Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

A simple plant polytene chromosome system,and its use for in situ hybridisation

When the cotyledons of Pisum sativum are cultured in the presence of 2–4D, large populations of cells with polytene chromosomes are stimulated to enter prophase; the use of these chromosome for studies of in situ hybridisation with nucleic acids is described.     

Non-random distribution of Alu-family repeats in human chromosomes

A phage lambda recombinant clone containing at least 8 Alu-family repeats (AFRs) has been isolated from a human genomic library, and DNA from the phage was used as a probe for in situ hybridization on G-banded human metaphase chromosomes of healthy donors and leukemic patients. Some chromosome bands show prominent clusters of silver grains in all individuals examined: 1p34, 1q23, 2q21–22, 10p14, 11p14, 10q21 and 11q14. The data suggest non-random distribution of AFRs in the human genome.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

核酸杂交技术在微生物生态学上的应用

由于自然界中大部分的微生物种类到目前为止仍不可培养,传统基于培养和纯种分离的技术在研究微生物生态时面临很大障碍。分子生物学的进展为诸多长期困扰微生物学研究的问题提供了解决办法。核酸杂交技术已被证实在微生物系统分类的微生物生态等领域的研究具有巨大潜力并已被广泛应用。综述核酸杂交技术的基本原理、实际应用及其主要优点和局限性,以及提高其检测灵敏度和特异性的方法,并对该技术的应用前景作了探讨。     

核酸杂交技术在微生物生态学上的应用

由于自然界中大部分的微生物种类到目前为止仍不可培养,传统基于培养和纯种分离的技术在研究微生物生态时面临很大障碍。分子生物学的进展为诸多长期困扰微生物学研究的问题提供了解决办法。核酸杂交技术已被证实在微生物系统分类和微生物生态等领域的研究具有巨大潜力并已被广泛应用。综述核酸杂交技术的基本原理、实际应用及其主要优点和局限性,以及提高其检测灵敏度和特异性的方法,并对该技术的应用前景作了探讨。     

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

肽核酸在分子生物学技术中的应用

肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同。PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构。与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点。简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望。     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

核酸探针传感器的构建

依据石英振子的晶体谐振频率是其表面沉积物的函数,建立了简便、特异、敏感性达100pg,并能对受检样品相对定量的核酸杂交检测新方法,即压电晶体式核酸探针传感器,该方法的主要特点是以快速、敏感的频变信息作为核酸杂交的显示系统.     

Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine

Reserpine evokes transsynaptic impulse activity by depleting catecholaminergic neurotransmitters in the rat brain. Previous studies suggest a relationship between catecholaminergic activity and calmodulin concentration. In this report we employ Northern blot analysis to examine the effect of a single subcutaneous injection of reserpine on levels of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Regional differences in mRNA levels were also investigated byin situ hybridization and drug-induced changes were noted particularly in specific regions of the rat brain stem. The riboprobe used in thein situ hybridization study recognized a 4.0 kilobase neuronal calmodulin mRNA species (NGB1), which was derived from the rat CaM1 gene. A calmodulin radio-immunoassay was utilized to demonstrate a drug-induced increased in calmodulin protein levels in a region which included the brain stem.     

Detection of a single-copy gene on plant chromosomes by in situ hybridization

Summary DNA recombinant technology, combined with improvements in hybridization efficiency and quality of chromosome spreads, has made the method of in situ hybridization a reliable tool for gene mapping used by mammalian cytogeneticists to complement other methods. By appropriate alterations of the method, we demonstrate that detection of unique genes can be achieved along plant chromosomes despite some inherent disadvantages of the plant material. Using genomic subclones homologous to 6.6 kb of the single-copy chalcone synthase gene in parsley, we report the first example of chromosomal detection and localization of a unique endogenous gene in plants.