极端嗜盐古生菌启动子序列缺失突变的微量热研究

用微量热方法和DNA缺失突变技术研究了来源于极端嗜盐古生菌R1上的一个推测的启动子片段(RM10)在大肠杆菌中的启动子功能. 启动子片段融合到质粒pKK232-8上无启动子的氯霉素乙酰转移酶(CAT)基因前来检测它驱动基因表达的能力, 缺失分析RM10启动子片段定位具有启动活性的重要功能区. 实验结果从热动力学角度揭示, 这个启动子片段上含有－35区和－10区特征的1382～1517 bp(碱基对)区段是它在大肠杆菌中具有启动子功能的关键部分; 在1～1382 bp区段或1571～1848 bp区段上还存在它的负调控区. 该研究为基因启动子功能研究提供了一种新的、更加灵敏便捷的、化学与生物学相结合的方法.     

在一个中国人家庭中发现联锁的三个α-珠蛋白基因

本文以重组质粒pRB_α1 PstI-1.5Kb片段为α-珠蛋白基因探针,应用限制性内切酶图谱和印迹杂交技术,分析了一个中国人家庭的α-珠蛋白基因在染色体上的排列。该家庭的父母在临床上均无明显的贫血症状,而其一对孪生女患有HbH病。基因分析表明,母亲在一条染色体上具有三个紧密连锁的α-基因,而在另一染色体上完金缺失α-基因(ααα/--),父亲为右侧缺失α-地贫2杂合子(α-/αα),两个女儿均为右侧缺失α-地贫2与α-地贫1双重杂合子(α-/--)。     

Azorhizobium caulinodans ORS571结瘤位点5(nod locus 5)的鉴定

用克隆的含A.caulinodans ORS 571 nodABC基因启动子区域(P_1)的800bp AccⅠ-pUC Ⅱ DNA做探针,在染色体上探测到另一个与P_1同源的8.4kb DNA片段,从ORS571 pLAFR1基因文库中分离到带有该片段的克隆子pRG90,8.4 kb DNA的内切酶图谱分析及DNADNA杂交实验表明同源区(P_2)位于450bp SmaⅠ-SphⅠ片段内。用Ω因子对8.4 kb DNA进行缺失插入突变,鉴定出一个使S.rostrata结瘤延迟的突变株ORS571-5。该突变株结瘤延迟的表型可由pRK84质粒(nod locus 5)互补。     

无胶筛分毛细管电泳分离盐生盐杆菌DNA片段

 由羟乙基纤维素和聚吡咯烷酮混合组成筛分介质 ,在涂敷聚硅氧烷的毛细管柱上 ,研究了LambdaDNA/EcoRⅠ +HindⅢ片段分离的最佳条件。实验表明 ,混合筛分介质与单一的羟乙基纤维素筛分介质相比 ,改变了筛分介质的孔径大小 ,抑制了毛细管壁对DNA的吸附 ,从而改善了分离 ,并首次在同一条件下将所含的 13个片段完全分离。方法简便、快速 ,曾应用于两组盐生盐杆菌DNA片段的分离及其碱基对数目的推测。     

具有原核基因启动子活性的家蚕核多角体病毒的DNA片段

本文利用大肠杆菌启动子克隆用质粒pHE5,克隆和分离了家蚕核多角体病毒基因组的8种HindⅢ酶解DNA片段和一种SalⅠ酶解DNA片段。我们发现它们都具有指导抗四环素基因表达的启动子活性。测定了含有这些DNA片段的重组质粒的菌株抗四环素能力。其中最高的可以抗四环素至每毫升约30μg。我们分析了一个DNA片段的部分核苷酸顺序。发现它具有与原核基因启动子极相似的顺序结构。     

微量热法研究环境NaCl浓度对盐生盐杆菌生长代谢的影响

采用TAM air微量热系统和安瓿法测定了盐生盐杆菌在不同NaCl浓度中生长的生长产热曲线, 拟合得到盐生盐杆菌在不同NaCl浓度中生长的热动力学方程和热动力学参数, 并分析了盐生盐杆菌生长的各种热动力学参数与环境NaCl浓度的关系. 由此发现, 盐生盐杆菌生长的最适NaCl浓度并不是传统认为的一个宽泛的范围——3.5 mol·L^-1至约5.2 mol·L^-1 (NaCl饱和), 而是约3.9 mol·L^-1. 在环境NaCl浓度由3.9 mol·L^-1逐步升高至饱和的过程中, 盐生盐杆菌的生长代谢持续减弱. 进一步的透射电镜观察发现在近饱和的NaCl浓度中生长的盐生盐杆菌细胞发生了质壁分离, 较好地解释了微量热研究的结果. 由此对NaCl浓度变化导致嗜盐古生菌表面结构改变提出了新的解释.     

我国高效杀蚊球形芽孢杆菌BS10的43kd毒素蛋白基因的定位

从国内高效杀蚊球形芽孢杆菌BS10的质粒pFWI,克隆杀蚊毒素基因的～1.4kb Hind Ⅲ DNA片段,在大肠杆菌表达杀蚊幼虫活性的43kd毒素蛋白,首次为球形芽孢杆菌杀蚊毒素基因定位在质粒上提供了直接证据。通过Southern blot和Western blot对10株BS无杀蚊活性菌株进行分析,揭示出无毒株为杀蚊毒素基因缺失突变株,这是从基因及蛋白质水平对杀蚊和不杀蚊球形芽孢杆菌的区别的首次报道。本文得到的pFL109克隆可作为探针区分杀蚊和不杀蚊球形芽孢杆菌,具有应用价值。     

应用生物技术向小麦导入黄矮病抗性的研究

小麦黄矮病是我国小麦的主要病害之一,迄今在普通小麦中还未发现抗性材料.经酶联免疫吸附分析,发现在小麦族中有13个种具不同程度抗性.中间偃麦草、小麦-中间偃麦草的八倍体衍生物无芒中4和TAF46及由TAF46育成的小麦附加系L_1均高抗黄矮病.以L_1为抗源,采用CSph突变体和组织培养诱导分别育成了抗病的易位系119880和119899.资料表明其抗性由一个显性基因所控制.筛选出pEleAcc3和pPJN8(E_1-T_1)两个互补DNA分子探针,可探测出在普通小麦遗传背景中的中间偃麦草DNA,在Southern杂交中,中间偃麦草及其衍生物的DNA清晰显示出小麦DNA所缺乏的一条特异带,通过比较其相对深浅程度可判断易位系所获的外源染色体片断的大小,作为黄矮病抗性的选择标志.     

产甘油基因工程菌的构建

利用途径工程的基本原理,在大肠杆菌中构建一条产甘油的新代谢途径。从酿酒酵母Sacchdromyces cerevisiae INVSc1菌株总DNA克隆3-磷酸甘油脱氢酶基因(gpdl)和3-磷酸甘油酯酶基因(hot2),构建由两个杂合启动子trc启动基因的双表达盒的重组质粒pGEM-Cgpd1-Chor2,后者转入E．coli JM109菌株,构建的重组菌株就具有一条直接将葡萄糖转化为甘油的新代谢途径,将该重组菌株以葡萄糖为底物进行摇瓶发酵,甘油产率为1．18g／L。该研究结果为进一步构建生产1,3-丙二醇工程菌打下了基础。     

氧化锆/聚亚甲基蓝修饰电极检测CaMV35S启动子基因

制备了基于氧化锆(ZrO_2)/聚亚甲基蓝(PMB)修饰电极的无标记DNA传感器,用于转基因植物CaMV35S启动子基因的检测。探针DNA(ssDNA)通过ZrO_2和DNA的磷酸基的相互作用修饰到电极表面,以PMB氧化峰的示差脉冲伏安响应为检测信号,传感器和完全互补的DNA片段杂交后,PMB的氧化峰电流明显降低,当和完全不匹配的DNA片段杂交时,峰电流无明显变化。对于完全互补的DNA片段,在2.0×10~(-12)~2.0×10~(-8) mol/L浓度范围内峰电流的变化值和浓度的对数成良好的线性关系,检测限为4.1×10~(-13) mol/L(S/N=3)。所制备的传感器具有良好的稳定性、再生性和重现性,用于样品检测,结果令人满意。     

Microcalorimetric Studies on the Promoter Function in E. Coli TG1 from P. Maltophilia AT18 Chromosome DNA

In this paper, a promoter-probe plasmid pKK232-8 was used as a vector, which functioned in Escherichia coli TG₁ host. The plasmid DNA fragments from Pseudomonas maltophilia AT18 chromosome DNA active as promoter inEscherichia coli TG₁, the promoter function was studied by means of microcalorimetry, the promoter is about 800 bp DNA, it can promote the chloramphenicol (Cm) gene in plasmid pKK232-8, the Cm resistance level is about 80 μg mL^–1, the promoter activity is high. It implicates that there are probably many promoters in Pseudomonas maltophilia AT18 chromosome. All these information is readily obtained by an LKB 2277-204 heat conduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile method for microbiological genetic research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase （CAT） gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL^-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.     

利用微生物直接生产羟基丁酸单体

利用重组大肠杆菌E.coli HB101来进行直接生产羟基丁酸(HB)手性单体,研究了含pUCAB质粒的重组体E.coli HB101在各种条件下生长及积累HB单体的情况,研究了HB单体的积累随pH值变化的规律。结果表明,pH=6.8时,48 h内细菌可生产0.5 g/L以上的HB单体。     

重组质粒对大肠杆菌HB101生长影响的微量热研究

用微量热方法研究了嗜麦芽假单胞菌AT18, 受体菌大肠杆菌HB101, mel基因工程菌——大肠杆菌HB101/pWSY8和携带克隆载体pUC18质粒的大肠杆菌HB101等的生长代谢过程. 实验结果从热化学和热动力学上阐明了细菌的生长速率常数与其所含质粒的大小呈负相关. 探讨了低温处理对含不同质粒大肠杆菌生长的影响, 发现低温处理对工程菌生长影响最大.     

Analysis of plasmid DNA synthesis by double tracer method--differential effects of rifampicin and chloramphenicol on the replication of pSC101 and ColEl-amp in Escherichia coli K-12

An Escherichia coli strain, CR34 , harboring both pSC101 and ColEl -amp plasmids was exposed to media containing rifampicin (100 micrograms/ml) and/or chloramphenicol (180 micrograms/ml) and the cells were labeled for 20 min with 3H-thymine at 3, 25 and 50 min after exposure to drug(s). The plasmid DNA synthesis was assayed by DNA-DNA hybridization with 14C-labeled pSC 134 DNA as internal marker. In the presence of rifampicin, the replication of pSC 101 was from 57 to 104% that in its absence, and that of ColEl -amp was from 17 to 26%. The DNA replication of pSC 101 after addition of chloramphenicol was reduced to 35 to 75%, and that of ColEl -amp was reduced to 39% and then restored to 92%. This restoration was not observed in the presence of rifampicin.     

Molecular cloning and expression of Bacillus thuringiensis subsp. galleriae insecticidal crystal protein genes in Escherichia coli

The location of the toxin gene of B. thuringiensis subsp. galleriae (H5ab) on the Mr-130Md plasmid is determined by molecular cloning. Double digestion fragments (BamHI and SalI) and PstI restriction fragments as well, from the 130 Md plasmid, of B. thuringiensis subsp. galleriae, are ligated with the cloning vector pAT 153 respectively and transformed into E. coli strain HB 101. Out of 208 transformants, three colonies (FG2, FG9, FG19) give positive hybridization reaction using the HD-1 delta-endotoxin gene as a probe. They are presumed to contain the delta-endotoxin gene of B. thuringiensis subsp. galleriae. Western blot assays indicate that Mr-130 kDal and 68 kDal, crystal proteins produced by clone FG2 react with anticrystal protein antibody. The protein extracts of clone FG2 are lethal to Ostrinia furnacalis (Guenee). This is the first report with regard to the cloning and expression of the B. thuringiensis subsp. galleriae (H5ab) delta-endotoxin gene.     

THE PHOTOTOXICITY OF 8-M-ETHOXYTHIONEPSORALEN AND 6-M-ETHYLTHIONECOUMARIN

The phototoxicity of 8-methoxythionepsoralen (8-MOTP) and 6-methylthione coumarin (6-MTC) when activated by UV-A has been investigated using a variety of Escherichia coli strains, Haemophilus influenzae transforming DNA and Escherichia coli pBR322 plasmid DNA. The results demonstrate that 8-MOTP is a strictly oxygen independent photosensitizer that is about 500-fold less efficient in forming lesions leading to equivalent lethality than is the parent compound from which it is derived (8-MOP). As is true for 8-MOP, 8-MOTP is capable of inducing histidine independent mutations in E. coli and inactivating transforming DNA consistent with DNA being a target for lesions induced by this molecule in the presence of UV-A. 6-MTC is a strongly oxygen dependent photosensitizer activated by UV-A when tested with either E. coli cells or transforming DNA in contrast to the parent compound (6-methylcoumarin; 6-MC) which is not phototoxic when treated with UV-A. These results imply that the membrane may be an important target leading to lethality. 6-MTC in the presence of UV-A can inactivate pBR322 plasmid and Haemophilus influenzae transforming DNA activity in vitro suggesting that DNA is a potential target for this molecule when activated by UV-A.     

Cranberry changes the physicochemical surface properties of E. coli and adhesion with uroepithelial cells

Cranberries have been suggested to decrease the attachment of bacteria to uroepithelial cells (UC), thus preventing urinary tract infections, although the mechanisms are not well understood. A thermodynamic approach was used to calculate the Gibbs free energy of adhesion changes (DeltaG(adh)) for bacteria-UC interactions, based on measuring contact angles with three probe liquids. Interfacial tensions and DeltaG(adh) values were calculated for Escherichia coli HB101pDC1 (P-fimbriated) and HB101 (non-fimbriated) exposed to cranberry juice (0-27 wt.%). HB101pDC1 can form strong bonds with the Gal-Gal disaccharide receptor on uroepithelial cells, while HB101-UC interactions are only non-specific. For HB101 interacting with UC, DeltaG(adh) was always negative, suggesting favorable adhesion, and the values were insensitive to cranberry juice concentration. For the HB101pDC1-UC system, DeltaG(adh) became positive at 27wt.% cranberry juice, suggesting that adhesion was unfavorable. Acid-base (AB) interactions dominated the interfacial tensions, compared to Lifshitz-van der Waals (LW) interactions. Exposure to cranberry juice increased the AB component of the interfacial tension of HB101pDC1. LW interactions were small and insensitive to cranberry juice concentration. The number of bacteria attached to UC was quantified in batch adhesion assays and quantitatively correlated with DeltaG(adh). Since the thermodynamic approach should not agree with the experimental results when specific interactions are present, such as HB101pDC-UC ligand-receptor bonds, our results may suggest that cranberry juice disrupts bacterial ligand-UC receptor binding. These results help form the mechanistic explanation of how cranberry products can be used to prevent bacterial attachment to host tissue, and may lead to the development of better therapies based on natural products.