–2 hr prior to cytokinesis. Tubulin synthesis, in this case, persists at relatively high levels (～5% of the total protein synthesis) for the next 9 hr, i.e., through the entire period of cell division to a time just before the liberation of fully flagellated daughter cells at hr 20 of the cell cycle. Changes in the programming of protein synthesis, and of tubulin synthesis in particular, are discussed in relation to specific physiological and cytological transitions that occur during the growth and differentiation of C. reinhardi.     

Influence of temperature, photoperiod and nutrition on growth and consumption of Chaetopteryx villosa (Trichoptera)

Chaetopteryx villosa Fabr. is uni- or semivoltine. The species was reared at 6°C and 10°C (8 h light, 16 h dark = LD 8/16) and at 10°C and 14°C (LD 14/10). Larval instar duration and pupal period decreased with increasing temperature. Instars II-IV were completed within 2-5 wk. Vth instar larvae pupated after two to twelve months. Pupal development required between one and three months. At 6°C no specimens pupated. At 10°C, LD 14/10 accelerated the development of instars II, IV, V and pupae. Head capsule widths were different between laboratory and field specimens. Relative consumption rate (RCR) ranged between 3.07 ± 1.12 (instar II, 14°C) and 0.09 ± 0.05 (instar V 6°C) mg mg^-1 d^-1. Growth rate, relative growth rate and efficiency of conversion were calculated. In a field experiment fine particulate versus coarse particulate organic material were tested as food sources. FPOM-fed specimens were smaller than those fed with FPOM + CPOM. Pupation was initiated earlier in the FPOM fed group and lasted longer than in the other groups.