原位DNA合成技术

原位ＤＮＡ合成技术李子银,胡会庆（华中农业大学农学系,武汉４３００７０）关键词原位ＤＮＡ合成自六十年代放射标记探针应用于原位分子杂交技术后,人们就可以对特异的核苷酸在原位进行检测,然而,直到八十年代由于非放射性标记探针的使用,原位分子杂交技术才得到了...     

以地戈辛标记反义RNA为探针的组织原位杂交

原位分子杂交是在液相和固相分子杂交基础上发展起来的,从染色体、细胞或整体水平在原位研究特异核酸的分布、数量、表达形态及其同细胞的分化、生理或病理状态、形态特征演化之间关系的分子生物学技术。随着技术的发展,原位杂交不仅被用来研究特定DNA、RNA的细胞内的分布,而且被用来探索被检mRNA相关基因的表达。可以说,原位杂交技术在分子生物学与形态学研究之间架起了一座桥梁。     

原位分子杂交法检测恙螨体内肾综合征出血热病毒核酸的研究

为探讨肾综合征出血热病毒（ＨＦＲＳＶ）在恙螨体内的分布和定位,采用原位分子杂交技术检测恙螨体内ＨＦＲＳＶＲＮＡ。结果发现：原位分子杂交技术的检出阳性率较ＩＦＡＴ高;ＨＦＲＳＶ阳性信号颗粒呈弥散分布,多见于恙螨幼虫和若虫的腹部组织细胞内,该部位是恙螨的卵巢细胞、中肠及支囊上皮细胞。前体组织细胞内少见;个别幼虫和若虫的前足和中足细胞内亦可见到ＨＦＲＳＶＲＮＡ阳性信号。在原位分子杂交中,若虫组织细胞内的ＨＦＲＳＶＲＮＡ阳性信号较幼虫密集而且量多,表明ＨＦＲＳＶ在恙螨体内可传递并有增殖现象     

植物细胞核纤层的研究

应用细胞成分选择性抽提方法,结合非树脂包埋去包埋电镜技术显示悬浮培养的胡萝卜细胞和银杏雄性生殖细胞均具有核纤层结构,免疫印迹反应证明这两种细胞的核纤层由A型和B型核纤层蛋白组成;至少分别含有66ku,84ku和66ku,86ku多肽,免疫胶体金标记将这些蛋白定位在核周缘,光镜和电镜原位分子杂交显示植物细胞具有与动物细胞核纤层蛋白cDNA同源的序列存在;其mRNA分选的部位主要分布在靠近核膜周围的胞质部分,实验结果证明植物细胞确实存在核纤层.     

电镜技术在核酸分子杂交研究中的应用

电子显微镜是目前唯一能直接看到DNA或RNA分子的工具,而电泳、同位素标记等技术只能间接地测定这些分子,电镜就是以它这种得天独厚的优点在核酸分子研究中崭露头角。基因的准确定位,核酸复制模型的完善,真核基因DNA倒转重复序列“十字架”结构的观 察及内含子的二发现等等,近四十年来,核酸分子杂交技术取得如此可喜的成绩,电镜所作的贡献是不能低估的。     

结构生物学研究方法的重大突破——电子直接探测相机在冷冻电镜中的应用

近年来,科学家应用冷冻电镜技术(cryo-EM)解析出了低对称性生物大分子的高分辨率(3~5魡)三维结构,并用其密度图直接进行了分子建模。与传统的X-射线和NMR方法相比,冷冻电镜技术具有适用于分子量较大的生物分子、样品不需结晶且用量很少等优势。尤其是电子直接探测相机(electron direct detection device,DDD)在冷冻电镜技术中的应用,使高分辨率的结构研究变得更加简单、应用更为广泛,是一个重大突破。文章介绍DDD相机的原理和技术优势,及其在解决冷冻电镜技术困难中的一些应用,进而展望了DDD相机可能给冷冻电镜技术应用带来的突破性进展。     

肾综合征出血热人体组织中病毒包涵体的性质及意义的进一步研究

应用免疫组化、原位分子杂交、电镜及免疫电镜等方法进一步对肾综合征出血热人体尸检组织中病毒包涵体（ＩＢ）的抗原、核酸性质和超微结构特点作了进一步观察。结果,在３９例中的２０例尸检病例组织中显示出病毒核蛋白抗原和血凝素抗抗原阳性的ＩＢ,其中包括１６例陕西尸检病例组织中的６例休克期和１例多尿期病例,１７例上海病例中的３例休克期和９例少尿期病例及３例江西病例中的１例休克期病例。ＩＢ主要分布在呼吸道和肺泡、肾远曲小管和集合管、胃肠道、腺垂体、扁桃体、胰腺、前列腺等组织的粘膜上皮和腺上皮细胞及肝细胞和睾丸生精上皮细胞胞浆中,阳性细胞形态基本正常。应用原位分子杂交,可在该组细胞小同时检测到病毒ＲＮＡ,多为胞浆内弥漫阳性,仅少数组织中显示出病毒ＲＮＡ阳性ＩＢ结构。电镜观察阳性组织细胞中出现由大量微丝微管及颗粒样结构组成的ＩＢ结构,其小的病毒颗粒状结构、内质网及纤维丝状结构呈病毒抗原阳性,上述结构位于高尔基体区。结果说明该病毒有感染上皮细胞的特性,对其宿主细胞的致细胞病变作用是极其温和的,且多表现在亚细胞水平。ＩＢ可能是病毒过量表达抗原的堆积或病毒复制部位,而微丝微管结构可能参与病毒的感染过程。     

“缺口翻译”法制备~(32)P 标记DNA探针和几种核酸杂交技术

定性或定量检测天然DNA或重组DNA中某些特殊的顺序片段,分子杂交是最常用的方法。近几年,核酸的分子杂交技术日趋完善,以不同材料为支持物的固相杂交技术取得了更为迅速的发展。核酸分子杂交方法的灵敏性主要取决于同位素标记杂交探针的比放射性强度。因此制备高比放射性探针是提高灵敏性的关键。用缺口翻译方法制备较稳定的高比放射性强度的杂交探针,大肠杆菌DNA聚合酶Ⅰ是一个理想的工具。本文介绍我们建立并改进“缺口翻译”制备P标记的探针方法,以及几种重要的核酸固相杂交技术。     

始于努力,续于真诚,臻于共赢

2010年第7期,<学报>为在生物物理研究所举办的电镜三维重构学习班出版了一期专刊,其中除了两篇与电镜三维重构相关的研究论文之外,在朝仡夕拾栏目发表了北京大学医学部尹长城老师撰写的一篇介绍运用电子显微镜研究生物人分子起源及进展的文章,另外四篇综述分别讨论了电镜三维重构、单颗粒电镜、冷冻电镜断层成像技术及三维电镜自动化...     

介绍一种非放射性标记的原位杂交新方法

在病理诊断和科研工作中,原位杂交是一种常用的检测技术。原位杂交组织化学方法是将核酸分子杂交技术与组织细胞原位显示某种特定ＤＮＡｍＲＮＡ及其产物的定性定量及变化规律相结合的一种新技术。目前,在检测特异ｍＲＮＡ的原位杂交组织细胞化学中多采用同位素标记的探...     

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.     

Analysis of viral infections by in situ hybridization

In situ hybridization provides a versatile means of analysis of the life cycles of viruses in single cells. This kind of analysis in "real life" situations has provided considerable insight into the spread of viruses, mechanisms of tissue damage by viruses, and virus-host cell interactions in chronic diseases. In this article I describe refinements in technology underlining these advances, especially developments that have made the technique such a sensitive and quantitative one. I also describe a method for combined macroscopic and microscopic in situ hybridization, new assays for the simultaneous detection of genes and gene products in a single cell, and a double-label in situ hybridization technique. These methods have already proved useful in analyzing the molecular ecology of viral infections, and should find wide application to problems of genetic regulation in many other systems.     

In Situ Hybridization of Cytospin Preparations: A Rapid Nonisotopic Screening Method for Isolated Cells

A simple and rapid method for light microscopic in situ hybridization on cytospin preparations is described and demonstrated for detection of viral nucleic acid in a virus-infected cell line. Cells were fixed by acetone followed by chloroform, denatured by heat, hybridized at 37 C, and hybridized sites detected with a multiple step procedure (primary anti-biotin antibody, biotinylated second antibody, streptavidin-peroxidase). This method can be used for screening studies at the light microscope level, and offers a useful and simple way to determine optimum hybridization conditions for subsequent electron microscopic investigations.     

Ultrastructural distribution of growth hormone and prolactin mRNAs in normal rat pituitary cells: a comparison between preembedding and postembedding methods

In situ hybridization (ISH) at the electron microscopic level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our electron microscopic ISH method using biotinylated oligonucleotide probes for rat growth hormone and prolactin mRNAs and compare the preembedding method with the postembedding method. Preembedding electron microscopic ISH localized rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticulum (RER). Rat growth hormone mRNA was distributed diffusely on the RER, whereas rat prolactin mRNA was scattered and distributed focally. Thus there might be a specific translational site for prolactin mRNA on the RER. Rat growth hormone mRNA signals were also recognized on the polysomes of the RER, using the postembedding method with streptavidin gold conjugate. The hybridization signal intensity using the postembedding method was lower, and non-specific signals were more frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of utility and preservation of mRNA. Electron microscopic ISH is considered to be an important tool for evaluating the intracellular localization of mRNA and the site of specific hormone synthesis on the RER.     

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells

The present review evaluates methods for electron microscopic immunocytochemistry and in situ hybridization, using post-embedding techniques and colloidal gold as a label. Special emphasis is given to double labeling immunocytochemistry and double in situ hybridization and to their combined application on the same specimen. Brief guidelines are presented for fixation, embedding media, the use of polyclonal and monoclonal antibodies and nucleic acid probes. Conditions for labeling and binding of antibody and nucleic acid probes to the target and protocols for direct and indirect immunodetection are discussed. Combinations of direct and indirect immunodetections in multiple labeling experiments are summarized.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

Transmission electron microscopic study of maize pachytene chromosome 6

Cytoplasm-free chromosomes are frequently obtained in meiotic chromosome spreads prepared from mildly-fixed maize microsporocytes. These chromosomes are suitable for detailed structural analysis using a published electron microscopic technique. In the electron micrograph, the knobs and heterochromatin regions that have been used for karyotype analyses in the light microscope are clearly visible. Therefore, the electron microscopic map can be easily aligned with the traditional cytological map. In addition to these prominent structural features, numerous electron-dense bands also are observed. To determine whether the bands can be used as markers for the identification of each chromosomal subregion, the banding pattern of chromosome 6 is analyzed. Chromosome 6 is frequently associated with the nucleolus and can be easily recognized. We observed that at the zygotene stage in prophase I, electron-dense regions are detected on each homolog of the synapsing chromosome. During synapsis, the electron-dense regions on both homologs are brought into register to form more conspicuous bands. At the early pachytene stage, the banding pattern is stable and reproducible. Chromosome 6 contains eight dark bands, 19 medium bands and 14 light bands. The bands can be used as intrachromosomal markers for regional assignment of genes in detailed in situ hybridization mapping or cytogenetic studies. As the pachytene stage progresses, condensation of the chromosome bivalents is accompanied by fusion of adjacent bands.     

rRNA sequence-based scanning electron microscopic detection of bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.