酵母LAG1同源的人类长寿保障新基因LASS2的克隆和功能研究

目的　分离和克隆肝癌相关基因。方法　我们用高通量功能筛选和RACE(rapidamplificationofcDNAends)方法从人肝cDNA文库中克隆到一个与酵母长寿保障基因LAG1高度同源的人类新基因LASS2 (homosapienslongevityassurancehomologue 2ofyeastLAG1)。结果　LASS2在正常人肝组织和肾组织中高表达 ,其表达谱不同于早先在人中克隆到的另一个与酵母LAG1高度同源的人类长寿保障基因LAG1Hs- 1。放射杂交基因定位显示LASS2位于人类染色体 1q11,酵母双杂交和GST结合实验发现LASS2蛋白能与细胞中的几个膜相关受体或转运体相互作用 ;另外 ,LASS2在体外对人肝癌细胞的克隆形成具有抑制作用 ,提示该基因可能参与细胞的生长调控。结论　LASS2是一个新的肝癌相关基因。     

The Yeast Two-hybrid System and Its Pharmaceutical Significance

The detected phenotypes in many diseases are caused from dysfunction in protein-protein, protein-DNA and receptor-ligand interactions. Therefore, determination of these molecular interactions followed by designing or screening the compounds to target these interactions provides a significant challenge in drug development. This review aims to highlight the yeast two-hybrid system in determination of protein-protein interactions and its possible outcomes in pharmaceutical research. The variations of the basic methodology as one- and three-hybrid systems are also disussed in relation to their potential pharmaceutical applications.     

Formation and stability of linear plasmids in a recombination deficient strain of yeast

Summary Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation in yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A)], a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52 ^– cells.     

Antisuppression of class I suppressors in an isopentenylated-transfer RNA deficient mutant of Saccharomyces cerevisiae

Summary The effect of a previously isolated antisuppressor mutation from bakers' yeast, that reduced the efficiency of the tyrosine-inserting ochre suppressor, SUP7-o, on other tyrosine-inserting ochre suppressors has been determined. As expected, the antisuppressor mutation, mod5-1, restricted the capacity of all eight tyrosine-inserting ochre suppressors to suppress nonsense mutations. Based on the suppression of five ochre alleles in the presence of mod5, the eight class I suppressors can be grouped into three subclasses. The most efficient subclass had only one member, SUP4-o. Members of the second group included SUP2-o, SUP3-o, SUP7-o, and SUP8-o. The third and least efficient subclass included SUP5-o, SUP6-o, and SUP1 1-o. These differences in efficiencies are a function of the relative expression of the eight genes encoding tRNA^TYR.     

A novel class of vector for yeast transformation

Summary We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 m plasmid to completely eliminate the bacterial moiety upon introduction into yeast. A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth. These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression.     

The polymerase (L) protein of rinderpest virus interacts with the host cell protein striatin

Rinderpest virus (RPV) is a morbillivirus that causes a highly contagious disease affecting members of the order Artiodactyla. The viral L protein is the catalytic subunit of the RNA-dependent RNA polymerase. To search for host cell proteins with which L interacts, a library screen was performed using the yeast two-hybrid system. Several host cell proteins were recovered from the library screen as putative L-interactors; one of these was identified as striatin. A direct interaction between RPV L and striatin was confirmed using both co-immunoprecipitation assays and co-localisation studies using confocal microscopy. Striatin was also shown to co-localise with the RPV L protein in infected cells. The L proteins of morbilliviruses consist of three long highly conserved domains separated by short unconserved stretches of amino acids. The L domain with which striatin interacts was investigated by co-immunoprecipitation and striatin was shown to interact primarily with the central conserved domain.     

蛋白质酵母双杂交系统的建立及其在大肠癌肝转移研究中的意义

目的 建立酵母双杂交系统,筛选与FasL相互作用的蛋白,探讨FasL与大肠癌肝转移的关系。方法 以FasL基因构建诱饵蛋白质粒,筛选人胎肝cDNA文库,鉴定与FasL相互作用的蛋白,通过生物信息学分析FasL及其相互作用蛋白在大肠癌肝转移中的作用。结果 筛选出10个与FasL特异性相互作用的蛋白,包括金属硫蛋白1K、1G、2A,组织蛋白酶B,脂肪酸合成酶,干扰素α诱导蛋白27,磷脂清除酶,丝氨酸／苏氨酸样激酶,锚着黏附蛋白以及纤维微丝蛋白-5等。结论 成功建立了筛选FasL相互作用蛋白的酵母双杂交系统,并初步证明FasL与组织蛋白酶、金属硫蛋白、锚着黏附蛋白等之间的相互作用与大肠癌肝转移密切相关。     

Mason-Pfizer monkey virus Gag proteins interact with the human sumo conjugating enzyme, hUbc9

Retroviral Gag proteins function during early and late stages of the viral life cycle. To gain additional insight into the cellular requirements for viral replication, a two-hybrid screen was used to identify cellular proteins that interact with the Mason-Pfizer monkey virus Gag protein. One of the cellular proteins found was identified as hUbc9, a nuclear pore-associated E2 SUMO conjugating enzyme. In vitro protein interaction assays verified the association and mapped the interaction domain to the CA protein. In vivo, hUbc9 and Gag colocalized in the cytoplasm as discrete foci near the nuclear membrane. In addition, overexpression of hUbc9 in cells caused a fraction of Gag to colocalize with hUbc9 in the nucleus. These experiments demonstrate that hUbc9 and Gag interact in cells, strengthen the hypothesis that Gag proteins transiently associate with the nuclear compartment during viral replication, and suggest that hUbc9 plays a role in this process.     

酵母RNA对老龄大鼠生理机能和肝、脑形态学的影响

目的 探讨外源核酸对老龄大鼠的生理机能和肝、脑形态学影响。方法 选取健康老龄(20月龄)Wistar雄性大鼠随机分为高、中、低3个剂量组和老龄对照组、另取青龄(3月龄)作为青龄对照组,高、中、低剂量组在标准饲料基础上,每日每只分别添加酵母RNA93．75mg、46．88mg和9．38mg,饲喂4周。结果 高剂量组超氧化物歧化酶(SOD)活性、血清高密度脂蛋白(HDL)和生长激素水平均高于老龄对照组,酵母RNA补充各组丙二醛含量均低于老龄对照组,高、中剂量组血清睾酮达到青龄对照组水平,而老龄动物各组间雌二醇水平未见统计学差异;补充酵母RNA组的老龄大鼠脑神经元数与老龄对照组大鼠相比有增多趋势并存在剂量效应关系,但没有统计学差异;肝细胞核面积与核浆比均大于老龄对照组,其中核浆比具有统计学差异。结论 酵母RNA可能具有改善老龄大鼠生理机能和肝、脑组织形态学改变的作用,并可能具有量-效关系。