Effect of High Oxygen Tension on Potassium Retentivity and Colony Formation of Bakers' Yeast

The ability of yeast cells to retain potassium and to form colonies was studied after exposure to pressures ranging from 2 to 143 atmospheres of oxygen. The investigations allow comparison of these responses with those found after x-ray exposure. Exposure to 2 to 8 atmospheres of oxygen for 2, 20, and 40 hours showed decreased potassium leakage as measured by an elution technique. Further experiments using 0.5 to 22 hour exposures to 10 to 143 atmospheres of oxygen showed decreased potassium leakage when glucose was present in the test media, but increased leakage (as did x-ray effects) in the absence of substrate. There was increased potassium leakage into the suspending media (distilled water) during oxygen exposure but this usually did not affect the leakage rates measured subsequently. Marked inability to form colonies was observed after 20 hour exposures to 100 atmospheres of oxygen, with a much smaller response at lower pressures. Increased oxygen concentrations, not pressure, evidently caused these effects, since comparable pressures of nitrogen produced almost no change. The ratio of potassium leakage to survival sensitivity was found to be approximately unity when comparing exposures causing 50 per cent damage. This is quite different from that seen with x-ray or ultraviolet irradiation.     

High-Temperature Microbial Sulfate Reduction Can Be Accompanied by Magnetite Formation

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was found to be capable of lithoautotrophic growth on medium containing molecular hydrogen, sulfate, and amorphous Fe(III) oxide. During the growth of this microorganism, amorphous Fe(III) oxide was transformed into black strongly magnetic precipitate rich in magnetite, as shown by Moessbauer studies. Experiments involving inhibition of microbial sulfate reduction and abiotic controls revealed that magnetite production resulted from chemical reactions proceeding at elevated temperatures (83°C) between molecular hydrogen, amorphous Fe(III) oxide, and sulfide formed enzymatically in the course of dissimilatory sulfate reduction. It follows that magnetite production in this system can be characterized as biologically mediated mineralization. This is the first report on magnetite formation as a result of activity of sulfate-reducing microorganisms.     

Formation of 5-Hydroxy-4-Ketohexanoic Acid by Yeast

When grown on medium containing ethanol as the sole carbon source, three of five strains of yeast tested produced a keto acid which was demonstrated by paper chromatography. This compound was isolated and its structure was examined by elementary analysis, infrared spectrum, nuclear magnetic resonance and periodate oxidation. The compound was proved to be identical with 5-hydroxy-4-ketohexanoic acid. Formation of this compound by cell suspensions of Hansenula miso IFO 0146 was achieved by addition of acetaldehyde, although the presence of α-ketoglutaric acid enhanced the formation of the keto acid from acetaldehyde during a short incubation period. Added 5-hydroxy-4-ketohexanoic acid was exhausted by the cell suspension.     

Environmental and Genetic Determinants of Colony Morphology in Yeast

Nutrient stresses trigger a variety of developmental switches in the budding yeast Saccharomyces cerevisiae. One of the least understood of such responses is the development of complex colony morphology, characterized by intricate, organized, and strain-specific patterns of colony growth and architecture. The genetic bases of this phenotype and the key environmental signals involved in its induction have heretofore remained poorly understood. By surveying multiple strain backgrounds and a large number of growth conditions, we show that limitation for fermentable carbon sources coupled with a rich nitrogen source is the primary trigger for the colony morphology response in budding yeast. Using knockout mutants and transposon-mediated mutagenesis, we demonstrate that two key signaling networks regulating this response are the filamentous growth MAP kinase cascade and the Ras-cAMP-PKA pathway. We further show synergistic epistasis between Rim15, a kinase involved in integration of nutrient signals, and other genes in these pathways. Ploidy, mating-type, and genotype-by-environment interactions also appear to play a role in the controlling colony morphology. Our study highlights the high degree of network reuse in this model eukaryote; yeast use the same core signaling pathways in multiple contexts to integrate information about environmental and physiological states and generate diverse developmental outputs.     

酵母过氧化物酶体的形成机制

过氧化物酶体是细胞中一种重要的细胞器.过氧化物酶体在细胞功能的发挥和人体健康方面有着重要作用.目前,以酵母过氧化物酶体为模型,研究过氧化物酶体的形成机制是研究热点.从过氧化物酶体起源、生成方式介绍最新研究进展,总结在酵母细胞中参与过氧化物酶体形成的必需基因(pex),及其编码Peroxin蛋白在过氧化物酶体形成过程中的...     

一种简单高效的酵母单菌落 PCR 方法

目的:为快速简便地挑选出酿酒酵母重组克隆,探索建立一种经济、直接、高效的酵母单菌落 PCR 方法.方法:以 Leu2MX6基同重组或重组质粒转化得到的酵母突变菌为材料,分别采用传统的提取基组或质粒的方法、煮沸法及化学试剂处理法等制备 PCR 模板进行重组克隆鉴定,并对6种 PCR 模板制备方法的效果进行比较与分析;对加热提取法进行优化并进行重组子的提取和验证.结果与结论:直接以1 mm2单克隆菌株95℃处理5 min 后的酵母菌落水悬浮液为模板进行单菌落 PCR,是一种简单高效的酵母重组克隆鉴定方法.该方法能弥补传统方法的不足,且简便快速、结果稳定,可作为筛选和鉴定阳性克隆的有效手段.同时,这种单菌落 PCR 法也可应用重组毕赤酵母的阳性克隆筛选.     

Allele-Specific Suppression by Formation of New Protein-Protein Interactions in Yeast

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have conducted a biochemical analysis of the interaction between various combinations of wild-type and mutant actin and Sac6 proteins. Previously, we showed that actin mutations that are suppressed by sac6 mutations encode proteins with a reduced affinity for wild-type Sac6p. In the present study, we have found that mutant Sac6 proteins bind more tightly to mutant actin than does wild-type Sac6p, and thus compensate for weakened interactions caused by the mutant actin. Remarkably, we have also found that mutant Sac6 proteins bind more tightly to wild-type actin than does wild-type Sac6p. This result indicates that suppression does not occur through the restoration of the original contact site, but rather through the formation of a novel contact site. This finding argues against suppression occurring through a ``lock-and-key' mechanism and suggests a mechanism involving more global increases in affinity between the two proteins. We propose that the most common kind of suppressors involving interacting proteins will likely occur through this less specific mechanism.     

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.     

Auxin-Induced Expansion Growth of Cells and Protoplasts of Yeast

Using an auxin-responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts.

• 1 Indole-3-acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred.
• 2 The auxin-induced expansion growth was inhibited by an antiauxin, trans-cinnamic acid.
• 3 Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin-induced cell expansion growth.
• 4 Protoplasts did not expand in response to auxin under the condition where intact cells did.
• 5 The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion.
• 6 High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

     

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.     

Cellular Fatty Acid and Ester Formation by Brewers′ Yeast

The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were in-corporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers′ yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane.     

发酵培养基的模式识别法优化

应用模式识别法优化发酵培养基,以培养基组成构筑模式空间,线性降维揭示可视优化区,再以多因素轮换优化实现由低维到高维的回复,得到最佳培养基组成。