新型隐球酵母U6启动子的鉴定、克隆及功能验证

【目的】鉴定及克隆新型隐球酵母(Cryptococcus neoformans)中PolⅢ型的U6启动子(Cn U6启动子),并验证CnU6启动子能否有效转录shRNA及CRISPR/Cas9系统中gRNA。【方法】结合Gen Bank数据库已公布的新型隐球酵母基因组信息和本实验室RNA-seq文库数据,利用生物信息学技术分析得到新型隐球酵母中具有高转录水平的U6RNA序列。使用重叠PCR和Easy Geno方法将预测的CnU6启动子分别克隆到sh RNA及gRNA的上游区域,通过观察shRNA对靶基因的RNAi效果及gRNA引导Cas9核酸酶对靶位点的切割结果,确定CnU6启动子能否转录短RNA。【结果】CnU6启动子能够转录形成shRNA对靶基因进行沉默,并且能转录形成g RNA引导Cas9核酸酶对靶点进行切割。【结论】新型隐球酵母的CnU6启动子被成功鉴定及克隆,它能有效驱动shRNA和gRNA的转录。     

新型隐球酵母非编码小RNAs的研究进展

新型隐球酵母是一种担子菌病原真菌,主要感染免疫功能缺陷的人群,例如HIV-1感染病人,最终会引起致命隐球菌性脑膜炎。非编码小RNAs一般指长度为20-30nt的小RNAs,具有调节功能。新型隐球酵母能够产生大量的小RNAs,但是其生成（biogenesis）过程以及生物学功能尚未完全阐述。本文就新型隐球酵母中小RNAs的特征和产生、以及在新型隐球酵母中的生物学作用和机制进行综述。     

人类致病菌新型隐球酵母Snf1/AMPK 蛋白激酶调节细胞壁的完整性

摘要：【目的】Snf1/AMPK在真核生物中是重要的且高度保守的一类蛋白激酶。在新型隐球酵母中,SNF1 基因在调节致病因子的生物合成和细胞毒力方面具有重要作用。本文进一步报道了该基因在维持细胞壁完整方面的新功能,这一功能在其他微生物中未见报道。【方法】利用荧光增白剂染料（Calcofluor white dye）染色,荧光显微观察细胞分离、胞壁完整性;利用恒定流速和压力水流冲击菌落,测定细胞黏附琼脂糖表面能力;在含有十二烷基硫酸钠（Sodium dodecyl sulfate,SDS）,刚果红（Congo red）染料和增白剂（Fluorescent Brightener 28）的培养基上观察突变株的生长情况,以验证细胞壁完整性。【结果】SNF1 基因突变菌株对细胞壁抑制剂SDS等敏感,表明细胞壁完整性的损坏;在葡萄糖固体培养基上表现为细胞与琼脂间的黏附力丧失;在热击压力下,该菌株不能正常生长,而这种生长缺陷能够被渗透平衡抑制。【结论】新型隐球酵母SNF1 基因对于维持细胞壁完整性是非常重要的,并且影响细胞与琼脂间黏附作用以及细胞对抗热的能力。     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

耐低温酵母Curvibasidium rogersii菌株在松萝样品中的首次分离鉴定及基于基因组分析的生物特性探究

【目的】对西藏松萝地衣来源的两株非常规酵母进行分离鉴定,并通过基因组序列分析探究其生物学特性和应用潜力。【方法】从西藏来源的松萝地衣样品内部分离得到2株耐低温酵母菌株,通过26S D1/D2和ITS序列比对分析以及生理生化实验进行菌种鉴定;通过全基因组序列分析和验证探究菌株的生物特性。【结果】两株酵母菌株经鉴定均为Curvibasidium rogersii,可以在10℃低温良好生长,在20℃生长最佳,25℃及以上温度生长缓慢或不生长。对其进行基因组测序和基因组挖掘,测序结果发现,其基因组注释出功能的部分与产油脂的低温酵母白冬孢酵母Leucosporidium creatinivorum具有最高相似性,尼罗红染色发现两株酵母都能够生产油脂,另外在基因组序列中还发现了可能参与木糖代谢的相关蛋白编码基因,实验证明两个酵母菌株可以利用木糖生长。【结论】首次分离鉴定了来自西藏松萝的酵母C. rogersii,为充分开发利用松萝和其他地衣来源微生物,以及利用可代谢木糖的新资源酵母生产微生物油脂提供了基础。     

真菌miRNA的发现

microRNAs (miRNAs)在植物和动物中大量存在,但是否在真菌中存在一直是个未解之谜.本研究组在担子菌新型隐球酵母Cryptococcus neoformans中发现了miRNA.两个miRNA,miR1和miR2,长度分别是22nt和18nt,前体是70nt,和动物miRNA相近.通过报告基因,证实miRl/2具有沉默功能.真菌miRNA的发现为研究其进化、功能等提供有用知识.     

伞形酮产生菌的构建

【背景】伞形酮为香豆素类化合物,具有较广泛的药用价值,也是重要的工业制品原料。传统的植物提取成本较高,应开发更有效的获取技术。【目的】利用微生物为宿主异源合成伞形酮。【方法】对不同植物来源香豆素类化合物合成基因进行整合;以酿酒酵母为宿主,通过对宿主酪氨酸代谢通路的改造构建表达宿主,进一步将表达基因转化到改造后的宿主中。【结果】获得产伞形酮的酵母菌株,产量为(67.39±4.87)μg/mL。【结论】通过整合生物合成基因可获得香豆素类天然产物表达菌株。     

新生隐球菌半胱氨酸转运蛋白Mup1鉴定及其表达调控研究

【目的】鉴定新生隐球菌(Cryptococcus neoformans)的半胱氨酸转运蛋白及其对致病性的影响。【方法】构建候选基因敲除株,检测突变株以半胱氨酸为唯一硫源的生长情况;检测半胱氨酸转运蛋白Mup1对新生隐球菌毒力因子表达和不同胁迫条件下生长的影响;通过新生隐球菌大蜡螟(Galleria mellonella)和小鼠感染模型分析Mup1对致病性的影响;通过转录组分析和酵母单杂交研究硫代谢核心转录因子Cys3与Mup1的调控关系。【结果】Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的能力。基因MUP1缺失不影响毒力因子表达和细胞对应激的反应。大蜡螟和小鼠隐球菌感染模型表明Mup1对新生隐球菌的致病性无显著影响。转录组分析和酵母单杂交实验显示Cys3可能间接调控MUP1的转录。【结论】新生隐球菌Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的功能,但不影响致病性,基因转录可能受Cys3的间接调控。     

利用根癌农杆菌T-DNA 插入突变寻找参与漆酶葡萄糖阻遏的关键基因

【目的】新型隐球酵母(Cryptococcus neoformans)是人类重要致病真菌,主要毒性因子之一漆酶的表达受葡糖糖阻遏,机制未知。本文拟寻找参与葡萄糖阻遏的关键基因。【方法】建立根癌农杆菌介导的转化方法(Agrobacterium tumefanciens-mediated transformation,ATMT)建立一个容量约200000的随即插入突变文库,在高浓度葡萄糖条件下从中筛选葡萄糖去阻遏的突变株。通过Southern确定突变株中T-DNA的拷贝数,利用反向PCR获得依赖葡萄糖的漆酶阻遏基因序列。【结果】筛选到了30株葡萄糖去阻遏突变株,Southern blot发现83%的葡萄糖去抑制突变株含有单个T-DNA拷贝。初步鉴定了可能参与漆酶阻遏的10个不同生物学功能基因,如参与碳水化合物的代谢,固醇的合成,几丁质的合成,GPI脂锚钩的合成等等。【结论】ATMT突变策略可以找到一些参与漆酶葡萄糖阻遏的关键基因,为理解漆酶在致病过程中的作用机制和工业改进漆酶活性提供参考。     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Shapes of curves of pH-dependence of reactions

A simple case is considered in which the rate of a two-step reaction depends on pH because the intermediate formed in the first step has to gain (or lose) a proton before it can react in the second step, and in which the rate-determining step therefore changes with pH. The curves of reaction rate against pH are shown to be symmetrical, and the sharpest peak possible has a width at half its height of 1.53pH units, i.e. of 2log(3+2 radical2). Any particular curve for this situation proves to be identical with a curve that could be generated for the pH-dependence of a single-step reaction in which the rate is proportional to the concentration of a particular ionic form of a reactant. Curves for the latter situation, however, can have forms impossible for the former case in which the rate-determining step changes, but only if the protonations that activate and deactivate the reactant are co-operative. The peak can then become even sharper, and its width at half its height can fall to 1.14pH units, i.e. to 2log(2+ radical3).     

Overview of mechanisms of action of lycopene

Dietary intakes of tomatoes and tomato products containing lycopene have been shown to be associated with decreased risk of chronic diseases such as cancer and cardiovascular diseases in numerous studies. Serum and tissue lycopene levels have also been inversely related to the risk of lung and prostate cancers. Lycopene functions as a very potent antioxidant, and this is clearly a major important mechanism of lycopene action. In this regard, lycopene can trap singlet oxygen and reduce mutagenesis in the Ames test. However, evidence is accumulating for other mechanisms as well. Lycopene at physiological concentrations can inhibit human cancer cell growth by interfering with growth factor receptor signaling and cell cycle progression specifically in prostate cancer cells without evidence of toxic effects or apoptosis of cells. Studies using human and animal cells have identified a gene, connexin 43, whose expression is upregulated by lycopene and which allows direct intercellular gap junctional communication (GJC). GJC is deficient in many human tumors and its restoration or upregulation is associated with decreased proliferation. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibits a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression in the HL-60 promyelocytic leukemia cell line, suggesting some interaction at a nuclear or subcellular level. The combination of lycopene and lutein synergistically interact as antioxidants, and this may relate to specific positioning of different carotenoids in membranes. This review will focus on the growing body of evidence that carotenoids have unexpected biologic effects in experimental systems, some of which may contribute to their cancer preventive properties in models of carcinogenesis. Consideration of solubility in vitro, comparison with doses achieved in humans by dietary means, interactions with other phytochemicals, and other potential mechanisms such as stimulation of xenobiotic metabolism, inhibition of cholesterogenesis, modulation of cyclooxygenase pathways, and inhibition of inflammation will be considered. This review will point out areas for future research where more evidence is needed on the effects of lycopene on the etiology of chronic disease.