利用粗线期染色体和DNA纤维的FISH分析水稻端粒序列

利用粗线期染色体和DNA纤维的荧光原位杂交(FISH)技术分析了水稻广陆矮四号(Oryzasativassp.indicacv.GuangluaiNo.4)的端粒序列。粗线期染色体荧光原位杂交结果表明,大多数染色体的末端都有端粒串联重复,但信号的强度在不同染色体上是不同的。伸展DNA纤维荧光原位杂交结果显示,端粒最长的线状信号长度为6.55μm,最短的为1.82μm,依据2.51kb/μm的标准,它们分别相当于16.44kb和4.56kb。端粒的平均信号长度为3.62±1.32μm,相当于9.09±3.31kb。由此可以估计,最长的、最短的和平均长度的端粒拷贝数约为2349、651和1298±473。     

水稻染色体DNA的交变脉冲电泳分析

从水稻完整的细胞核中释放出来的染色体DNA在交变脉冲电泳申表现为一种“240kb单位”的形式。这种单位,经限制性内切酶消化,可产生连续分布的大至1500kb的染色体DNA片段。文章讨论了“240kb单位”作为水稻染色体DNA基本结构单位的可能性。     

水稻根尖染色体制备的一些体会

水稻的染色体比较小 ,不象小麦那样容易分散 ,尤其在大量检测染色体倍性的时候 ,要快速准确地对其倍性进行检测 ,需要花费大量的时间和精力。我们结合以往水稻染色体倍性检测的经验 ,谈一些体会。　　材料的准备　水稻根尖的准备非常重要 ,我们的做法是这样的 :在田间选择生长旺盛的植株 ,然后将根尖用剪刀去掉老根 ,放在清水中 ,可以适当加些肥料如尿素等 ,但注意不能过量 ,经常换水 ,待根尖长到 1 cm时 ,选择生长粗壮的根尖剪下 ,用 0 .0 0 2 %的 8-羟基喹啉预处理 2～ 3 h(处理时温度不要太高 ,最好保持在 2 5℃以下 ) ,再把根尖转移到 …     

显微镜光度计扫描测量人中期染色体的面积与DNA相对含量

应用显微镜光度计扫描测量人中期染色体的面积与DNA相对含量,结果显示出人染色体相对面积与相对长度之间密切的相关性。染色体DNA相对含量的高低,与染色体面积大小呈正相关。此外,DNA在染色体二维图形的分布是不均匀的,边缘与着丝粒部位的含量低,在纵向范围内变化较大,在横向范围内的变化较小。     

快速制备水稻基因组DNA PCR模板的煮沸法

文章发展了一个采用煮沸法快速制备水稻基因组DNA PCR模板的程序,成功地从水稻基因组扩增到了相应基因。     

酿酒酵母染色体DNA样品制备和CHEF分析

采用国产溶壁酶蛊酿酒酵母菌株Ｘ８的原生质体，将４×１０＾８／ｍｌ的原生持体悬浮液与等体积的１％低融点琼脂糖混匀后，凝固的原生质体包埋块在含１ｍｇ／ｍｌ蛋白酶Ｋ的ＮＤＳ缓冲中处理２４ｈ，获得适宜于ＣＨＥＦＪ民泳的染色体ＤＮＡ样品。采用了上述与前人不同的方法制备酿酒酵母染色体ＤＮＡ样品，为广泛研究真菌电泳核型和制备巨型ＤＮＡ分子量标记样品奠定了基础。ＣＨＥＦ电泳结果显示，供试菌株Ｘ８与对照菌７５５之间     

在植物粗线期染色体和DNA纤维上的荧光原位杂交技术

介绍了两种荧光原位杂交技术的详细实验步骤。第一种技术是在减数分裂粗线期染色体上的荧光原位杂交,包括从花粉母细胞中制备粗线期染色体和在这种染色体上定位ＤＮＡ序列,其分辨率水平能够达到１００ｋｂ。第二种技术是从植物细胞核中制备ＤＮＡ纤维,并在上面进行原位杂交,能够直接分析ＤＮＡ序列的分子排列关系,其分辨率水平能达到几个ｋｂ。为了说明这两种原位杂交技术在研究基因组和染色体结构、构建高分辨率的ＤＮＡ物理图谱上的能力,将展示用该技术直接分析番茄染色体端粒重复序列和端粒联接重复序列的染色体定位和ＤＮＡ分子排列。     

利用微波炉快速制备水稻基因组DNA PCR模板

建立了利用微波炉法快速制备水稻基因组DNA PCR模板的程序,成功地从水稻基因组扩增到了相应基因。     

酿酒酵母染色体DNA样品制备和CHEF分析

等高锁状均匀电场（contolllsclampedhomogeneouselectncfield,CHED电泳技术与分子杂交、酵母人工染色体（yeastartificialchromosome,YAC）等分子生物学技术的有机结合,推动了真菌电泳核型、染色体作图和染色体DNA长度多型性（chromosomalDNAlellgthpolymorphism,CLP...     

小鼠中期染色体制备方法探讨

目的:寻找小鼠细胞中期染色体制备过程中理想的取材方法和低渗的最佳时间,以及制片时滴片的最佳高度。方法:根据以往小鼠染色体制备方法的基本步骤,在取材方面进行对比,同时分别设置4个加入低渗液的时间和4个滴片高度进行试验对比。结果:各低渗时间段和各滴片高度所制备的结果存在一定的差异性,制备成功率和良好率最高的低渗时间为25 min,滴片高度为20 cm。结论:取材骨髓比较容易和简洁制备中期染色体,最佳低渗时间为25 min,最佳滴片高度为20 cm。     

Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.     

Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.     

红莲型细胞质雄性不育水稻线粒体DNA的AP-PCR分析

为了研究红莲型细胞质雄性不育与线粒体基因组的关系。以水稻红莲型粤泰细胞质雄性不育系A和保持系B及杂种一代F1为材料。应用AP-PCR分析,用10个单引物对其线粒体DNA进行扩增。实验结果表明,不同的引物在3种材料间均有不同程度的差异。为红莲型细胞质雄性不育分子机理的研究提供了线索;此外,在引物6F1的扩增图谱中找到一条在YTA和F1中特异的带TAF6F2,Sounthern分析TAF6F2不育胞质的特异性,可能与红莲型水稻细胞质雄性 不育性状的形成有关。     

Cell cycle and DNA content of mitotic cells in brain ganglia ofdrosophila larvae

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae ofDrosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and ofDrosophila nasuta were examined by autoradiography of [³H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [³H]thymidine for 24 hin vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae ofDrosophila melanogaster andDrosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

高效回收苏云金杆菌质粒DNA的方法研究

介绍三种简易、快速和高效回收苏云金杆菌（Bacillus thuringiensis,简称Bt)质粒DNA的方法。这些方法省时、经济、适用范围广,回收的Bt质粒DNA质量高,可直接用于各种分子克隆操作。     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.