Alteration of xylose reductase coenzyme preference to improve ethanol production by Saccharomyces cerevisiae from high xylose concentrations

A K270R mutation of xylose reductase (XR) was constructed by site-direct mutagenesis. Fermentation results of the F106X and F106KR strains, which carried wild type XR and K270R respectively, were compared using different substrate concentrations (from 55 to 220 g/L). After 72 h, F106X produced less ethanol than xylitol, while F106KR produced ethanol at a constant yield of 0.36 g/g for all xylose concentrations. The xylose consumption rate and ethanol productivity increased with increasing xylose concentrations in F106KR strain. In particular, F106KR produced 77.6g/L ethanol from 220 g/L xylose and converted 100 g/L glucose and 100g/L xylose into 89.0 g/L ethanol in 72h, but the corresponding values of F106X strain are 7.5 and 65.8 g/L. The ethanol yield of F106KR from glucose and xylose was 0.42 g/g, which was 82.3% of the theoretical yield. These results suggest that the F106KR strain is an excellent producer of ethanol from xylose.     

A comparison of within-plant karyological heterogeneity between agamospermous and sexualTaraxacum (Compositae) as assessed by the nucleolar organiser chromosome

Morphological variation for the NOR chromosome was studied for four half-siblings of a sexual outbreedingTaraxacum, for three siblings of the obligate agamospermT. pseudohamatum, and for two individuals of the agamospermT. brachyglossum. No rearrangement was detected for the 113 chromosomes of sexuals, or for 41 chromosomes of two agamospermous individuals. In the other three agamospermous individuals, 3/16, 5/50, and 5/20 chromosomes showed evidence of chromosomal rearrangement. The majority of rearrangement events (10/13) occurred to the satellite rather than to the body of the NOR-chromosome. It is considered that such high levels of somatic chromosomal rearrangement in agamospermousTaraxacum may be the result of activity by transposable genetic elements. This recombination may be of selective advantage to asexual plants which cannot generate genetic variability through the sexual process.     

Spatio-temporal variation in the structure of a chromosomal polymorphism zone in the house mouse

Several long-term temporal analyses of the structure of Robertsonian (Rb) hybrid zones in the western house mouse, Mus musculus domesticus, have been performed. Nevertheless, the detection of gradual or very rapid variations in a zone may be overlooked when the time elapsed between periods of study is too long. The Barcelona chromosomal polymorphism zone of the house mouse covers about 5000, km(2) around the city of Barcelona and is surrounded by 40 chromosome telocentric populations. Seven different metacentrics and mice with diploid numbers between 27 and 40 chromosomes and several fusions in heterozygous state (from one to seven) have been reported. We compare the present (period 2008-2010) and past (period 1996-2000) structure of this zone before examining its dynamics in more detail. Results indicate that there is not a Rb race in this area, which is consistent with the proposal that this zone was probably originated in situ, under a primary intergradation scenario. The lack of individuals with more than five metacentrics in heterozygous state in the current period suggests that selection acted against such mice. By contrast, this situation did not occur for mice with fewer than five fusions in heterozygous condition. Changes in human activity may affect the dynamics of gene flow between subpopulations, thus altering the chromosomal composition of certain sites. Although these local variations may have modified the clinal trend for certain metacentrics, the general staggered structure of the zone has not varied significantly in a decade.     

Broadening the insecticidal spectrum of Lepidoptera-specific Bacillus thuringiensis strains by chromosomal integration of cry3A

A TnpI-TnpIA-mediated and thermosensitive recombination system was developed to construct genetically modified Bacillus thuringiensis strains encoding a crystal protein particularly active against Coleopteran species. Based on B. thuringiensis transposon Tn4430, an integrative vector, pBMB-R14E, was constructed, by which the cry3A delta-endotoxin gene highly toxic to Lepidoptera was delivered into a wildtype B. thuringiensis subsp. kurstaki strain YBT1520. The cry3A gene was integrated into the chromosome of the host strain. Then the integrative vector was eliminated by moving recombinant cultures to 46 degrees C. Two recombinant B. thuringiensis strains, BMB1520-S and BMB1520-T, were obtained. In recombinant strains, the cry3A gene was stably expressed in measurable amounts and did not reduce the expression of endogenous crystal protein genes. Bioassay results showed that BMB1520-S and BMB1520-T, in addition to the activity against lepidopteran Plutella xylostella third-instar larvae present in the parental strains, exhibited a high level of activity against coleopteran Rhyllodecta vulgatissima third-instar larvae, absent from the parental strains.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

直结肠癌BUBR1的过度表达与TP53突变及微卫星状态关系提示其过度表达与染色体不稳定表型有关

在有丝分裂过程中BUBR1监视微管与着丝点的结合,是保证染色体均等分离的重要分子机制之一.BUBIB变异家谱研究及其敲除模型的研究表明,BUBR1缺陷与染色体不稳定性及肿瘤的发生直接相关.近来在数种人类肿瘤,对BUBR1蛋白过度表达有所报道.但在直结肠癌,BUBR1的过度表达是否与染色体不稳定性的发生有关目前仍无定论.在人类结直肠癌的遗传不稳定性主要表现为两种类型,染色体不稳定性及微卫星不稳定性,它们提示了两条独立的肿瘤发生路径.一般认为不存在高频度微卫星不稳定性表型的肿瘤通过染色体不稳定途径癌变.P53蛋白通过多种机制对维护遗传稳定性起到重要的作用,TP53基因突变经常与染色体不稳定现象并存.DNA倍体情况也是染色体不稳定研究不可缺少的指标.本研究采用免疫组织化学法检测了一组93例进展期散发结直肠癌BUBR1蛋白的表达情况,直接测序法检测TP53变异.高分辨率荧光标记微卫星不稳定检测技术检测微卫星状态,固相激光扫描细胞仪技术检测DNA倍体情况.我们分析了BUBR1表达与三种反映遗传背景的因子的关系.BUBR1蛋白过度表达在人结直肠癌较为常见.在非高频度微卫星不稳定的结直肠癌,BUBR1蛋白过度表达率明显为高(P＜0.01),在TP53基因突变的病例其过度表达率亦较高(P＜0.05).BUBR1蛋白的过度表达与DNA异倍体无统计学相关,但DNA异倍体病例的BuBRl过度表达有偏高倾向.BuBRl表达情况与常用的临床病理学指标无统计学相关.BuBRl过度表达同微卫星状态及TP53突变的关系明确的提示,在人类散发结直肠癌,BUBR1蛋白过度表达与染色体不稳定状态有关.BUBR1过度表达作为一种常见的分子异常,对于肿瘤的早诊预防提供新的标志物.并可能成为治疗的新靶点.     

Mitotic drug targets

Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti‐cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti‐mitotic drugs induce tumor cell death. J. Cell. Biochem. 111: 258–265, 2010. © 2010 Wiley‐Liss, Inc.     

酿酒酵母基因组位置效应对外源基因表达的影响

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程,而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响,以绿色荧光蛋白为报告基因,通过双交换同源重组方法,对酿酒酵母单基因敲除库进行高通量转化,构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析,构建高表达位点库和低表达位点库,共发现促进绿色荧光蛋白表达的位点428个,抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布,从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考,对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。