DNA催化活性的研究：Ⅱ.DNA分子催化机理探讨

通过乙酸萘酯水解产物的显色反应，研究了ＤＮＡ分子二级结构对催化萘酯水解活性的重要性，结果表明：双链ＤＮＡ（ｄｓＤＮＡ）分子具有良好催化乙酸－α－萘酯、乙酸－β－萘酯水解的活性，而单链ＤＮＡ（ｓｓＤＮＡ）则完全不具备这种活性，ＤＮＡ完全酶解产物也不能使α－萘酯产生显色反应。因此认为，ＤＮＡ分子是具有催化活性的生物高分子。从ＤＮＡ二级结构角度探讨了其催化萘酯水解的反应机理。     

牦牛角真皮乳头cDNA文库的构建

构建cDNA表达文库用于克隆和研究牦牛角生长相关基因.Trizol提取牦牛角真皮乳头总RNA并纯化mRNA,用ZAP Express cDNA Synthesis Kit合成双链DNA,进行末端修饰,接入ZAP表达载体.经Gigapack Ⅲ Gold Packaging extract体外包装,转染XL1-Blue MRF'宿主菌,形成初级文库,初级文库进一步扩增形成稳定的cDNA文库.结果表明,文库库容量为4×107,重组率达93%,为进一步从文库中筛选相关基因提供了有效工具.     

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.     

单增李斯特菌溶源性噬菌体的诱导及鉴定

利用丝裂霉素C诱导获得单增李斯特菌溶源性噬菌体,并确定其核酸类型。PCR及其扩增产物的序列测定检测原噬菌体,透射电子显微镜观察丝裂霉素C诱导获得的溶源性噬菌体颗粒,DNase I、RNase A和绿豆核酸酶分别消化噬菌体基因组,确定噬菌体的核酸类型。结果显示,38株单增李斯特菌分离株中的16株在tRNAArg位点携有原噬菌体,其中1株可诱导出噬菌体颗粒,该噬菌体为长尾噬菌体,核酸为dsDNA。噬菌体的成功诱导为噬菌体及其功能基因的开发利用奠定了基础。     

A potassium channel protein encoded by chlorella virus PBCV-1

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.     

BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure

Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.     

基于DNA双链电荷转移的Hg2+电化学生物传感器的研究

基于DNA双链电荷转移原理,利用胸腺嘧啶(thymine)与Hg2的特异性识别和计时电量法构建了一种高灵敏检测水溶液中Hg2的电化学生物传感器.该传感器将含有1个T-T碱基错配对的DNA互补双链通过Au-S键自组装在金电极表面,运用计时电量法在含有亚甲基蓝的铁氰化钾溶液中进行测定.T-T错配阻断了DNA双链内部电荷转移,而Hg2通过T-Hg2-T配位作用与双链DNA特异性结合并形成DNA双链内部电荷转移通路,引起电极表面计时电量的变化.计时电量测定结果显示:在亚甲基蓝的还原峰电位(-380mV)附近,计时电量随着溶液中的Hg2+浓度的增大而增加,Hg2+浓度在1.0 nmol/L～ 104 nmol/L范围内,计时电量的变化量与Hg2浓度的对数呈良好的线性关系,线性相关系数(R2)为0.997,检测限为0.5 nmol/L(S/N=3).干扰实验表明,该传感器对Hg2具有良好的特异性和选择性.     

2种不同方法制备玉米赤霉烯酮单克隆抗体适配子的ssDNA文库的比较

体外合成全长为71个碱基的随机ssDNA文库,采用SELEX技术,以玉米赤霉烯酮单克隆抗体为靶蛋白,微孔板为筛选介质进行筛选。通过对比不对称PCR法和生物素-链霉亲和素磁珠2种分离方法制备ssDNA文库的效果,确定了以生物素-链霉亲和素磁珠分离方法制备ssDNA为优选方法。ssDNA文库扩增为双链DNA(dsDNA)文库的PCR反应条件为:退火温度50℃,循环数20次。不对称PCR退火温度为50℃,最佳循环数为30次,引物Ⅰ与引物Ⅱ的比例为80∶1。此反应条件下,ssDNA文库PCR扩增的条带清晰、稳定、特异性高。生物素-链霉亲和素磁珠法为有效筛选特异性强的玉米赤霉烯酮单克隆抗体适配子的优选技术方案,为适配子SELEX筛选奠定基础。     

牛睫毛毛囊生长期毛乳头细胞cDNA表达文库的构建

为构建高质量的牛睫毛毛囊生长期毛乳头细胞cDNA表达文库,以新生荷斯坦牛上眼睑为组织材料,显微解剖分离出高纯度的生长期牛睫毛乳头,Trizol一步法提取总RNA后纯化mRNA;用ZAP Ex-press cDNA Synthesis Kit合成双链cDNA,进行末端修饰后接入ZAP表达载体。经GigapackⅢGold Pack-aging extract体外包装,转染XL1-Blue MRF′宿主菌,形成初级文库后进一步扩增,形成稳定的cDNA文库,文库库容量为3.3×107pfu/mL,重组率达95%。为进一步筛选、克隆生长期睫毛毛乳头特异性表达的基因、阐明睫毛毛囊发育的基因调控之分子机制奠定了研究基础。     

灵芝中一种新的脱氧核糖核酸酶的纯化及特征

通过(NH4)2SO4沉淀,利用Blue Sepharose 6 Fast Flow和SP Sepharose Fast Flow层析方法从灵芝中分离纯化了一种脱氧核糖核酸酶,命名为GLDNase.通过质谱分析确定GLDNase精确分子质量为13807 u.SDS-PAGE电泳表明GLDNase为单亚基多肽,其N-端氨基酸序列为PLDTGRYHIYTW/T/CDGG.GLDNase可作用于ssDNA和dsDNA,是一种非限制性内切酶,酶活性依赖于二价金属阳离子Mg2+,10 mmol.L-1EDTA可完全抑制其活性.最适pH值为8.4,40℃时相对活性最高.GLDNase水解DNA的产物末端的基团为3′-OH、5′-磷酸.     

Deoxyribonucleic acid in germinal vesicles of oocytes of Rana pipiens

The amount of DNA isolated from germinal vesicles of Rana pipiens oocytes is similar to that isolated from whole unfertilized eggs, and this suggests that oocyte nuclei are the source ofmuch of the DNA obtained from whole eggs. Fluorometric determinations of the isolated DNA show the presence of 0.004 microgram of DNA equivalents per germinal vesicle. Similar values were obtained by estimations from comparisons of the cesium chloride densitygradient profiles of sample DNA and a known amount of reference DNA from Pseudomonas aeruginosa. The buoyant density of DNA prepared from isolated germinal vesicles is the same as that of frog-liver DNA, 1.706 grams per cubic centimeter, as determined by equilibrium sedimentation in a cesium chloride density gradient.     

黄鳝体表粘液中基因组DNA的提取与分析

黄鳝基因组DNA一直采用抽血或剪取肌肉来提取,但是采血或剪取肌肉后黄鳝成活率很低。为了保留亲本,尝试从黄鳝体表粘液中提取DNA,并采用紫外分光光度计对提取到的DNA的纯度和浓度进行了检测。结果表明,用蛋白酶K消化,酚仿抽提后从粘液中得到的DNA的纯度与从肌肉中提取到的相差不大,但是提取到的量较少。为了验证提取的DNA是不是黄鳝基因组DNA,用黄鳝actin引物和18S r RNA基因引物扩增之后检测到了粘液中的较亮的DNA片段。为了进一步确认检测到的是黄鳝的DNA,我们又对得到的DNA进行了扩增,并得到的产物进行了克隆和测序。结果表明,粘液中提取到的DNA是黄鳝的基因组DNA。随后又选择了60尾黄鳝,提取其粘液之后对其成活率进行统计,养殖1个月后黄鳝的成活率都在95%以上。此黄鳝体表粘液提取DNA方法能提取高质量的DNA,可用于用于PCR鉴定。     

PCR法快速检测扩张青霉

 【目的】研究棒曲霉素扩张青霉菌（Penicillium expansum）的快速和特异性PCR检测方法。【方法】通过P.expansum的纯菌平板和液体培养以及纯菌接种于苹果、苹果果肉和苹果汁,用Cenis改良方法提取DNA,以基于isoepoxydon dehydrogenase和polygatacturonase编码基因的两对引物扩增P.expansum DNA,建立特异和快速的检测P.expansum的方法。【结果】基于polygatacturonase基因的引物具有很强的特异性,只有目标DNA片段被扩增,而基于isoepoxydon dehydrogenase基因的引物扩增特异性较差。纯培养可以检测到5×10-6 μｇDNA/每反应体系,整个检测时间约为5 h,比传统培养法（4～6 d）时间大大缩短。苹果、苹果果肉和果汁接种纯P.expansum后所提DNA都得到了很好的扩增,具有很高的特异性,不受苹果样品DNA的干扰。【结论】本研究建立的P.expansum的PCR检测方法特异、快速、灵敏,可用于商品果汁和苹果汁工业化生产中P.expansum的快速检测和质量控制,也可用于工艺过程对P.expansum污染的快速溯源。     

高粱总DNA不同提取方法的比较

[目的]探索高粱总DNA的快速、高效提取方法,为将其导入水稻提供大量、高质量的DNA。[方法]以高粱幼叶为材料,分别采用SDS法、CTAB法、尿素法和NaOH法提取高粱总DNA,并对不同方法提取DNA的纯度、浓度和产量进行检测。[结果]尿素法提取DNA纯度和质量最好,SDS法和CTAB法次之,NaOH法最差。采用尿素法、SDS法、CTAB法和NaOH法分别可从1g高粱样品中提取到约292.00、21.75、152.25和243.75μg的DNA。4种方法提取的高粱总DNA的琼脂糖凝胶电泳结果表明NaOH法不能有效提取高粱总DNA,其他3种方法在提取高粱总DNA时能较好地保证其完整性。高粱总DNA分子量约为50KB。[结论]尿素法提取DNA的纯度最好、产率最高,是一种理想的高粱总DNA提取方法。     

提取方法与部位对浙贝母基因组DNA提取质量的影响

探讨了改良CTAB法、SDS法、高盐低pH法和试剂盒法对浙贝母(Fritillaria thunbergii Miq.)基因组DNA的提取效果.综合比较了各方法提取的DNA浓度、纯度、完整性,结果表明高盐低pH法是比较适合浙贝母新鲜叶片基因组DNA提取的方法.比较了分别以叶片和鳞茎为材料提取的DNA质量,发现以新鲜叶片...     

Mitochondrial DNA in yeast and some mammalian species

Yeast DNA, in a cesium chloride density gradient, shows a minor or satellite band with a density lower than that of the main nuclear component. The DNA isolated from purified mitochondria of yeasts corresponds in density to this satellite band. In solution, this DNA more easily undergoes renaturation as compared to DNA from cell nuclei. The ease of this renaturation is presumably due to a homogeneity greater than that of nuclear DNA. Mitochondrial DNA isolated from several mammalian species has the same or higher density than nuclear DNA, but differs in its ready renaturability.     

几种提取植物DNA方法的比较

以玉米和草的叶片为材料,分别用CTAB,SDS和尿素法提取基因组DNA,通过紫外分光光度法、琼脂糖凝胶电泳、蒽酮比色法对所提DNA的产量、质量和糖含量进行了检测比较,并对鲜样采取了不同的干燥处理。试验结果表明,鲜样和硅胶干燥的叶片提取的DNA无降解或降解很少,45℃烘干样也能提取到大量的高分子DNA,自然干燥样的提取效果因植物材料而异。     

信号芋螺基因组DNA提取方法的优化

高质量的基因组DNA的获取是芋螺毒素基因克隆及基因组文库构建的关键。笔者采用4种方法提取信号芋螺（Conus litteratus)的不同组织器官的DNA，并综合比较了各组DNA的浓度和纯度、DNA片段的大小和完整性。结果表明，由改良的DNA快速提取试剂盒法提取的DNA纯度和浓度较好；4种组织器官中，腹足纯度较高，是更合适的提取组织；肝胰脏和毒管获取的DNA产率最高，但片段完整性差，污染严重；PCR反应获得了跟预期大小一致的特异扩增产物。因此，信号芋螺基因组DNA的提取最佳方法为改良的DNA快速提取试剂盒法，提取部位为腹足组织，可以获得高质量的基因组DNA，且符合后续实验要求。     

菊芋不同部位DNA提取的比较研究

分别以菊芋的茎段、叶片和块茎为材料,采用改良CTAB法分别提取菊芋3个部位的基因组DNA,用0.8%的琼脂糖凝胶电泳检测DNA质量,分别测定3个部位的DNA在A260和A280下的吸光值,根据A260/A280的值检测DNA的浓度和纯度,并以3个部位提取的DNA为模板进行ISSR扩增。结果表明：采用菊芋叶片提取DNA的产率最高,茎段次之,块茎最低。三个部位提取的DNA纯度均相近,提取叶片获得的DNA浓度最大,为163.2 ng·μL-1;其次是茎段,为137.4 ng·μL-1;块茎获得的DNA浓度最低,为95.5 ng·μL-1。菊芋不同部位提取的DNA模板对ISSR扩增没有明显影响,DNA浓度都能达到扩增的要求。菊芋3个部位提取的DNA质量均较好,可以进行后续的酶切和PCR扩增等实验。