BACKGROUND & AIMS: :A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.

背景与目标： ：已鉴定出来自模型生物酿酒酵母的内质网（ER）的磷脂翻转酶活性，并在功能上重构为蛋白脂质体。使用荧光停止流反向交换测定法分析酰基-N-硝基苯-2-氧杂-1,3-二氮杂-4-基（酰基-NBD）标记的磷脂酰胆碱在酵母微粒体中的跨双分子运动，显示了快速的ATP独立的触发器（半时，<2分钟）。由酵母微粒体膜的Triton X-100提取物制备的蛋白脂质体也能够以不依赖ATP的方式快速翻转NBD标记的磷脂类似物。 Flippase的活性对蛋白质修饰试剂N-乙基马来酰亚胺和焦碳酸二乙酯敏感。通过速度梯度离心分离Triton X-100提取物可鉴定出富含脂肪酶活性的约4S蛋白级分，以及脂肪酶活性被耗尽或无法检测到的其他组分。我们估计，flippase的活性是由于蛋白质代表了Triton X-100提取物中蛋白质的大约2％（wt / wt）。这些结果表明，需要特定的蛋白质来促进ER中不依赖ATP的磷脂触发器，并且它们的鉴定是可行的。 ER蛋白转运蛋白的结构表明它可以解释ER中的flippase活性。我们使用由温度敏感的酵母突变体制备的微粒体测试了这一假设，其中主要的转座子成分Sec61p被定量耗尽。我们发现，蛋白质的跨膜转运不是跨ER的磷脂的跨双层运动所必需的。我们的工作将酵母定义为一种有前途的模型系统，可用于未来鉴定ER磷脂翻转酶以及测试和纯化候选翻转酶的尝试。

BACKGROUND & AIMS: :Ethanol fermentation from sweet sorghum juice containing 240 g/l of total sugar by Saccharomyces cerevisiae TISTR 5048 and S. cerevisiae NP 01 immobilized on low-cost support materials, corncob pieces, was investigated. In batch fermentation, S. cerevisiae TISTR 5048 immobilized on 6 × 6 × 6 mm(3) corncobs gave higher ethanol production than those immobilized on 12 × 12 × 12 mm(3) corncobs in terms of ethanol concentration (P), yield (Y ( p/s )) and productivity (Q ( p )) with the values of 102.39 ± 1.11 g/l, 0.48 ± 0.01 and 2.13 ± 0.02 g/l h, respectively. In repeated-batch fermentation, the yeasts immobilized on the 6 × 6 × 6 mm(3) corncobs could be used at least eight successive cycles with the average P, Y ( p/s ) and Q ( p ) of 97.19 ± 5.02 g/l, 0.48 ± 0.02 and 2.02 ± 0.11 g/l h, respectively. Under the same immobilization and repeated-batch fermentation conditions, P (90.75 ± 3.05 g/l) and Q ( p ) (1.89 ± 0.06 g/l h) obtained from S. cerevisiae NP 01 were significantly lower than those from S. cerevisiae TISTR 5048 (P < 0.05), while Y ( p/s ) from both strains were not different. S. cerevisiae TISTR 5048 immobilized on the corncobs also gave significantly higher P, Y ( p/s ) and Q ( p ) than those immobilized on calcium alginate beads (P < 0.05).

背景与目标： ：研究了固定在低成本支撑材料玉米芯上的酿酒酵母TISTR 5048和酿酒酵母NP 01从甜高粱汁中所含的总糖为240 g / l的乙醇发酵。在分批发酵中，固定在6×6×6mm（3）玉米芯上的酿酒酵母TISTR 5048与固定在12×12×12mm（3）玉米芯上的酿酒酵母相比，乙醇浓度（P），产量（ Y（p / s））和生产率（Q（p））的值分别为102.39±1.11 g / l，0.48±0.01和2.13±0.02 g / l h。在重复分批发酵中，固定在6×6×6mm（3）玉米芯上的酵母至少可以连续使用八个循环，平均P，Y（p / s）和Q（p）为97.19±5.02 g / l，分别为0.48±0.02和2.02±0.11 g / l h。在相同的固定化和重复分批发酵条件下，从酿酒酵母NP 01获得的P（90.75±3.05 g / l）和Q（p）（1.89±0.06 g / l h）显着低于酿酒酵母NP。 TISTR 5048（P <0.05），两种菌株的Y（p / s）均无差异。固定在玉米芯上的酿酒酵母TISTR 5048也比固定在藻酸钙珠上的P，Y（p / s）和Q（p）高得多（P <0.05）。

BACKGROUND & AIMS: :Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

背景与目标： ：生物素原养是酿酒酵母菌株的一种罕见，尚未完全理解并且在工业上具有相关性的特征。单倍体实验室菌株CEN.PK113-7D的基因组包含完整的生物素生物合成基因，但在不含生物素的合成培养基中的生长极为缓慢（比生长率[μ]≈0.01 h-1）。在重复的分批培养和加速器中进行的四个独立进化实验得出的菌株在无生物素和补充生物素的培养基中的生长速率（μ≤0.36 h-1）相似。这些进化菌株的全基因组重测序揭示了BIO1的40倍扩增，该BIO1编码庚二酰辅酶A（CoA）合成酶。在不同的染色体上发现了BIO1的其他副本，其扩增与大量的染色体重排相符。 BIO1在菌株CEN.PK113-7D中的过表达证实了该基因扩增的关键作用，该菌株能够在不含生物素的培养基中生长（μ= 0.15 h-1）。在一些进化的菌株中发现了膜转运蛋白基因TPO1和/或PDR12的突变。在过表达BIO1的菌株中TPO1和PDR12的缺失将其比生长速率提高到0.25 h-1。这些基因中无效突变的作用是非可累加的，这些突变以前未与生物素代谢相关。这项研究表明，具有生物素合成基本遗传信息的酿酒酵母菌株可以进化为完整的生物素原营养型，并为将生物素原营养型工程化到该酵母的实验室和工业菌株中确定了新的目标。在所有生物体中发挥作用，并非所有生物体都能合成这种维生素。面包酵母的许多菌株是工业生物技术中的重要微生物，至少含有一些生物素合成所需的基因。然而，这些菌株中的大多数不能完全合成生物素，或者不能以足以维持快速生长和产物形成的速率合成生物素。因此，通常将这种昂贵的维生素添加到面包师的酵母培养物中。在这项研究中，实验室在无生物素生长培养基中的进化产生了新菌株，该菌株在不存在生物素的情况下的生长速度与在存在生物素的情况下一样快。通过分析进化的不依赖生物素的菌株的DNA序列，鉴定出有助于此能力的突变。这项工作证明了与工业相关的酵母具有完全的生物素独立性，并鉴定了突变，将其引入其他酵母菌株可以降低或消除其对生物素的需求。

BACKGROUND & AIMS: :Although microorganisms account for the largest fraction of Earth's biodiversity, we know little about how their reproductive barriers evolve. Sexual microorganisms such as Saccharomyces yeasts rapidly develop strong intrinsic post-zygotic isolation, but the role of extrinsic isolation in the early speciation process remains to be investigated. We measured the growth of F1 hybrids between two incipient species of Saccharomyces paradoxus to assess the presence of extrinsic post-zygotic isolation across 32 environments. More than 80% of hybrids showed either partial dominance of the best parent or over-dominance for growth, revealing no fitness defects in F1 hybrids. Extrinsic reproductive isolation therefore likely plays little role in limiting gene flow between incipient yeast species and is not a requirement for speciation.

背景与目标： ：尽管微生物是地球生物多样性的最大组成部分，但我们对其生殖屏障如何进化知之甚少。性微生物（例如酵母）迅速发展出强大的内在合子后分离，但是外源分离在早期物种形成过程中的作用仍有待研究。我们测量了两个Saccharomyces paradoxus初始物种之间的F1杂种的生长，以评估在32个环境中外部合子后分离的存在。超过80％的杂种显示出最佳亲本的部分优势或对生长的过度主导，表明F1杂种中没有适合性缺陷。因此，外源性生殖分离可能在限制初始酵母物种之间的基因流动方面几乎没有作用，并且不是物种形成的必要条件。

BACKGROUND & AIMS: :In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress.IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

背景与目标： ：在酿酒酵母中，Y家族DNA聚合酶Rev1参与通过病灶DNA合成（TLS）修复DNA损伤。在当前的研究中，为阐明Rev1在酿酒酵母中氧化应激诱导的DNA损伤中的作用，将REV1删除并过表达。对这些突变体以及野生型菌株进行转录组分析，以筛选在对DNA损伤的反应过程中可能与REV1相关的潜在基因。当用2μmMH2O2处理酵母细胞时，REV1的缺失导致存活率和突变频率分别降低1.5倍和2.8倍，而REV1的过表达使存活率和突变频率提高1.1倍和2.8倍。分别是野生型菌株的存活率和突变频率的2.9倍。转录组和表型分析确定Sml1通过抑制Rev1的活性加剧了酵母细胞中的氧化应激。这种抑制是由于Rev1的BRCA1 C末端（BRCT）结构域与Sml1的36至70位氨基酸残基之间的物理相互作用所致。当这种相互作用被阻止时，细胞存活率和突变频率分别增加了1.8倍和3.1倍。我们还发现，Sml1在氧化应激下抑制Rev1的磷酸化，SML1的缺失使Rev1的磷酸化增加46％，而SML1的过表达减少了Rev1的磷酸化。总的来说，这些发现表明Sml1可能是介导Rev1去磷酸化以抑制其在氧化应激过程中的活性的新型调节剂。氧化应激（2 mM H2O2）。此外，我们发现Sml1与Rev1发生物理相互作用并抑制Rev1的磷酸化，从而抑制Rev1 DNA的抗氧化活性。这些发现表明Sml1可能是Rev1的新型调节剂，以响应氧化应激对DNA的损伤。

BACKGROUND & AIMS: :Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast Saccharomyces cerevisiae. Multiple biosynthetic pathway variants, carrying only plant genes or a combination of plant and Escherichia coli genes, were implemented using a full factorial design of experiment. Through analysis of variances, the effect of each enzyme variant (factors), together with possible interactions between these factors, was assessed. The best pathway variant produced 2.95 ± 0.08 mg/L rosmarinic acid in mineral medium with glucose as the sole carbon source. Increasing the copy number of rosmarinic acid biosynthetic genes increased the titer to 5.93 ± 0.06 mg/L. The study shows the feasibility of producing rosmarinic acid by yeast fermentation.

背景与目标： ：迷迭香酸是常见于紫草科和唇形科植物家族中的羟基肉桂酸酯。它具有多种生物活性，包括抗氧化，抗炎，抗菌，抗过敏和抗病毒特性。迷迭香酸被用作食品和化妆品成分，并且已经提出了几种药物应用。迷迭香酸目前是通过从植物中提取或化学合成生产的。然而，由于植物来源的可获得性有限以及化学合成方法的复杂性，通过微生物发酵生产该化合物的兴趣日益浓厚。在这项研究中，我们旨在通过工程面包酵母酿酒酵母生产迷迭香酸。使用完整的因子设计实验，可以实现仅携带植物基因或植物和大肠杆菌基因组合的多种生物合成途径变体。通过方差分析，评估了每种酶变体（因子）的作用，以及这些因子之间可能的相互作用。最佳途径的变体在葡萄糖为唯一碳源的矿物培养基中产生了2.95±0.08 mg / L迷迭香酸。迷迭香酸生物合成基因的拷贝数增加将滴度提高到5.93±0.06 mg / L。研究表明通过酵母发酵生产迷迭香酸的可行性。

BACKGROUND & AIMS: :The monitoring of fermentation at low temperatures (12-15°C) is a current practice in the winery for retention and enhancement of the flavour volatile content of wines. Among Saccharomyces species, Saccharomyces uvarum and Saccharomyces kudriavzevii have revealed interesting industrial properties, including better adaptation at low temperatures. To gather deeper knowledge of the fermentative metabolism at a low temperature of these species together with S. cerevisiae, we performed a comparative metabolomic analysis using four representative strains. We used batch cultures to obtain an exhaustive and dynamic image of the metabolome of strains passing through the sequential stresses related to the winemaking environment. A great variety of intra- and extracellular metabolites (>500 compounds) were quantified across fermentation using distinct chromatographic methods. Besides a global decrease in the lipid composition of the four strains when they entered into the stationary phase, we reported some strain-specific high magnitude changes. Examples of these differences included divergent patterns of production of short-chain fatty acids and erythritol in the S. uvarum strain. Strains also differed in expression for aromatic amino acid biosynthesis and sulphur metabolism, including the glutathione pathway. These data will allow us to refine and obtain the most value of fermentations with this alternative Saccharomyces species.

背景与目标： ：在酿酒厂中，为了保持和提高葡萄酒的风味挥发物含量，目前在低温（12-15°C）下对发酵进行监控是目前的做法。在酿酒酵母种类中，葡萄酿酒酵母和库氏酿酒酵母显示出令人感兴趣的工业特性，包括在低温下更好的适应性。为了收集这些物种与酿酒酵母在低温下的发酵代谢的更深入的知识，我们使用四种代表性菌株进行了比较代谢组学分析。我们使用分批培养来获得菌株代谢组的详尽而动态的图像，这些菌株通过与酿酒环境有关的顺序应力传递。在整个发酵过程中，使用不同的色谱方法对各种各样的细胞内和细胞外代谢产物（> 500种化合物）进行了定量。除了当它们进入固定相时这四个菌株的脂质组成总体降低外，我们还报道了一些菌株特异性的高强度变化。这些差异的例子包括在葡萄链球菌菌株中短链脂肪酸和赤藓糖醇生产的不同模式。菌株在芳香族氨基酸生物合成和硫代谢（包括谷胱甘肽途径）的表达上也有所不同。这些数据将使我们能够精炼并获得这种替代酵母菌发酵的最大价值。

BACKGROUND & AIMS: :In this study we show that inactivation of Hsl1 or Hsl7, negative regulators of the Swe1 kinase, enhances the invasive behavior of haploid and diploid cells. The enhancement of filamentous growth caused by inactivation of both genes is mediated via the Swe1 protein kinase. Whereas Swe1 contributes noticeably to the effectiveness of haploid invasive growth under all conditions tested, its contribution to pseudohyphal growth is limited to the morphological response under standard assay conditions. However, Swe1 is essential for pseudohyphal differentiation under a number of nonstandard assay conditions including altered temperature and increased nitrogen. Swe1 is also required for pseudohyphal growth in the absence of Tec1 and for the induction of filamentation by butanol, a related phenomenon. Although inactivation of Hsl1 is sufficient to suppress the defect in filamentous growth caused by inactivation of Tec1 or Flo8, it is insufficient to promote filamentous growth in the absence of both factors. Moreover, inactivation of Hsl1 will not bypass the requirement for nitrogen starvation or growth on solid medium for pseudohyphal differentiation. We conclude that the Swe1 kinase modulates filamentous development under a broad spectrum of conditions and that its role is partially redundant with the Tec1 and Flo8 transcription factors.

背景与目标： ：在这项研究中，我们显示Hsl1或Hsl7（Swe1激酶的负调节剂）的失活增强了单倍体和二倍体细胞的侵袭行为。由两个基因的失活引起的丝状生长的增强是通过Swe1蛋白激酶介导的。尽管在所有测试条件下，Swe1均对单倍体侵袭性生长的有效性做出了显着贡献，但其对假菌丝生长的贡献仅限于标准测定条件下的形态学响应。但是，Swe1对于在许多非标准测定条件（包括温度变化和氮增加）下的假菌丝分化必不可少。在不存在Tec1的情况下，假菌丝的生长也需要Swe1，并且通过丁醇（相关现象）诱导丝化也需要Swe1。尽管Hsl1的失活足以抑制由Tec1或Flo8失活引起的丝状生长缺陷，但在没有这两种因素的情况下，不足以促进丝状生长。此外，Hsl1的灭活不会绕过氮饥饿或在假菌丝分化的固体培养基上生长的需求。我们得出的结论是，Swe1激酶在广泛的条件下调节丝状发育，并且其作用与Tec1和Flo8转录因子部分重叠。

BACKGROUND & AIMS: :The FCY2 gene of Saccharomyces cerevisiae encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine. A structure-function model for this PCP has been recently proposed. In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.

背景与目标： ：酿酒酵母的FCY2基因编码嘌呤-胞嘧啶通透酶（PCP），介导嘌呤和胞嘧啶的主动转运。最近已经提出了用于该PCP的结构功能模型。在这项研究中，我们开发了一个基于质粒的系统，该系统生成了许多亲和突变的等位基因，使我们能够定义对通透酶功能至关重要的新氨基酸。

BACKGROUND & AIMS: :The chromatin remodeling complex RSC from Saccharomyces cerevisiae is a DNA translocase that moves with directionality along double-stranded DNA in a reaction that is coupled to ATP hydrolysis. To better understand how this basic molecular motor functions, a novel method of analysis has been developed to study the kinetics of RSC translocation along double-stranded DNA. The data provided are consistent with RSC translocation occurring through a series of repeating uniform steps with an overall processivity of P = 0.949 +/- 0.003; this processivity corresponds to an average translocation distance of 20 +/- 1 base pairs (bp) before dissociation. Interestingly, a slow initiation process, following DNA binding, is required to make RSC competent for DNA translocation. These results are further discussed in the context of previously published studies of RSC and other DNA translocases.

背景与目标： 酿酒酵母的染色质重塑复合物RSC是一种DNA转位酶，它在与ATP水解反应中沿着双链DNA定向移动。为了更好地了解这种基本分子运动的功能，已经开发了一种新颖的分析方法来研究RSC沿双链DNA转运的动力学。提供的数据与通过一系列重复的统一步骤发生的RSC易位一致，总体生产率为P = 0.949 /-0.003；该分离性对应于解离前的平均移位距离为20 -1个碱基对（bp）。有趣的是，DNA结合后需要缓慢的启动过程才能使RSC具有DNA易位的能力。这些结果将在先前发表的RSC和其他DNA转位酶研究的背景下进一步讨论。

BACKGROUND & AIMS: :The human fungal pathogen Candida albicans displays a very high degree of plasticity, including the types of genomic changes frequently observed with cancer cells, such as gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity. Despite its relevance to every aspect of genetics and evolution of this pathogen, our understanding of the mutation process and its bearing on organismal fitness remains quite limited. Here, we have evaluated and compared two approaches to estimate the mutation frequency at three ORFs/regions (HIS4, CEN4 and EST2) of the C. albicans genome. Sequencing of individual DNA molecules (clone-by-clone sequencing) identified de novo mutations at these DNA regions, whose frequency was similar to that observed for S. cerevisiae at homolog sites following the same approach. However, mutations were not detected when the same regions were directly sequenced from the pooled DNA. In addition, in the absence of the homologous recombination protein Rad52, mutation frequency within these sites remained unaltered. The use of an alternative polymerase also found mutations. These results suggest that at least some mutations are artifacts caused by the polymerase used, advising that post-PCR procedures might generate mutations which may become undistinguishable from the genuine mutations and thus may interfere with mutational analysis. Furthermore, we recommend that new mutations found in the sequences of cloned alleles used for the determination of haplotypes should be contrasted with the sequence yielded by the pooled DNA.

背景与目标： ：人类真菌病原体白色念珠菌具有很高的可塑性，包括癌细胞经常观察到的基因组变化类型，例如总体染色体重排，非整倍性和杂合性丧失。尽管它与这种病原体的遗传学和进化的各个方面都相关，但我们对突变过程及其对机体适应性的了解仍然十分有限。在这里，我们评估和比较了两种方法来估计白色念珠菌基因组的三个ORF /区域（HIS4，CEN4和EST2）的突变频率。单个DNA分子的测序（逐个克隆测序）在这些DNA区域识别出从头突变，其频率与酿酒酵母在同源位点处观察到的频率相似。但是，当从合并的DNA直接对相同区域进行测序时，未检测到突变。另外，在缺少同源重组蛋白Rad52的情况下，这些位点内的突变频率保持不变。使用替代的聚合酶也发现了突变。这些结果表明，至少某些突变是由使用的聚合酶引起的伪像，建议PCR后操作可能会产生突变，这些突变可能与真正的突变无法区分开，因此可能会干扰突变分析。此外，我们建议将用于确定单倍型的克隆等位基因序列中发现的新突变与汇集的DNA产生的序列进行对比。

BACKGROUND & AIMS: :High-affinity K(+) transporters play an important role in K(+) absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WDelta3, AlHAK performed high-affinity K(+) uptake with a K(m) value of 8 muM, and the growth of transformants was dramatically inhibited by 150 mM Rb(+) and 150 mM Cs(+) but less affected by 300 mM Na(+). AlHAK may thus improve the capacity of plants to maintain a high cytosolic K(+)/Na(+) ratio at high salinity.

背景与目标： ：高亲和力K（）转运蛋白在植物对K（）的吸收中起重要作用。我们从线虫类盐生植物长尾鸢中分离了一个HAK基因。 AlHAK的氨基酸序列与从稻（82％）和大麦（82％）获得的HAK转运蛋白显示出高度同源性。当在酿酒酵母WDelta3中表达时，AlHAK表现出高亲和力K（）吸收，K（m）值为8μM，转化子的生长受到150 mM Rb（）和150 mM Cs（）的明显抑制，但受影响较小300 mM Na（）。因此，AlHAK可以提高植物在高盐度下保持高细胞溶质K（）/ Na（）比的能力。

BACKGROUND & AIMS: :The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast Saccharomyces cerevisiae show that Ipl1/Aurora B kinase and a centromere-associated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2ARts1, and we found that rts1Δ mutants exhibited premature SAC silencing as well. We further revealed that sgo1 mutants with abolished binding to H2A or PP2ARts1 displayed premature SAC silencing. Together, our results suggest that, in budding yeast S. cerevisiae, the Bub1-H2A-Sgo1-PP2ARts1 axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.

背景与目标： ：主轴组件检查点（SAC）监视动粒-微管相互作用中的错误，其激活可防止后期进入。 SAC保持活动状态，直到所有染色体都达到双极性附着，从而在动植物上施加张力。我们以前在酿酒酵母中的数据表明，在产生张力之前，需要Ipl1 / Aurora B激酶和着丝粒相关蛋白Sgo1来防止SAC沉默，但我们认为该调控网络是不完整的。 Bub1激酶是SAC的成分之一，Bub1依赖性H2A磷酸化触发酵母和人类细胞中H2A着丝粒募集Sgo1。尽管缺少Bub1激酶结构域的酵母细胞显示出有效的SAC激活，但我们发现，在无张力附着的情况下，突变细胞无法维持延长的检查点停滞期。 H2A中Bub1磷酸化位点的突变也会导致酵母细胞中SAC过早沉默。先前的数据表明Sgo1蛋白与PP2ARts1结合，并且我们发现rts1Δ突变体也表现出过早的SAC沉默。我们进一步揭示，已取消与H2A或PP2ARts1的结合的sgo1突变体显示过早的SAC沉默。总之，我们的结果表明，在酿酒酵母中，Bub1-H2A-Sgo1-PP2ARts1轴可防止SAC沉默，并有助于延长在动植物建立张力之前的检查点停滞。

BACKGROUND & AIMS: :Meiotic progression requires precise orchestration, such that one round of DNA replication is followed by two meiotic divisions. The order and timing of meiotic events is controlled through the modulation of the phosphorylation state of proteins. Key components of this phospho-regulatory system include cyclin-dependent kinase (CDK) and its cyclin regulatory subunits. Over the past two decades, studies in budding and fission yeast have greatly informed our understanding of the role of CDK in meiotic regulation. In this review, we provide an overview of how CDK controls meiotic events in both budding and fission yeast. We discuss mechanisms of CDK regulation through post-translational modifications and changes in the levels of cyclins. Finally, we highlight the similarities and differences in CDK regulation between the two yeast species. Since CDK and many meiotic regulators are highly conserved, the findings in budding and fission yeasts have revealed conserved mechanisms of meiotic regulation among eukaryotes.

背景与目标： ：减数分裂的进行需要精确的编排，这样一轮DNA复制后会进行两次减数分裂。减数分裂事件的顺序和时间是通过调节蛋白质的磷酸化状态来控制的。该磷酸调节系统的关键成分包括细胞周期蛋白依赖性激酶（CDK）及其细胞周期蛋白调节亚基。在过去的二十年中，有关发芽和裂变酵母的研究极大地帮助了我们了解CDK在减数分裂调控中的作用。在这篇综述中，我们概述了CDK如何控制发芽和裂变酵母中的减数分裂事件。我们讨论通过翻译后修饰和细胞周期蛋白水平的变化来调节CDK的机制。最后，我们强调了两种酵母物种在CDK调控方面的异同。由于CDK和许多减数分裂调节剂是高度保守的，所以在发芽和裂变酵母中的发现揭示了真核生物中减数分裂调节的保守机制。

BACKGROUND & AIMS: :Repression of an essential nucleolar small nuclear RNA (snRNA) gene of Saccharomyces cerevisiae was shown to result in impaired production of 18S rRNA. The effect, observed for an snRNA species of 128 nucleotides (snR128), was evident within one generation after the onset of SNR128 gene repression and correlated well with depletion of the snRNA. The steady-state mass ratio of 18S RNA to 25S RNA decreased eightfold over the course of the analysis. Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor. This effect appears to result from impairment of processing of the 35S rRNA transcript at sites that define the 20S species coupled with rapid turnover of unstable intermediates. Possible bases for the effects observed are discussed. A common U14 designation is proposed for the structurally related yeast snRNA and 4.5S hybRNAs from amphibians and mammals.

背景与目标： ：酿酒酵母的基本核仁小核RNA（snRNA）基因的抑制显示导致18S rRNA的生产受损。在SNR128基因阻抑发作后的一代人中，观察到了对128个核苷酸的snRNA物种（snR128）产生的影响，并且该效应与snRNA的消耗密切相关。在分析过程中，18S RNA与25S RNA的稳态质量比降低了八倍。脉冲追踪分析的结果表明，失衡的基础是18S RNA及其20S前体的积累不足。此作用似乎是由于在定义20S物种的位点处35S rRNA转录物加工过程受到损害以及不稳定中间体的快速更新所致。讨论了观察到的影响的可能依据。对于来自两栖动物和哺乳动物的与结构相关的酵母snRNA和4.5S hybRNA，提出了通用的U14名称。