In situ localization and in vitro induction of plant COPI-coated vesicles

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.     

Biosorption of nickel and cadmium by metal resistant bacterial isolates from agricultural soil irrigated with industrial wastewater

Agricultural soil irrigated with industrial wastewater (more than two decades) analysed for heavy metals revealed high levels of Fe, Cr, Cu, Zn, Ni and Cd. Out of a total of 40 bacterial isolates obtained from these soils, 17 belonged to the family enterobacteriaceae and 10 were Pseudomonas spp. A maximum MIC of 200 for Cd, 400 for Zn and Cu, 800 for Ni, and 1600 microg/ml for Pb was observed. Biosorption of Ni and Cd studies over a range of metal ion concentrations with Escherichia coli WS11 both in single and bi-metal systems showed that the adsorption of Cd and Ni was dependent on the concentrations and followed the Freundlich adsorption isotherm. The biosorption of Ni increased from 6.96 to 55.31 mg/g of cells, and Cd from 4.96 to 45.37 mg/g of cells at a concentration ranging from 50 to 400 microg/ml after 2h of incubation in a single metal solution. A further increase in incubation time had no significant effect on the biosorption of metals.     

A new insight into lead (II) tolerance of environmental fungi based on a study of Aspergillus niger and Penicillium oxalicum

Environmental microorganisms have been widely applied in heavy metal remediation. This study explored the mechanisms of lead tolerance of two typical filamentous fungi, Aspergillus niger and Penicillium oxalicum. It is shown that the mechanisms of reducing Pb toxicity by these two fungi have three major pathways. The secreted oxalic acid can react with Pb (II) to form insoluble Pb minerals, primarily lead oxalate. Then, the enhanced biosorption via forming new border of cell wall prevents the transportation of Pb (II) into hypha. In addition, the fungal activity could be maintained even at high Pb concentration due to the intracellular accumulation. It was confirmed that A. niger has the higher Pb tolerance (up to 1500 mg l⁻¹ Pb level) compared with P. oxalicum (up to 1000 mg l⁻¹). Meanwhile, Pb levels below 1000 mg l⁻¹ partially stimulate the bioactivity of A. niger, which was confirmed by its elevated respiration (from 53 to 63 mg C l⁻¹ medium h⁻¹). This subsequently enhanced microbial functions of A. niger to resist Pb toxicity. A better understanding of Pb tolerance of these two fungi sheds a bright future of applying them to remediate lead-contaminated environments.     

Effects of Cu2+, CrO4(2-), Co2+ and Pb2+ on the monovalent ion content of goldfish (Carassius auratus gibelio) tissues studied by X-ray microanalysis

Carassius auratus gibelio was chosen as an organism with a high level of tolerance against heavy metal to investigate changes in monovalent ions content in its tissues. Fish were kept in 10 ppm Cu2+ (3h), CrO4(2-) (96h), Co2+ (96h), Pb2+ (8h) and control (96h) solutions, then tissues were dissected and prepared for X-ray microanalysis. K+, Na+ and Cl- concentration was measured and calculated. Short periods of time of fish acclimation to Cu2+ (3h) and Pb2+ (8h) caused fish to suffocate as a consequence of heavy metal ions binding to gill mucopolisaccharides. Cl- and Na+ content decreased after Cu2+ treatment in kidney cells and muscle fibers, and so did K+ concentration in gill cells in comparison to control. After that CrO4(2-) ions acclimation changes in all tissues and in all measured ions were observed. Similar effects were observed in Co2+ ions treatment but not for muscle fibers. Pb2+ ions caused an elevation of Cl- and Na+ ions content in gill cells and muscle fibers but decreasing in liver and kidney cells in comparison to control. Changes in monovalent ions concentration are probably related to heavy metal ions influence on ionic pump activity, their interaction with metabolic enzymes, ATP production or membrane phospholipids.     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Biosorption of Pb(II) and Cu(II) by activated sludge in batch and continuous-flow stirred reactors

Biosorption of Pb(II) and Cu(II) ions in single component and binary systems was studied using activated sludge in batch and continuous-flow stirred reactors. In biosorption experiments, the activated sludge in three different phases of the growth period was used: growing cells; resting cells; dead or dried cells. Because of the low adsorption capacity of the non-viable activated sludge especially in the case of Pb(II) ions, biosorption of the Cu(II) and Pb(II) ions from the binary mixtures was carried out by using the resting cells. The biosorption data fitted better with the Freundlich adsorption isotherm model. Using a mathematical model based on continuous system mass balance for the liquid phase and batch system mass balance for the solid phase, the forward rate constants for biosorption of Pb(II) and Cu(II) ions were 0.793 and 0.242 1 (mmolmin)(-1), respectively.     

耐铅镉菌株的分离鉴定及其吸附能力

堆肥中添加生物钝化剂是当前降低粪便中重金属生物毒性最为有效的方式之一,为了进一步提高其钝化重金属的能力,文中获得了复合重金属高耐性的钝化剂菌株,并探究其生物学特性和吸附特征。采集猪粪堆肥样品并在改良的牛肉膏培养基中分离和筛选耐铅又耐镉的高耐性菌株,通过形态结合分子生物学鉴定该菌株。该菌株分别在不同pH、温度和盐浓度条件下培养获得其最适的生长条件,进而在该条件下分析其对铅镉吸附的特性。结果获得一株耐铅浓度为600 mg/L、镉浓度为120 mg/L的铅镉复合耐性菌株SC19,该菌株为西地西菌属,其最适生长环境为pH值7.0、温度37℃、盐浓度0.5%。培养36 h的稳定期SC19菌株在吸附时间30min时铅的去除率最高,对铅的最大去除率和吸附量分别为60.7%和329.13mg/g;培养8h的对数期在吸附时间30min时镉的去除率最高,对镉的最大去除率和吸附量分别为51.0%和126.19 mg/g。红外光谱分析显示,SC19菌株对铅镉离子的吸附主要是细胞表面的多种活性基团与金属离子发生络合作用。该研究揭示了SC19菌株有较好的二价态铅镉离子吸附能力,可为生物钝化重金属提供重要的微生物种质资源。     

Cadmium-induced apoptosis in C6 glioma cells: Influence of oxidative stress

Cadmium has recently been shown to induce apoptosis in C6 glioma cells via disruption of the mitochondrial membrane potential and subsequent caspase 9-activation. Here we show that both H₂O₂ and CdCl₂ induced apoptotic DNA fragmentation in C6 cells. The employment of glutathione as an antioxidant prevented the induction of apoptotic DNA fragmentation by cadmium completely and catalase strongly reduced cadmium-induced DNA fragmentation suggesting that cadmium exerts its apoptotic effects at least partly via the production of H₂O₂. Apoptosis may be induced by cadmium indirectly through formation of oxidative stress, e.g., by inhibition of antioxidant enzymes. After incubation of C6 cells with cadmium for short times (up to 4 h), we analyzed the formation of intracellular reactive oxygen species and cellular lipid peroxidation. After 1 h of incubation with inreasing concentrations of CdCl₂ (1–500 M), no increase in dichlorofluorescein fluorescence was found. At variance, lipid peroxidation was slightly elevated after 2 h incubation with cadmium (50–100 M). Furthermore, we analyzed the modulation of markers for oxidative stress after prolonged (24 h) exposure to cadmium. The intracellular glutathione content as measured using the fluorescent probe monobromobimane was decreased after incubation with CdCl₂ (0.5–10 M) for 24 h. Furthermore, we measured the effect of cadmium on the level of oxidized DNA lesions (predominantly 8-hydroxyguanine) using the bacterial Fpg-DNA-repair protein. After 24 h of incubation with 5 M CdCl₂ we found a sixfold increase in Fpg-sensitive DNA-lesions. We conclude that short time incubations with cadmium (up to 4 h) caused only slight or insignificant effects on the generation of reactive oxygen species (formation of thiobarbituric acid reactive substances, fluorescence of dichlorofluorescein), whereas incubation with this heavy metal for 24 h lead to a decrease in intracellular glutathione concentration and an increase in oxidative DNA-lesions. Our data demonstrate that cadmium as similar to H₂O₂ is a potent inducer of apoptosis in C6 cells. Even if cadmium unlike Fenton-type metals can not produce reactive oxygen species directly, the apoptotic effects of cadmium at least in part are mediated via induction of oxidative stress. Because both apoptosis and oxidative stress are thought to play important roles in neurodegenerative diseases, low concentrations of cadmium that initiate programmed cell death may lead to a selective cell death in distinct brain regions via generation of oxidative stress.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells

The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances.     

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.     

Biosorption characteristics of Aspergillus flavus biomass for removal of Pb(II) and Cu(II) ions from an aqueous solution

The Pb(II) and Cu(II) biosorption characteristics of Aspergillus flavus fungal biomass were examined as a function of initial pH, contact time and initial metal ion concentration. Heat inactivated (killed) biomass was used in the determination of optimum conditions before investigating the performance of pretreated biosorbent. The maximum biosorption values were found to be 13.46 +/- 0.99 mg/g for Pb(II) and 10.82 +/- 1.46 mg/g for Cu(II) at pH 5.0 +/- 0.1 with an equilibrium time of 2 h. Detergent, sodium hydroxide and dimethyl sulfoxide pretreatments enhanced the biosorption capacity of biomass in comparison with the heat inactivated biomass. The biosorption data obtained under the optimum conditions were well described by the Freundlich isotherm model. Competitive biosorption of Pb(II) and Cu(II) ions was also investigated to determine the selectivity of the biomass. The results indicated that A. flavus is a suitable biosorbent for the removal of Pb(II) and Cu(II) ions from aqueous solution.     

Biosorption Potentiality of Living Aspergillus niger Tiegh in Removing Heavy Metal from Aqueous Solution

The species of Aspergillus niger Tiegh isolated from estuarine sediments has been studied for tolerance to heavy metals such as Hg and Pb and for its capacities to uptake metals. A. niger was allowed to grow in monometal- as well as bimetal-containing media (25 mg L^?1) to determine the biosorption capacity of the organism. The effects of temperature and pH on biosorption were studied to elucidate the biosorption property and optimum growth conditions for the organism. Results revealed that 91.1% of Pb and 97.1% of Hg were removed from the monometal solutions, and there was a reduction of 96.9% of Hg and 89.3% of Pb from the bimetal solution after 92 h of fungal growth. The binding mechanism involved between metal ion and functional groups present on the cell surface of the biomass was studied using Fourier transform infrared (FTIR), which confirms the presence of amine, hydroxyl, carboxyl, and phosphate groups. The adsorption of metal ions on the biomass surface was confirmed using scanning electron microscopy–energy dispersive x-ray (SEM-EDAX) studies. The experimental study proved that A. Niger can be used as a suitable biosorption agent for removing metal ions when present in low concentration.     

微生物胞外多糖的合成及其在重金属修复中的作用机制与应用

重金属的生物不可降解性使其在环境中长期存在,导致严重的环境污染,对人类健康和生态系统构成威胁。与传统的物化修复技术相比,微生物修复具有成本低廉、环境友好和高效等特点。在面对重金属胁迫或营养不均衡时,微生物会被激发以分泌合成胞外多糖(exopolysaccharides, EPS)。由此可见,EPS的产生是微生物对抗重金属胁迫的重要策略之一。EPS不仅能保护微生物在低温、高温、高盐等极端环境或受毒性化合物胁迫的条件下存活,并且在细胞内外进行信息和物质的交流与传递,既作为保护屏障限制重金属离子进入细胞,又作为介质进行交流。EPS结构中含有多个带负电荷的官能团,能够与重金属离子发生络合、离子交换、氧化还原等反应,从而降低重金属的生物有效性并减轻其毒性。微生物EPS在重金属胁迫环境中的修复具有重要意义。然而,目前缺乏关于微生物EPS合成过程、与重金属互作机制及其在重金属胁迫环境中应用现状的系统综述。本文概述了微生物EPS及其分类,详细阐述了细菌EPS胞内及胞外的生物合成机制,并探讨了微生物EPS与重金属互作机制,以及微生物EPS修复水、土环境中重金属污染方面的研究进展。最后,展望了EPS合成及其在重金属修复中的作用机制研究,可为微生物EPS进一步应用于环境重金属污染修复提供支持。     

Sclerotial biomass and carotenoid yield of Penicillium sp. PT95 under oxidative growth conditions and in the presence of antioxidant ascorbic acid

AIMS: To determine the effect of oxidative stress and exogenous ascorbic acid on sclerotial biomass and carotenoid yield of Penicillium sp. PT95. METHODS: In this experiment, high oxidative stress was applied by the inclusion of FeSO(4) in the growth medium and exposure to light. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous ascorbic acid (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 48 h), decrease of sclerotial biomass (up to 40%) and reduction of carotenoid yield (up to 91%). On the contrary, the exogenous ascorbic acid also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus. CONCLUSIONS: Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 305 mg and 32.94 microg, which were 1.23 and 3.71 times higher, respectively, than those at low oxidative stress growth condition. These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that strain PT95 may be used for solid-state fermentation of carotenoid production under high oxidative stress growth conditions.     

Membrane-Elasticity Model of Coatless Vesicle Budding Induced by ESCRT Complexes

The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.     

Biosorption of cadmium and lead ions by ethanol treated waste baker's yeast biomass

The biosorption of cadmium and lead ions from artificial aqueous solutions using waste baker's yeast biomass was investigated. The yeast cells were treated with caustic, ethanol and heat for increasing their biosorption capacity and the highest metal uptake values (15.63 and 17.49 mg g(-1) for Cd(2+) and Pb(2+), respectively) were obtained by ethanol treated yeast cells. The effect of initial metal concentration and pH on biosorption by ethanol treated yeast was studied. The Langmuir model and Freundlich equation were applied to the experimental data and the Langmuir model was found to be in better correlation with the experimental data. The maximum metal uptake values (qmax, mg g(-1)) were found as 31.75 and 60.24 for Cd(2+) and Pb(2+), respectively. Competitive biosorption experiments were performed with Cd(2+) and Pb(2+) together with Cu(2+) and the competitive biosorption capacities of the yeast biomass for all metal ions were found to be lower than in non-competitive conditions.     

Biosorption of cadmium(II), lead (II) and copper(II) with the filamentous fungus Phanerochaete chrysosporium

The biosorption from artificial wastewaters of heavy metals (Cd(II), Pb(II) and Cu(II)) onto the dry fungal biomass of Phanerochaete chryosporium was studied in the concentration range of 5-500 mg l(-1). The maximum absorption of different heavy metal ions on the fungal biomass was obtained at pH 6.0 and the biosorption equilibrium was established after about 6 h. The experimental biosorption data for Cd(II), Pb(II) and Cu(II) ions were in good agreement with those calculated by the Langmuir model.     

Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

The aim of this study was to evaluate melatonin cytotoxicity by measuring its effects on various cellular targets. Cell viability, intracellular reduced glutathione (GSH) level, and reactive oxygen species (ROS) production were assessed in the human liver cell line (HepG2), after incubation with increasing melatonin concentrations (0.1-10,000 microM). The incubation times tested were 24, 72, and 96 h for cell viability and intracellular GSH level, and 15 and 45 minutes for ROS production. Cellular target evaluations were possible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, and ROS overproduction with, respectively, neutral red, monochlorobimane (mBCl), and 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1-10 microM) and for a relatively short incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability improvement. In contrast, after longer incubation (96 h), cell viability significantly decreased with these lowest melatonin concentrations (0.1-10 microM). Moreover, high melatonin concentrations (1,000-10,000 microM) induced GSH depletion. This oxidative stress is associated with ROS overproduction from 10 microM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the human liver cell line, depending on the concentration and incubation time.     

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.