一种快速、有效、经济的提取酵母质粒的方法

目的：建立一种经济有效、快速简便、稳定的提取酵母质粒的方法。方法：用葡糖苷酸酶消化酵母细胞壁以获取原生质体,然后采用碱裂解法裂解原生质体以获得质粒。结果：与采用商品化离心柱法试剂盒所提取的质粒相比,用该法获取的酵母质粒在PCR分析及转化效果方面没有差异。结论：建立了一种经济有效、快速简便、稳定的提取酵母质粒的方法。     

酵母双杂交相关方法的改良及应用

对酵母双杂交实验过程中较为耗时的阳性克隆鉴定过程进行改进,以期建立一种快速有效的鉴定方法。分别采用液氮冻融法、超声破碎法、渗透压破壁法以及煮沸裂解法裂解酵母细胞,获得质粒作为PCR模板,直接测序鉴定筛选到的相互作用蛋白。以液氮冻融法和超声破碎法裂解细胞获得的质粒为模板进行PCR,得到特异的产物,测序鉴定结果明确,与经典的鉴定方法相比效果相当,但更加经济快捷;而渗透压破壁法和煮沸裂解法则效果不好。说明前两种方法可代替常规方法用于阳性克隆的鉴定,从而加快酵母双杂交实验中大量阳性克隆的筛查工作。     

TNF-α转化酶酵母双杂交系统"诱饵"质粒的构建及鉴定

目的：构建酵母双杂交系统“诱饵”质粒载体pGBKT7-TACE,并检测其是否具有自身激活及毒性作用。方法：从小鼠肝组织提取总RNA,用RT-PCR的方法获得TACE,克隆于pGBKT7载体上,酶切鉴定及序列分析,并检测pGBKT7-TACE在MATH-MAKER GAL4 Two-Hybrid System 3中的自激活和毒性作用。结果：本实验成功构建了“诱饵”质粒载体pGBKT7-TACE,并证明其在酵母双杂交系统中无自激活及毒性作用。结论：构建的诱饵质粒pGBKT7-TACE可应用在酵母双杂交系统中,为筛选小鼠cDNA文库中与TACE相互作用的蛋白奠定实验基础。     

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.     

FOXL2酵母双杂交诱饵载体构建及检测

目的:用FOXL2编码区片段构建酵母双杂交诱饵质粒并对诱饵质粒进行自激活活性及毒性检测.方法:PCR扩增FOXl2编码区DNA片段,克隆入pGBKT7质粒,转化至DH-5α大量扩增后提取质粒,经PCR、酶切和测序验证,再转化至Y187酵母菌株中用缺陷培养基筛选并进行自激活和毒性检测.结果:获得FOXL2基因1137bp片段,并成功将人类正常和一突变型FOXL2编码区克隆人pGBKT7中,经检测在SD/-Trp/X-α-Gal单缺培养基平板上无蓝色菌斑出现,在SD/-Ade/-Trp培养基平板无生长.同时在SD/-Trp/Kana(20μg/ml)培养24h后测得菌液OD600≥0.8,说明重组诱质粒无自激活活性和酵母毒性,满足作为诱饵质粒的条件.结论:成功构建了正常及突变型的FOXL2酵母杂交诱饵质粒;为进一步利用酵母双杂交技术研究与FOXL2相互作用的蛋白打下坚实基础.     

组蛋白酵母双杂交诱饵载体的构建及其自激活作用检测

目的:构建组蛋白 H2A、H2B、H3、H4的酵母双杂交诱饵载体,检测其在酵母细胞中的表达和自激活活性,为应用酵母双杂交系统筛选与组蛋白相互作用的蛋白奠定基础.方法:扩增 H2A、H2B、H3、H4蛋白基编码区 cDNA序列,克隆至酵母表达载体 pGBK-T7中,再将其转化至酵母细胞 AH109中,提取酵母总蛋白,检测各种组蛋白的表达,并检测表达的组蛋白在酵母细胞中对报告基有无自激活作用.结果:将 H2A、H2B、H3、H4基的 cDNA 克隆至酵母表达载体 pGBK-T7中,其中组蛋白 H4得到正确表达,且对报告基 LacZ、HIS3、Ade 无激活作用.结论:可以利用酵母双杂交系统筛选与 H4相互作用的蛋白质.     

酵母双杂交系统

酵母双杂交系统是研究细胞内蛋白质之间相互作用的一种分子遗传学技术,用已知的蛋白质作为诱饵来筛选其可以相互作用的伙伴蛋白。本文简要叙述了酵母双杂交系统的原理、基本方法,以及这个技术的发展和应用。     

中华蜜蜂幼虫膜蛋白酵母双杂交文库的构建

【目的】本研究旨在利用位点特异性重组技术(FullCoV)将中华蜜蜂 Apis cerana cerana 幼虫膜蛋白cDNA连接到pPR3-N载体上,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库。【方法】提取2-3日龄中华蜜蜂工蜂幼虫总RNA;分离mRNA后,在反转录酶的作用下合成幼虫膜蛋白cDNA第1链,并合成双链cDNA。在双链cDNA的5′端加上带有重组序列的接头后,通过FullCoV技术与载体pPR3-N进行连接,然后将连接产物电转化到DH10B感受态细胞,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,并对该文库插入片段大小和文库滴度进行检测。【结果】通过FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,经检测,中华蜜蜂幼虫膜蛋白酵母cDNA文库的总库容量为1.5×10^7 cfu,文库滴度为3×10^6 cfu/mL,重组率达到100%。【结论】本研究利用FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母cDNA文库,为进一步探究感染中华蜜蜂的病原微生物与宿主蛋白互作研究奠定了基础。     

酵母双杂交筛选类固醇激素合成急性调节蛋白的相互作用蛋白质

目的:利用酵母双杂交技术筛选人肝cDNA文库中与类固醇激素合成急性调节蛋白(STAR)相互作用的蛋白质。方法:将全长STAR基因克隆到酵母表达载体pDBLeu中,形成诱饵;将构建好的诱饵质粒转化至AH109酵母感受态中,利用酵母双杂交技术筛选人肝cDNA文库中与其相互作用的蛋白质,并进行相互作用的回转验证。结果:构建了酵母诱饵蛋白表达质粒pDBLeu-STAR,并筛选到6个猎物,其中有5对相互作用回转验证阳性。结论:为进一步研究STAR的功能和作用机制提供了新的线索。     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii. Received April 16, 1998; accepted June 30, 1998.     

利用细胞质导入法选育嗜杀啤酒酵母

利用细胞质导入(Cytoduction)法中的核融合缺陷细胞融合技术,在对酿酒酵母(Saccharomyces cerevisiae)D518菌株不做任何遗传标记,将嗜杀酵母5045菌株的嗜杀质粒转移到受体菌D518中,获得了具有两亲株优良性状的融合子KD102菌株。对融合子分析表明:融合子遗传性状稳定,不仅含有供体菌5045的嗜杀质粒,而且受体菌D518的核基因被原封不动地保留下来,为异质体细胞(Heteroplasmon)。将融合子KD102菌株用于小型、中型及生产性酿酒试验,结果表明,具有与亲株D518同样的酿造特性。在发酵过程中,能抑制野生酵母污染,净化发酵体系。对于保证啤酒纯种酿造及提高成品酒的生物稳定性具有明显效果。     

Transformation of Yeast by Agitation with Glass Beads

We have found that agitation of Saccharomyces cerevisiae with glass beads and plasmid DNA using a vortex mixer results in genetic transformation of the yeast cells. This method is less efficient, but considerably more convenient, than other yeast transformation procedures. The fact that the minimal requirements for transformation are simply physical damage and the presence of DNA in an osmotically supportive environment suggests that this process may occur in nature.     

碱裂解提取质粒DNA的改进

碱裂解提取质粒DNA是分子生物学实验中常用的方法,但通常方法所提取的质粒往往含有大量的RNA和其他杂质.本文适时加入较高浓度的RNA酶和适当延长冰浴时间,结果得到了几乎没有RNA和其他杂质的高纯质粒DNA,不仅达到了分子生物学实验要求,而且可用作抗原检测抗dsDNA抗体.该法操作简单、经济、实用.     

A Rapid Method for Protein Extraction from Fission Yeast

Researchers working with fission yeast conduct protein extraction widely and frequently, but this includes the handling of glass beads, and hence is laborious and cumbersome, especially when dealing with a large number of samples. Here we describe a rapid and reliable method for preparing protein extract from fission yeast, one which is applicable to routine western blotting.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

应用酵母单杂交系统分离水稻蜡质基因5'调控区结合蛋白的基因

在水稻蜡质基因５’上游区中一段３１ｂｐ 核苷酸序列能与水稻未成熟种子核蛋白特异结合。为了克隆这一核蛋白基因,以此３１ ｂｐ 序列构建成“鱼饵”质粒,从水稻ｃＤＮＡ文库中筛选到１３ 个阳性克隆。根据这些阳性克隆中插入ｃＤＮＡ 片段的相互杂交结果,对这些克隆进行了分组。     

DNA plasmid transmission in yeast is associated with specific sub-nuclear localisation during cell division

Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division. A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2. In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions. Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein. We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation. We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites. These sites are distinct from both the telomeres and from sites of cohesin binding. Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid. We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast.     

A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli

A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.