用原生质体融合法优化啤酒酵母的凝絮性和发酵性能

以发酵度较高、凝絮性较差的啤酒酵母菌株H38的原生质体为受体,以凝絮性较强、发酵度较低的啤酒酵母菌株N1热灭活的原生质体为供体进行融合.用正交试验法分别优化两亲株的原生质体制备和再生的条件以及原生质体融合的条件.用制霉菌素抗性为遗传标记选择融合子.融合子初筛时以凝絮性为指标,复筛时以发酵度和发酵液中的主要风味物质的含量为指标.对得到的融合子进行了异核体和遗传稳定性的检验,结果得到两株凝絮性以及发酵特性较好的菌株HN31和HN40.     

酵母双杂交筛选血液中与PERIOD1相互作用的新蛋白

采用酵母双杂交方法,以人PER1的PAS结构域为诱饵,在人血液cDNA文库中筛选能与之相互作用的蛋白.经酶切和核苷酸序列测定,证实重组诱饵载体hper1PAS/pGBKT7构建成功.血cDNA文库转化效率为1.4×106/3μg pGADT7-Rec.酵母双杂交文库营养缺陷筛选得到114个阳性克隆,β-半乳糖苷酶检测报告基因获得46个蓝色克隆.     

Molecular characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> PHO80-like proteins,a novel class of CDKA;1-interacting cyclins

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids (cyclin box) displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.Received 9 February 2004; received after revision 18 March; accepted 19 April 2004     

Mitochondrial DNA mutators

In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis. The 3–5 exonuclease proofreading activity of Pol ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated. The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion. Proofreading-deficient mutator human cell lines and knockin mice have been created. They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.Received 25 May 2004; received after revision 21 June 2004; accepted 7 July 2004     

Effect of GbKTN1 from Gossypium barbadense on cell elongation of fission yeast（Schizosaccharomyces pombe）

The GbKTN1 gene was isolated from 10 DPA fiber cells of Gossypium barbadense using 5′RACE/3′RACE.Full-length cDNA of this gene is 2006 bp, including a 113 bp of 5′untranslated region, a 1563 bp of an open reading frame(ORF), and a 327 bp of 3′untranslated region (excluding the stop codon TAA). The ORF of GbKTN1 encodes a 521-amino acid protein with a predicted size of 55 kD. Near C-terminal of the deduced protein there is a putative ATP binding site between amino acid residues from 233 to 414. Southern blot analysis indicated that the GbKTN1 was a single copy gene in G barbadense. Combining semi-quantitative RT-PCR with Southern blot hybridization revealed that GbKTN1 expressed in all the organs detected such as roots, stems, leaves and fibers. However, the mRNA of GbKTN1 was the most abundant in fiber cells, while it was the lowest in leaves. The GbKTN1 cDNA was transformed into S. pombe to verify its function on cell elongation. Results showed that most yeast cells over expressing GbKTN1 gene were elongated dramatically with an average length increase of 2.18 times than that of the non-induced cells. Even the morphology of some yeast cells appeared irregularly. To the best of our knowledge this is the first evidence that KTN1 is correlated with cell elongation in vivo.     

Identification and analysis of ARS function of six plant MARs

Six plant MARs isolated from tobacco and Arabdiposis were investigated for their ARS activity in yeast. The results showed that among the six plant MARs, only TM1 and AM4 had strong ARS activity which was almost the same as that of ARS from yeast chromosome. In order to further identify the core region of the two MARs for the ARS activity, a series of subclones were created by PCR strategy, and the corresponding subclones were designated as TM1-1, TM1-2, TM1-3, AM4-1, AM4-2 and AM4-3, respectively. Our studies revealed that TMI-3 and AM4-3 not only had higher ARS activity, but also displayed higher transformation frequency, plasmid stability and growth rate compared to their intact MARs, TM1 and AM4. These data present an important clue for further elucidating the relationship between MAR and ARS.     

固定化酵母细胞生物合成谷胱甘肽的研究

利用酵母细胞自身ＧＳＨ合成酶和糖酵解途径再生的ＡＴＰ能够合成ＧＳＨ,在含葡萄糖０．７ｍｏｌ／Ｌ,硫酸镁１０ｍｍｏｌ／Ｌ,０．３ｍｏｌ／Ｌ磷酸钾缓冲液和谷氨酸、半胱氨酸、甘氨酸各２０ｍｍｏｌ／Ｌ的１０ｍＬ反应液中,加入１０ｇ（湿重）固定化酵母细胞,３０℃振荡反应８ｈ产生０．９１ｇ／Ｌ的ＧＳＨ。加入腺苷会降低ＧＳＨ的产量;当腺苷开始转化生成ＡＴＰ时加入前体,可明显提高ＧＳＨ的产量。实验结果初步表明：Ａ     

木薯酒精酵母菌种工程化选育的思路和方向

我国木薯酒精企业的发酵水平低,而国内酵母研究技术水平较高,但其选育的优良酵母菌种未能在企业得到推广应用,原因是高酒度等性能的酵母菌种需要改变现有工艺条件。因而,在不改变现有工艺条件下提高菌种的优良性能是木薯酒精酵母菌种选育的原则。本文在总结我国木薯酒精发酵技术现状的基础上,分析木薯酒精生产企业对酵母性能的要求,提出了现有工艺条件下酵母菌种工程化选育的思路和方向,为木薯酒精酵母菌种选育提供参考。     

酵母Mn-transporter基因研究进展

在酵母中有两个锰离子的转运系统 :即高亲和力系统和低亲和力系统 .本文简要介绍了酵母中与锰离子转运有关的基因 :SMF1、BSD2、ATX2、CCC1、PMR1和MNR1及它们所编码蛋白的大小、细胞定位、跨膜域及他们在锰转运中的功能 .文中还比较了几种蛋白之间的关系 .     

基于信噪比的蛋白质相互作用的预测

蛋白质间的相互作用在生命体中扮演着关键的角色.将改进的共鸣识别模型应用于预测酵母蛋白质间的相互作用,并改用信噪比为判别参数.此判别参数与之前的判别参数——峰值相比,在保证较高预测精度的基础上,还可以很好地区分阳性数据和随机数据,从而也就能较好地处理过度拟合的问题.