Transcription rates of yeast genes are influenced by the distribution of introns

A comparative analysis on the intron sequence oligonucleotide usages in two sets of yeast genes with higher and lower transcription frequencies, respectively, has shown that the intron sequence structures of the two sets of genes are different. There are more potential binding sites for transcription factors in the introns of the genes with high transcription frequencies. So it is speculated that introns regulate the transcription of genes. But more evidences are needed to favor this speculation. The detailed comparative analyses on the distribution ( length and position) of introns and exons in the two sets of gene sequences also show that there is an obvious boundary between the lengths of the two sets of introns. There is no boundary between the lengths of the two sets of exons, although the means of their lengths are of discrepancy. The situation of the gene lengths ( length of intron and exon) is similar to exon lengths. As far as the relative position, the introns in two sets of genes all have a bias toward the 5' ends of genes. But as the actual position is considered, more introns in high transcription genes have a tendency to be located toward the 5' ends of genes, some even located at 5'-UTR. These results suggest that the gene transcription rates are related to the length of intron, but not to the lengths of exons and genes sequences. The positions of introns may also influence the transcription rates. The transcriptional regulation of introns may be correlative with the transcriptional regulation of the upstream of genes, or be its continuous action.

Statistical analysis of sequence features of introns with positive transcriptional regulation in yeast genes

A great deal of experimental studies have shown that many introns of eukaryotic genes function as regulators of transcription. However, comprehensive studies of this problem have not yet been conducted. After checking the transcription frequencies of some Saccharomyces cerevisiae (yeast), genes and their introns, a remarkable phenomenon was discovered that generally the introns of the genes with higher transcription frequencies are longer, and the introns of the genes with lower transcription frequencies are shorter. This suggests that the longer introns of genes with higher transcription frequencies may contain some characteristic sequence structures, which could enhance the transcription of genes. Therefore, two sets of introns of yeast genes were chosen for further study. The transcription frequencies of the first set of genes are higher (>30), and those of the second set of genes are lower (less than or equal to10). Some oligonucleotides are detected by statistically comparative analyses of the occurrence frequencies of oligonucleotides (mainly tetranucleotides and pentanucleotides), whose occurrence frequencies in the first set of introns; are significantly higher than those in the second set of introns, and are also significantly higher than those in the exons flanking the introns of the first set. Some of these extracted oligonucleotides are the same as the regulatory elements of transcription revealed by experimental analyses. Besides, the distributions of these extracted oligonucleotides in the two sets of introns and the exons show that the sequence structures of the first set of introns are favorable for transcription of genes.

Detection of potential positive regulatory motifs of transcription in yeast introns by comparative analysis of oligonucleotide frequencies

We conducted a comparative statistical analysis of tetra- through hexanucleotide frequencies in two sets of introns of yeast genes. The first set consisted of introns of genes that have transcription rates higher than 30 mRNAs/h while the second set contained introns of genes whose transcription rates were lower than or equal to 10 mRNAs/h. Some oligonucleotides whose occurrence frequencies in the first set of introns are significantly higher than those in the second set of introns were detected. The frequencies of occurrence of most of these detected oligonucleotides are also significantly higher than those in the exons flanking the introns of the first set. Interestingly some of these detected oligonucleotides are the same as well known "signature" sequences of transcriptional regulatory elements. This could imply the existence of potential positive regulatory motifs of transcription in yeast introns. (C) 2003 Elsevier Ltd. All rights reserved.

Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit OR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants. (c) 2005 Elsevier B.V. All rights reserved.

Evaluation of soot particles of biomass fuels with endocrine-modulating activity in yeast-based bioassay

Exposure to indoor air pollution (IAP) from the combustion of biomass fuels is an important cause of morbidity and mortality in developing countries. In the work discussed in this paper we evaluated the endocrine activity of soot particles from biomass fuels by using yeast bioassay. These pollutants could have beta-galactosidase activity with a relative potency (RP) about 10(-7)-10(-9) that of estradiol. Soot particles from wood and straw combustion only partially induced beta-galactosidase activity whereas others produced fully inductive activity in the yeast assay system. These pollutants did not have estrogen antagonist and progesterone agonist activity within the defined concentration range. However, these pollutants require 2-4 orders of magnitude higher IC50 to inhibit the activity of progesterone in a similar dose-response manner to mifepristone. We therefore propose that the endocrine activity of some environmental pollutants may be because of inhibition of the progesterone receptor (hPR). GC-MS results showed that substituted polycyclic aromatic hydrocarbon (PAH) compounds, substituted phenolic compounds and derivatives, aromatic carbonyl compounds, and phytosteroids in these soot particles may be mimicking endogenous hormones.

Differing estrogen activities in the organic phase of air particulate matter collected during sunny and foggy weather in a Chinese city detected by a recombinant yeast bioassay

Total air suspended particles (PM 100) collected from an urban location near a traffic line in Wuhan, China, were examined for estrogen using a recombinant yeast bioassay. Wuhan, located at the central part of China, is the fourth biggest city in China with 7 million populations. Today, Wuhan has developed into the biggest city and the largest traveling center of central China, becoming one of the important bases of industry, education and research. Wuhan is right at the confluent point of Yangzi River, the third longest river in the world, and its largest distributary Hanjiang, with mountains and more than 100 takes in downtown area. Therefore, by its unique landscape, Wuhan has formed clear four seasons with relatively long winter and summer and short spring and autumn. Foggy weather usually happen in early spring. The yeast line used in this assay stably expresses human estrogen receptor-alpha. Weak but clear estrogenic activities were detected in the organic phase of crude extracts of air particle materials (APM) in both sunny and foggy weather by 0.19-0.79 mug E2/gPM(100) which were statistically significantly elevated relative to the blank control responding from 20% to 50% of the maximum E2 response, and the estrogenic activity was much higher in foggy weather than in sunny weather. The estrogenic activities in the sub-fractions from chromatographic separation of APM sampled in foggy days were also determined. The results indicated that the responses of the fractions were obviously higher than the crude extracts. Since there is no other large pollution source nearby, the estrogenic material was most likely from vehicle emissions, house heating sources and oil fumes of house cooking. The GC/MS analysis of the PM100 collected under foggy weather showed that there were many phenol derivatives, oxy-PAHs and resin acids which have been reported as environmental estrogens. These results of the analysis of estrogenic potency in sunny and foggy weather in a subtropical city of China indicate that further studies are required to investigate the actual risks for the associated health and atmospheric system. (C) 2004 Elsevier Ltd. All rights reserved.

Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay

Estrogenic activities of emission samples generated by fossil fuel combustion were investigated with human estrogen receptor (ER) recombinant yeast bioassay. The results showed that there were weak but clear estrogenic activities in combustion emissions of fossil fuels including coal, petroleum, and diesel. The estrogenic relative potency (RP) of fossil fuel combustion was the highest in petroleum-fired car, followed by coal-fired stove, diesel-fired agrimotor, coal-fired electric power station. On the other hand, the estrogenic relative inductive efficiency (RIE) was the highest in coal-fired stove and coal-fired electric power station, followed by petroleum-fired car and diesel-fired agrimotor. The estrogenic activities in the sub-fractions from chromatographic separation of emitted materials were also determined. The results indicated that different chemical fractions in these complex systems have different estrogenic potencies. The GC/MS analysis of the emission showed that there were many aromatic carbonyls, big molecular alcohol, PAHs and derivatives, and substituted phenolic compounds and derivatives which have been reported as environmental estrogens. The existence of estrogenic substances in fossil fuel combustion demands further investigation of their potential adverse effects on human and on the ecosystem. The magnitude of pollution due to global usage of fossil fuels makes it imperative to understand the issue of fossil fuel-derived endocrine activities and the associated health risks, particularly the aggregated risks stemmed from exposure to toxicants of multiple sources. (C) 2003 Elsevier Science Ltd. All rights reserved.

Global transposable characteristics in the complete DNA sequence of the yeast

Global transposable characteristics in the complete DNA sequence of the Saccharomyces cevevisiae yeast is determined by using the metric representation and recurrence plot methods. On the basis of the correlation distance of nucleotide strings, 16 chromosome sequences of the yeast, which are divided into 5 groups, display 4 kinds of the fundamental transposable characteristics: a short increasing period, a long increasing quasi-period, a long major value and hardly relevant.

非粮原料燃料乙醇发酵关键技术的研究

生物质燃料乙醇是一种高度清洁的交通液体燃料，是减少温室气体排放，缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全，有利于产业的可持续发展。在众多的非粮原料中，甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90％。同时，甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的，因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇，其技术水平低，发酵强度低，一般在0.7－2.5g/（L•h），乙醇浓度低，甘薯发酵乙醇为6－8％(v/v)，能耗高，环境负荷大，污染严重。针对上述问题，本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中，大型发酵罐底部高压条件下，CO2对酵母乙醇发酵的影响，我们通过CO2 加压的方法进行模拟试验，研究结果表明，发酵时间随压强的升高而逐渐延长，高压CO2 对乙醇发酵效率影响不大，在0.3 MPa 以下时，发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下，酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下，酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下，乙醇发酵途径中多个基因表达量下调，同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾，实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本，实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1，进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵，发酵33h，最终乙醇浓度达到10.7%（w/w），发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右，显示菌株具有一定的耐酸性能力。同时观察到40 ℃下，菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化，乙醇发酵途径中关键酶基因表达下调，而海藻糖合成酶与热激蛋白基因表达量上调，这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料，本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂，研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液，采用分批补料的发酵方式，乙醇浓度最高达到了10.26% (w/w)，发酵时间为42 h。同时观察到在糖蜜发酵中，乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下，采用10L 发酵罐进行分批乙醇发酵，经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%，平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时，拥有快速生产乙醇的能力，适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点，传统液化糖化处理很难在短时间内充分糖化原料；高粘度的醪液也难以进行管道输送，容易堵塞管路；同时，也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析，进而对预处理条件进行了研究，在最佳预处理条件下，糖化2h 后，醪液葡萄糖值最高可达99.3，粘度4.5×104 mPa.s，而采用传统糖化工艺，醪液DE 值仅为85.8，粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明，通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上，进一步将发酵规模扩大到500L，在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模，采用边糖化边发酵（SSF）工艺，在料液比为3∶1，发酵醪液最高粘度为6×104mPa.s 条件下，发酵37h，乙醇浓度达到了12.7%（v/v），发酵效率91%，发酵强度为2.7 g/（L•h）。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成，采用了高效液相色谱—蒸发光散射检测法，对乙醇发酵残糖进行了分析。结果表明，甘薯、木薯乙醇发酵残糖均为寡聚糖，主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长，寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖，过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株，对甘薯乙醇发酵残糖的降解利用率均达到了40%以上，而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析，确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发，以期形成乙醇转化率高，能耗低，生产效率高、季节适应性好，原料适应性广，经济性强，符合清洁生产机制的燃料乙醇高效转化技术，为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%（w/w）at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.

A quick isolation method for mutants with high lipid yield in oleaginous yeast

A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 mu mol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol-chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism.

Inactive and mutagenic effects induced by carbon beams of different LET values in a red yeast strain

To evaluate biological action of microorganism exposed to charged particles during the long distance space exploration. Induction of inactivation and mutation in a red yeast strain Rhodotorula glutinis AY 91015 by carbon beams of different LET values (14.9-120 0 keV mu m(-1)) was investigated It was found that survival curves were exponential, and mutation curves were linear for all LET values The dependence of inactivation cross section on LET approached saturation near 120 0 keV mu m(-1) The imitation cross section saturated when LET was higher than 582 keV mu m(-1) Meanwhile, the highest RBEI for inactivation located at 120 0 key mu m(-1) and the highest RBEm for mutation was at 58.2 key mu m(-1) The experiments imply that the most efficient mutagenic part of the depth dose profile of carbon ion is at the plateau region with intermediate LET value in which energy deposited is high enough to Induce mutagenic lesions but too low to induce over kill effect in the yeast cells (C) 2010 Elsevier B V All rights reserved

~(12)C离子束辐照对酵母发酵能力的影响；Improved ethanol fermentation of a yeast mutant by C-12 ion beam irradiation

利用100MeV/u的12C6+离子束辐照酵母Saccharomyces cerevsiea YY,选育出一株高产突变菌株C03A,考察C03A发酵过程中不同温度、pH、糖汁浓度对发酵的影响。通过正交实验确定最佳发酵条件为:糖汁浓度24%、温度35℃、pH5.0。在10L发酵罐实验中,C03A发酵速率相对原始菌株高,36h发酵完全,比原始菌株缩短12h;发酵产酒率达到13.2%(V/V),比原始菌株高1.6%(V/V)。