Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.     

Cloning and sequencing of cDNA for mouse liver metallothionein-I

Metallothionein mRNA was purified from liver of mice injected with cadmium. The corresponding double-stranded cDNA was prepared and inserted into the PstI site of plasmid pBR322. The resulting recombinant DNA was used to transform the RR1 strain of $\text{Escherichia}\text{coli}$. Clones resistant to tetracycline and sensitive to ampicillin were screened for the presence of metallothionein-specific restriction fragments in their plasmids. One plasmid, called M135, contains a cDNA insert covering the entire length of the mRNA for mouse liver metallothionein-I, except for the first 18 bases at the 5′ end.     

The relative effectiveness of 6-thioguanine and 8-azaguanine in selecting resistant mutants from two V79 Chinese hamster cells in vitro

The frequency of surviving colonies in two V79 cell lines exposed to either 6-thioguanine or 8-azaguanine was dependent on initial plating density. Different degrees of metabolic-co-operation were found to occur in the two cell lines and the loss of both spontaneous and added mutants occurred at a lower cell density when 6TG was used for selection than when 8 AZ was used in both cell lines. Both analogues were degraded on incubation in medium plus serum in the absence of added cells. Variation in serum batch had little effect on the rate of degradation or on the frequency of colonies recovered after treatment of V79 cell lines with 8AZ. The reasons for preferring 8AZ to 6TG as a selective agent are discussed.     

不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析

为鉴定不同抗性苹果(Malus domestica)品种响应轮纹病菌胁迫的抗性相关蛋白表达差异, 以抗病品种华月及易感品种金冠为试材, 采用高通量同位素标记定量(IBT)技术结合液相色谱-串联质谱(LC-MS)鉴定技术, 对病原菌处理前后抗、感病品种叶片的蛋白质组差异表达进行分析, 共鉴定出171个差异表达蛋白(DEPs)。GO富集及KEGG通路分析表明, 在细胞组分、分子功能和生物过程3类中共注释到686个GO条目, 其中52个DEPs注释于KEGG通路的18个显著差异途径(P<0.05)。亚细胞定位预测分析表明, 171个DEPs中有170个分别定位于8类细胞器。蛋白功能注释分析表明, 46个DEPs注释于7类抗性相关蛋白, 包括类甜蛋白、过氧化物酶、多酚氧化酶、过敏原蛋白、几丁质酶、内切葡聚糖酶以及主乳胶蛋白。此外, 还对抗性相关蛋白的表达特点及基因定量结果进行了分析。该研究结果可为进一步解析抗、感病苹果品种应答轮纹病菌胁迫的抗性机制提供参考。     

氮代谢参与植物逆境抵抗的作用机理研究进展

近年来,植物所受到的诸如干旱、盐、高温、低氧、重金属胁迫和营养元素缺乏等环境胁迫越来越多,严重影响了植物的生长发育及作物的质量和产量。氮素是植物生长发育所需的必需营养元素,同时也是核酸、蛋白质和叶绿素的重要组成成分,其代谢过程与植物抵抗逆境的能力息息相关。氮代谢是指植物对氮素的吸收、同化和利用的全过程,是植物体内基础代谢途径之一。氮代谢主要从氮素吸收、同化及氨基酸代谢等方面参与植物的抗逆性,并通过调节离子吸收和转运、稳定细胞形态和蛋白质结构、维持激素平衡和细胞代谢水平、减少体内活性氧(reactive oxygen species,ROS)生成以及促进叶绿素合成等生理机制来影响植物抵抗非生物胁迫的能力。因此,提高植物在逆境下的氮代谢水平是减轻外界胁迫对其损伤的一种潜在途径。该文从氮素同化的基本途径出发,分别阐述了氮代谢在干旱胁迫、盐胁迫和高温胁迫等多个方面的逆境抵抗过程中的作用机理,为氮代谢参与植物抗逆性研究提供了有利参考。     

老年慢性阻塞性肺疾病患者和坠积性肺炎患者肺内病原菌比较及感染影响因素分析

目的对比老年慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)患者和坠积性肺炎患者肺内一般病原菌和多重耐药菌的分布,同时分析COPD患者和坠积性肺炎患者多重耐药菌感染的影响因素。方法对入选COPD患者和坠积性肺炎患者下呼吸道痰标本进行培养和鉴定,对比两组患者病原菌以及多重耐药菌的检出率,同时比较两组患者性别、年龄、住院时间、糖尿病病史、有创机械通气治疗和留置尿管等因素对多重耐药菌感染的影响。结果 COPD患者革兰阴性菌的检出率(48.39%)显著低于坠积性肺炎患者(66.13%),COPD患者真菌的检出率(11.06%)显著高于坠积性肺炎患者(2.42%)。两组患者检出的多重耐药菌均以产超广谱β-内酰胺酶肠杆菌科细菌、耐甲氧西林金黄色葡萄球菌和多重耐药/泛耐药铜绿假单胞菌为主。COPD患者中感染多重耐药菌患者的年龄和住院时间显著高于非感染患者;坠积性肺炎患者中感染多重耐药菌患者的住院时间、机械通气治疗率和留置尿管使用率显著高于非感染患者。结论影响老年COPD患者和坠积性肺炎患者多重耐药菌感染的因素有区别,应有针对性地应用抗生素以减少多重耐药菌的感染。     

乳源耐甲氧西林金黄色葡萄球菌icaA/D基因缺失株的构建及其生物被膜形成能力与耐药性分析

【背景】耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus,MRSA)是一种具有多重耐药性的人畜共患病原菌,常引起奶牛乳房炎等疾病。形成生物被膜是MRSA重要的耐药机制之一。研究发现ica操纵子调控的胞间多糖黏附素(Polysaccharide Intercellular Adhesin,PIA)通过促进MRSA的黏附与聚集介导生物被膜形成,icaA和icaD基因共表达可显著提高MRSA的N-乙酰葡聚糖转移酶活性,但icaA/D蛋白对MRSA生物被膜形成和耐药性的影响仍不清楚。【目的】探讨icaA/D基因、MRSA生物被膜形成及耐药性三者之间的相关性,为寻找药物作用新靶点提供科学依据。【方法】以具有多重耐药性且生物被膜形成能力强的乳源MRSA M5分离株为研究对象,利用同源重组技术构建其icaA/D基因缺失株;利用FITC-ConA染色结合激光共聚焦显微镜观察野生株与icaA/D基因缺失株的生物被膜形成过程与能力;采用微量肉汤稀释法测定14种抗菌药物对野生株与icaA/D基因缺失株的最小抑菌浓度(MinimumInhibitoryConcentration,MIC)。【结果】构建了MRSA M5的icaA/D基因缺失株。激光共聚焦显微镜下观察到野生株在培养16 h后形成了一层厚的成熟生物被膜,随后开始解离,直至120 h完全解离;而icaA/D基因缺失株在培养后16 h仅形成一薄层生物被膜,48h完全解离。10种受试抗菌药物对缺失株的MIC较野生株减小,而且缺失株对8种药物的敏感性由原来的耐药或中介转变为中介或敏感。【结论】icaA/D基因缺失可明显降低MRSA的生物被膜形成能力与耐药性。