Effect of temperature on the duration of mitosis in mammalian cells cultivated outside the body

Nine cell strains of different origin were cultivated at 28--36 degrees with the interval of 2 degrees. During the phase of logarithmic culture growth, the duration of mitosis (Tm) was determined by means of colchicine method. A strict temperatural dependence Tm, obeyed to Arrenius' law was revealed. Temperature range within which Arreinius' law is valid in different cell strains is not alike. Cultivation of L cells and connective tissue cells from Chinese hamster to 39, 41and 42 degrees demonstrated their upper critical point Tm to be for L cells 39 degrees, for connective tissue cells from the Chinese hamster--41 degrees. Electron microscopic investigations demonstrated that cell cultivation within physiological (mitosis destroying) range of temperatures does not notably effect their ultrastructural organization.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

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

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

tsLA23-NRK cells need pp60v-src protein-tyrosine kinase activity in G2 phase to initiate mitosis in serum-free medium.

Lowering the temperature from 41 to 36 degrees C stimulates quiescent tsLA23-NRK rat cells (infected with the tsLA23 mutant of the Rous sarcoma virus) in serum-free medium to resume cycling and initiate DNA replication by reactivating the tsLA23-RSV's abnormally thermolabile pp60v-src protein-tyrosine kinase. Inactivating the enzyme in these pp60v-src-stimulated cells by again raising the temperature to 41 degrees C after the cells had initiated DNA replication did not prevent the completion of DNA replication and entry into the G2 phase, but it stopped the initiation of mitosis. Adding serum at the time of the temperature increase replaced the lost pp60v-src activity and the cells were able to continue to mitosis. The G2-arrested cells at 41 degrees C were able to initiate mitosis when pp60v-src was reactivated again by lowering the temperature to 36 degrees C. These observations suggest that protein-tyrosine kinase activity is needed to initiate mitosis and that the tsLA23-NRK cell is a good model for studying the function of this kinase activity in the initiation of mitosis.     

Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits

Saccharomyces cerevisiae strains of clinical and nonclinical origin were compared by pulse field gel electrophoresis. Complete separation between strains of clinical origin and food strains by their chromosome length polymorphism was not obtained even though there was a tendency for the clinical and food strains to cluster separately. All the investigated strains, except for one food strain, were able to grow at temperatures > or =37 degrees C but not at 42 degrees C. Great strain variations were observed in pseudohyphal growth and invasiveness, but the characters were not linked to strains of clinical origin. The adhesion capacities of the yeast strains to a human intestinal epithelial cell line (Caco-2) in response to different nutritional availabilities were determined, as were the effects of the strains on the transepithelial electrical resistance (TER) across polarized monolayers of Caco-2 cells. The yeast strains displayed very low adhesion capacities to Caco-2 cells (0.6-6.2%), and no significant difference was observed between the strains of clinical and nonclinical origin. Both S. cerevisiae strains of clinical and non-clinical origin increased the TER of polarized monolayers of Caco-2 cells. Based on the results obtained in this study, no specific virulence factor was found that clearly separated the strains of clinical origin from the strains of nonclinical origin. On the contrary, all investigated strains of S. cerevisiae were found to strengthen the epithelial barrier function.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27 degrees C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1 degrees C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28 degrees C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2 degrees C; maximum permissive temperature, from 29.8 to 38.0 degrees C; and maximum survival temperature, from 33.7 to 48.7 degrees C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.     

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

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

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.     

Effect of repeated cooling on the state of the adrenals and corneal epithelial proliferation in white rats]

The effect of repeated cooling on the adrenal glands, on the mitosis and the number of DNA-synthesising cells in the corneal epithelium of albino rats was studied. The animals were cooled by the contact method to 28 degrees C and were exposed to this temperature for an hour daily within 5 days. Strong activation of the suprarenal glands was observed: 2-fold increase in the suprarenal weight, 3-fold decrease in cholesterol content, 4-fold growth of 11-hydroxy-corticosteroid level in the blood and enhanced adrenaline excretion. The average number of mitoses in the corneal decreases 2-fold. Depression was not associated with the change in mitosis rate but with the inhibition of the cell entrance into mitosis in interphase. The level of pathological mitoses did not change. Chronic stress was not accompanied by the change in the number of DNA-synthesising cells or intensity of DNA-symthesis.     

Temperature-dependency of Betanodavirus infection in SSN-1 cell line

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.     

Pre-axonogenesis migration of afferent pioneer cells in the grasshopper embryo

In insects, afferent neurons arise primarily from the ectodermal epithelium in the periphery and differentiate at the site of their precursor mitosis. Here we describe ectodermally derived cells that migrate away from their site of origin and initiate axonogenesis at a distant location. In embryonic grasshopper limb buds, the first two pairs of afferents to differentiate are the pair of Ti1 pioneers at the limb tip and the pair of Cx1 cells found at the base of the limb. While the Ti1 pioneers arise from the mitosis of a pioneer mother cell at the limb tip, the Cx1 cells are shown to emerge from the epithelium at circumferential positions that are approximately 150 degrees apart and that belong to different embryonic compartments. The cells migrate into contact with each other before initiating axonogenesis, and their axons then extend in a new direction that is orthogonal to the route of cell migration.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

A CHO-cell mutant with a defect in cytokinesis.

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie. 34%) of polyploid cells at the permissive temperature of 34 degress C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5 degrees 39.5 degrees. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completion of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivty.     

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.     

Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.     

Temperature- and growth-phase-regulated changes in lipid fatty acid structures of psychrotolerant groundwater Proteobacteria

Cellular fatty acid compositions of five psychrotolerant groundwater isolates representing alpha- and beta-Proteobacteria were studied at temperatures ranging from 8 to 25 degrees C. Unsaturation of straight-chain fatty acids was the most common response to decreasing temperature and was detected in four of the isolates. On solid media, decrease of temperature resulted in a decrease of cyclopropane fatty acids in beta-proteobacterial isolates. The formation of cyclopropane fatty acids depended, however, to a greater extent on the growth phase than the temperature and increased drastically as the cells entered stationary phase. The alpha-proteobacterial isolates contained a branched C(19:1) fatty acid. The formation of the branched C(19:1) increased during growth in the same way as the cyclopropane fatty acids in beta-proteobacterial strains, indicating possibly an analogous formation of the branched fatty acid by methylation of the 18:1 fatty acid. Sphingomonas sp. K6 possessed a novel temperature-induced modification of lipid fatty acids. As temperature decreased from 25 to 8 degrees C, the fatty acid composition shifted from predominantly even-carbon fatty acids to odd-carbon fatty acids. The results show completely different fatty acid modifications in two strains of the same genus Sphingomonas.     

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.