酵母己糖转运蛋白Hxt7启动子突变体的构建与表达(英)

葡萄糖在酵母细胞中的转运是通过一个庞大的己糖转运子家族实现的.Hxt7为一种高亲和性转运蛋白，在这个家族中的表达丰度最高.它的表达为低浓度葡萄糖所诱导.为了研究葡萄糖转运对细胞生长和葡萄糖阻遏的控制作用，提高对糖酵解通路和糖代谢过程的可调控性，该研究对HXT7启动子进行逐步删除，并将含启动子区域长短不同的HXT7基因整合转入酿酒酵母己糖转运子缺失菌株hxtΔ(hxt1～hxt7, gal2缺失)的基因组，构建一组仅含HXT7且表达水平不一的酵母启动子突变株.将HXT7在突变株中的表达与在野生菌株（MC996A）中的表达进行比较，对它们在含50 mmol/L葡萄糖培养基中的生长速率、葡萄糖消耗性能、HXT7 mRNA及蛋白质表达水平等进行了实时测定.结果表明，当葡萄糖浓度低于5 mmol/L时，HXT7的表达量最高.高浓度葡萄糖环境下，HXT7在突变株中的表达高于在野生株中的表达，显示出不完全性葡萄糖阻遏效应.HXT7的表达水平与启动子长短和基因拷贝数有关.位于－495至－346之间的149 bp HXT7启动子区域对HXT7的表达至关重要.启动子短于346 bp，HXT7基本不表达.     

毕赤酵母己糖运载体HXT1基因缺失菌株的构建

葡萄糖在酵母胞内的转运是依靠己糖运载体家族成员完成的。目前在酿酒酵母中已鉴定了18个己糖运载体基因, 各自功能也已得到深入的研究。然而在作为优良蛋白表达系统的毕赤酵母中, 目前国内外尚未有此类基因的相关报道。本文基于酵母同源重组率高的特性, 以G418作为抗性筛选标记, 分别在其5￠和3￠端连接待缺失毕赤酵母基因HXT1开放阅读框ORF的上下游各200 bp作为同源重组区, 电转毕赤酵母GS115感受态, 通过不同浓度G418抗性平板筛选, 最终得到了HXT1基因缺失的毕赤酵母菌株GS115ΔHXT1。通过与野生株比较发现, HXT1基因缺失株的生长状况及葡萄糖利用都有所下降。     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β-半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYESZ中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4－3突变菌株。通过比较两株重组菌产生的β-半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β-卜半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4－3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β-半乳糖苷酶在pep4－3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

抗盐胡杨Na+/H+逆向转运蛋白基因PeNhaD1的功能

将胡杨Na /H 逆向转运蛋白基因PeNhaD1,分别转入对盐敏感的缺失质膜和缺失液泡膜Na /H 逆向转运蛋白基因的酵母突变菌株ANT3和GX1中。结果表明,在pH6.0、Na 浓度为80mmol/L(固体培养基)或400mmol/L(液体培养基)的条件下,转化具有目的基因的酵母ANT3具有更高的耐盐性,而将目的基因转化到突变株GX1时,却不能提高其耐盐性。实验结果说明PeNhaD1可能是通过编码质膜Na /H 逆向转运蛋白而提高酵母的耐盐性的,推测其在胡杨耐盐机制中的作用可能是提高拒盐性。     

酿酒酵母（Saccharomyces cerevisiae）半乳糖可诱导表达系统特性的研究

把大肠杆菌β－半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子ＧＡＬ１的穿梭表达质粒ｐＹＥＳ２中,并把得以的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失９０％以上的ｐｅｐ４－３突变菌株。通过比较两株重组菌产生的β－半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β－半乳糖苷酶少水平不仅均要高于另一对照菌株,并且ｐｅｐ４－３突变菌株表现受葡萄糖阻遏的严紧程     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β一半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GALl的穿梭表达质粒pYES2中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4-3突变菌株。通过比较两株重组菌产生的β一半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β一半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4-3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β一半乳糖苷酶在pep4-3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

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。     

毕赤酵母己糖运载体HXTl基因缺失菌株的构建

葡萄糖在酵母胞内的转运是依靠己糖运载体家族成员完成的.目前在酿酒酵母中已鉴定了18个己糖运载体基因,各自功能也已得到深入的研究.然而在作为优良蛋白表达系统的毕赤酵母中,目前国内外尚未有此类基因的相关报道.本文基于酵母同源重组率高的特性,以G418作为抗性筛选标记,分别在其5'和3'端连接待缺失毕赤酵母基因HXTl开放阅读框ORF的上下游各200 bp作为同源重组区,电转毕赤酵母GS115感受态,通过不同浓度G418抗性平板筛选,最终得到了HXTl基因缺失的毕赤酵母菌株GS115AHXTl.通过与野生株比较发现,HXTl基因缺失株的生长状况及葡萄糖利用都有所下降.     

单核细胞增生李斯特菌LadR蛋白对外排泵MdrL的调控机制研究

研究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)调控子LadR对外排泵MdrL的调控作用及方式。采用同源重组技术构建ladR基因缺失菌株ΔladR;通过实时荧光定量PCR检测mdrL基因在野生株EGD-e和突变株ΔladR中的转录水平;构建重组蛋白表达菌株BL21(DE3)-ladR并纯化重组蛋白His6-LadR;通过凝胶阻滞试验检测LadR蛋白与mdrL基因启动子区域的结合活性。成功构建ladR基因缺失菌株ΔladR。与野生株EGD-e相比,突变株ΔladR中mdrL基因的转录水平提高约51倍;成功构建重组蛋白表达菌株BL21(DE3)-ladR,纯化得到浓度为1 mg/mL的重组蛋白His6-LadR;凝胶阻滞实验结果显示,重组蛋白His6-LadR可与mdrL基因的启动子区域特异性结合。Lm的调控子LadR通过与mdrL基因启动子区域特异性结合实现对外排泵MdrL的负调控。     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.