Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.     

Alterations in nuclear phosphoproteins of a temperature-sensitive Chinese hamster cell line exposed to non-permissive conditions

Exposure of the temperature-sensitive Chinese hamster cell line K12 to its non-permissive temperature leads to a selective decrease in some of the nuclear phosphoproteins which are otherwise detectable in cells kept at the permissive temperature. No such temperature-dependent qualitative change is seen in cytoplasmic fractions of K12 cells or in the parent line Wg-1A from which the temperature-sensitive K12 cells are derived.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

Enhancement of the synthesis of specific cellular polypeptides in a temperature-sensitive Chinese hamster cell line (K12) defective for entry into S phase

Temperature-sensitive Chinese hamster cells (K12) have been shown to be defective for the initiation of new rounds of DNA replication when incubated at the restrictive temperature (40.5 degrees). By temperature shift experiments with synchronous cultures, we have marked out the step at which the mutation is expressed as the four hours preceding the initiation of DNA synthesis. The block imposed by the mutation has been shown to be irreversible. In order to approach the biochemical characterization of the temperature-sensitive function in K12 cells, we have analyzed the cellular proteins synthesized under permissive (35 degrees) and restrictive temperatures. The synthesis of three polypeptides is markedly enhanced when K12 cells are incubated at 40.5 degrees. One of them (band B) has turned out to be a useful biochemical marker of the expression of K12 mutation since its synthesis is not affected in other ts-mutants or in hybrids in which K12 mutation is complemented. In addition, the alteration in band B synthesis is irreversible and occurs during the same stage of the cell cycle at which the mutated function is expressed.     

Influence of prophage on the efficiency of plating of Escherichia coli K12

The efficiency of plating of Escherichia coli K12 and its derivatives K12 C600 and K12 C600(lambda) is diminished during the transition period from the lag of the exponential growth phase in cultures grown in tryptone broth. The phenomenon is suppressed in cultures of the strain K12 C600(lambda ind). The reduction in the efficiency of plating is transitory and not influenced by temperature between 30 and 42 degrees C.     

Constancy of the shift-up point in two temperature-sensitive mammalian cell lines that arrest in G1

Two cell cycle-specific temperature sensitive (ts) mutants of mammalian cell lines, AF8 and K12, are known to arrest in G1 when shifted to the non-permissive temperature. We have determined the entry into S of both AF8 and K12 cells in five different growth conditions, namely: (1) quiescent sparse cultures stimulated to proliferative by serum; (2) quiescent dense cultures stimulated by serum; (3) quiescent sparse cultures stimulated by trypsinization and replating; (4) quiescent, dense cultures stimulated by trypsinization and replating; and (5) mitotic cells collected by mitotic detachment. In addition, for each cell line and for each different growth condition, we have determined the shift-up time, i.e., the time at which a shift-up to the nonpermissive temperature no longer prevents the entry of cells into S. In no case did K12 or AF8 enter S at the nonpermissive temperature. At the permissive temperature, the average time of entry into S varied in different growth conditions, and so did the shift-up time. However, in both cell lines, the distance of the average shift-up time from the average time of entry into S was remarkably constant, regardless of the growth conditions. i.e., 1.8 hours in K12 and 8.6 hours in AF8.     

A defect of Kap104 alleviates the requirement of mitotic exit network gene functions in Saccharomyces cerevisiae

A subgroup of the karyopherin beta (also called importin beta) protein that includes budding yeast Kap104 and human transportin/karyopherin beta2 is reported to function as a receptor for the transport of mRNA-binding proteins into the nucleus. We identified KAP104 as a responsible gene for a suppressor mutation of cdc15-2. We found that the kap104-E604K mutation suppressed the temperature-sensitive growth of cdc15-2 cells by promoting the exit from mitosis and suppressed the temperature sensitivity of various mitotic-exit mutations. The cytokinesis defect of these mitotic-exit mutants was not suppressed by kap104-E604K. Furthermore, the kap104-E604K mutation delays entry into DNA synthesis even at a permissive temperature. In cdc15-2 kap104-E604K cells, SWI5 and SIC1, but not CDH1, became essential at a high temperature, suggesting that the kap104-E604K mutation promotes mitotic exit via the Swi5-Sic1 pathway. Interestingly, SPO12, which is involved in the release of Cdc14 from the nucleolus during early anaphase, also became essential in cdc15-2 kap104-E604K cells at a high temperature. The kap104-E604K mutation caused a partial delocalization of Cdc14 from the nucleolus during interphase. This delocalization of Cdc14 was suppressed by the deletion of SPO12. These results suggest that a mutation in Kap104 stimulates exit from mitosis through the activation of Cdc14 and implies a novel role for Kap104 in cell-cycle progression in budding yeast.     

Adaptation of potassium translocation into the shoot of maize (Zea mays) to shoot demand: Evidence for xylem loading as a regulating step

In order to manipulate the shoot demand for mineral nutrients per unit root weight, maize ( Zea mays L.) seedlings were grown in nutrient solution with different temperatures in the root zone and at the shoot base. The aerial temperature was kept uniform at 24/20°C day/night. At a root zone temperature (RZT) of 24°C, shoot growth was reduced by decreasing the shoot base temperature (SBT) to 12°C; at a RZT of 12°C, shoot growth was increased by raising the SBT to 24°C. At both RZT root growth was not affected by the SBT. Thus, the shoot demand for nutrients per unit root was either increased by raising, or decreased by lowering the SBT. The net uptake rate of potassium (K), as determined from accumulation rates between sequential harvests, was not affected within the first 3 days after lowering the SBT, whereas net translocation rates of K into the shoot and translocation rates in the xylem exudate of decapitated plants were markedly reduced. Obviously, translocation of K into the shoot seems to be regulated independently from K uptake into the root cells. Translocation rates of K in the xylem exudate of decapitated plants were markedly reduced when the nutrient solution was replaced by CaCl₂ solution during exudation. But, depending on the SBT before decapitation, significant differences remained in the translocation rates of K even when K uptake from the nutrient solution was prevented.
From the results it is suggested that xylem loading of K is regulated separately from K uptake from the external solution and that the adaptation of K translocation to shoot demand is coupled with an altered capacity of the root for xylem loading.     

Energy storage in the primary step of the photocycle of bacteriorhodopsin.

A pulsed-dye laser low temperature photocalorimeter is used to study the enthalpy differences between light-adapted bacteriorhodopsin (bR568) and its primary photoproduct (K) at 77 K. A key feature of our experimental method is the use of the laser-induced photostationary state as an internal reference. Analyses of the forward (bR leads to K), reverse (K leads to bR), and mixed (bR in equilibrium K) photoreactions were carried out to measure delta H12 = EK - EbR. All three experiments yielded identical values of delta H12 within experimental error (delta Have12 = 15.8 +/- 2.5 kcal mol-1). Accordingly, the primary event in the photocycle of light-adapted bacteriorhodopsin stores approximately 30% of the absorbed photon energy at the 568-nm absorption maximum. We observe that the quantum yields phi f1(bR leads to K) and phi r2(K leads to bR) add up to unity within experimental error: phi f1 + phi r2 = 1.02 +/- 0.19 for phi f1 in the range 0.28-0.33. A theoretical analysis of energy storage in K suggests that at least one-half of the enthalpy difference between K and bR is associated with charge separation accompanying chromophore isomerization.     

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45–55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.     

Control of temperature-dependent synthesis of K99 fimbriae

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.     

Viscosity B-coefficients and activation parameters for viscous flow of a solution of heptanedioic acid in aqueous sucrose solution

Viscosity and density data for the system of heptanedioic acid dissolved in aqueous sucrose solution at temperature range from 288.15 to 313.15 K have been measured. The viscosity B-coefficients for heptanedioic acid in aqueous sucrose solution has been calculated. The effect of temperature and sucrose concentration on the B-coefficients is discussed. On the basis of the Feakins equation, the activation parameters (Deltamu3++, DeltaH3++, DeltaS3++, DeltaG12(0)++, DeltaH12(0)++ and DeltaS12(0)++) for viscous flow of the solution have been evaluated, together with the Gibbs energy of transfer for the solute from the ground state solvent to the hypothetical viscous transition state solvent (DeltaG3++(1-1')). The effect of sucrose concentration and temperature on the activation parameters has been discussed.     

Temperature dependence of the conductivity of individual potential-dependent K+-channels in mollusk neurons]

Using the patch-clamp method temperature dependences of the chord conductance of single potential--dependent slow and fast K+ channels in mollusk neurons were studied. Under control conditions (20 degrees C, 0 mV, [K+]o = 1.5 mM and [K+]i = 100 mM) the conductances of the fast and slow K+ channels were equal to 20-25 pS and 30-40 pS, respectively. Besides, the temperature dependences of the currents through the K+ channels of lesser conductance (5-20 pS) were studied. Some of these channels may be regarded as subtypes of the fast and slow K+ channels named above. It was found that for the channels of all types single channel currents arise with temperature. However, in the range of 10-20 degrees C an anomalous conductance decrease at temperature elevation was observed. For all channels except for the fast one at temperatures above 20 degrees C activation energy (delta Ea) calculated from the Arrhenius plots of the currents was about 4 kcal/mol. At the temperatures below 10 degrees C delta Ea was equal to about 12 kcal/mol. In this temperature range delta Ea had a pronounced potential dependency. Temperature dependences of the fast K+ channel conductance were opposite to those of the slow K+ channel to some extent.     

Energy conversion efficiency in fish: The influence of food intake and temperature on K-lines at rations close to maintenance

Summary Data relating to growth in goldfish at ration levels very close to maintenance are used to calculate maintenance rations and food conversion efficiencies (K) in energetic terms. The relationship between maintenance metabolism and temperature is logarithmic, with a Q₁₀ of 2.2 (12–28°C). A significant positive relationship is apparent between ln K and ration on which the influence of temperature is equivocal. The meaning of this in relation to the findings of other workers is discussed.Abbreviation dbw dry body weight     

EXPERIMENTAL OBSERVATIONS ON THE INFLUENCE OF TEMPERATURE,LIGHT, AND SALINITY ON CELL DIVISION OF THE MARINE DIATOM,DETONULA CONFERVACEA (CLEVE) GRAN2

The influence of 116 combinations of temperature (2, 7, 12, 16 C), salinity (5–35‰ at 5‰ intervals) and light (5 levels) on the mean daily cell division rate ( K ) of the Narragansett Bay clone of Detonula confervacea was examined following appropriate preconditioning. Growth did not occur at 16 C, but was excellent (K = 1.2–1.5) under certain combinations of light and salinity at 2, 7, and 12 C, being somewhat better at the 2 highest temperature levels. At 32%, and 1100–1200 ft-c, K increased approximately 2.5 fold from 0.6 to 1.5 between 2 and 12 C. A light-temperature relationship was found which had the general trend of an increased optimal light intensity with increasing temperature. Within the optimal salinity range of 15–30‰, the optimal light intensity was 200–600 ft-c at 2 C, 600–1200 ft-c at 7 C, and 1200–1800 ft-c at 12 C. The light-temperature relationship was most pronounced at 2 and 12 C. At 2 C, K decreased with increasing light intensity, but was independent of this factor at higher temperatures. The optimal salinity range of 15–30‰ was independent of temperature negligible growth occurred at 5‰. In situ and in vitro responses of Detonula confervacea to salinity were in general agreement but its pronounced cryophilic preference in nature (usually reaching maximum abundance below 1 C) contrasts with its excellent growth at 12 C in culture. The experiments suggest that termination of the bloom of Detonula confervacea in Narragansett Bay and elsewhere is not solely temperature-dependent. Temperature does not satisfactorily account for its apparent exclusion from waters contiguous to Narragansett Bay and from other more northerly portions of the northeastern coast of the U.S, or, together with light, for its equally surprising apparent unimportance in Norwegian coastal waters.     

A precursor in the photolytic cleavage of the Co (III)-C bond of coenzyme B-12 unobservable by electron paramagnetic resonance.

Photolysis of a frozen (80--200 K) anaerobic solution of 5'-deoxyadenosyl-cobalamin in aqueous propan-1,2-diol produces only a small Co(II) signal detectable by electron paramagnetic resonance (EPR). Upon warming to room temperature and refreezing without further irradiation the Co(II) signal increases many-fold. The interpretation is that at low temperature there is an EPR-undetectable "incipient" homolysis of the Co-C bond of the coenzyme which is revealed at higher temperature. The possible implications of this observation for the coenzyme B-12-dependent enzymes are noted.     

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.     

DNA synthesis and mitosis in a temperature sensitive Chinese hamster cell line

Viability, DNA synthesis and mitosis have been followed in the temperature sensitive Chinese hamster cell mutant K12 under permissive and non-permissive conditions. On incubation at 40°C cells retained their ability to form colonies at 33°C for 15 to 20 hours, but viability was lost gradually during the following 20 hours. When random cultures of K12 were shifted to 40°C the rate of DNA synthesis was normal for three to four hours but then decreased markedly, reaching 95% inhibition after 24 hours. Under the same conditions mitosis was inhibited after 15 hours. If cultures which had been incubated at 40°C for 16 hours were placed at 33°C the rate of DNA synthesis increased five hours after the shift down and mitosis 18 hours after. These results can be interpreted on the assumption that K12 at 40°C is unable to complete a step in the cell cycle which is essential for DNA synthesis and which occurs three to four hours before the start of S at 33°C.     

Effect of sub-optimal root zone temperatures at varied nutrient supply and shoot meristem temperature on growth and nutrient concentrations in maize seedlings (Zea mays L.)

Maize seedlings were grown for 10 to 20 days in either nutrient solution or in soils with or without fertilizer supply. Air temperature was kept uniform for all treatments, while root zone temperature (RZT) was varied between 12 and 24°C. In some treatments the basal part of the shoot (with apical shoot meristem and zone of leaf elongation) was lifted up to separate the indirect effects of root zone temperature on shoot growth from the direct effects of temperature on the shoot meristem.Shoot and root growth were decreased by low RZT to a similar extent irrespective of the growth medium (i.e. nutrient solution, fertilized or unfertilized soil). In all culture media Ca concentration was similar or even higher in plants grown at 12 as compared to 24°. At lower RZT concentrations of N, P and K in the shoot dry matter decreased in unfertilized soil, whereas in nutrient solution and fertilized soil only the K concentration decreased.When direct temperature effects on the shoot meristem were reduced by lifting the basal part of the shoot above the temperature-controlled root zone, shoot growth at low RZT was significantly increased in nutrient solution and fertilized soil, but not in unfertilized soil. In fertilized soil and nutrient solution at low RZT the uptake of K increased to a similar extent as plant growth, and thus shoot K concentration was not reduced by increasing shoot growth rates. In contrast, uptake of N and P was not increased, resulting in significantly decreased shoot concentrations.It is concluded that shoot growth at suboptimal RZT was limited both by a direct temperature effect on shoot activity and by a reduced nutrient supply through the roots. Nutrient concentrations in the shoot tissue at low RZT were not only influenced by availability in the substrate and dilution by growth, but also by the internal demand for growth.