Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.     

Postdifferential display: parallel processing of candidates using small amounts of RNA

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.     

mRNA差异显示RT-PCR技术在昆虫学研究中的应用

mRNA差异显示RT- PCR是近 1 0年广泛使用的用于克隆和分析差异表达基因的有效技术。该文简要介绍了差异显示RT -PCR技术的原理、优缺点及其优化方法 ,重点综述了mRNA差异显示RT -PCR技术在昆虫学研究中的应用。     

运用多种策略改良差异显示PCR

目的：改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法：①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果：减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论：改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。     

差别显示mRNA法的改良

本文介绍了一种筛选和克隆差异表达基因的新方法——差别显示mRNA法。并在模板、引物、反应体系、反应条件等方面,对该方法作了深入的探索和改进,与原方法相比,改良法具有高效、可靠性高、经济等优点。实验表明,该方法能有效地筛选到一对细胞或组织在不同状态下表达差异的基因。为新基因的发现和克隆提供了新的手段。     

Male specific genes from dioecious white campion identified by fluorescent differential display

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.     

Differential display: Identifying genes involved in cardiomyocyte proliferation

An estimated 15,000 different mRNA species are expressed in a typical mammalian cell. The differential expression of mRNAs in both a temporal and cell-specific manner determines the fate of the cell and creates the organism. Analysis of this differential gene expression has become a central aim of many laboratories attempting to understand the mechanisms underlying various biological processes. Currently, we are using a technique called differential display to analyze the differential expression of genes in cardiomyocytes. Differential display is a rapid and powerful technique that was introduced by Liang and Pardee in 1992. Since that time, it has been successfully applied by several groups, and it is quickly becoming a standard method for studying differential gene expression. Here, we present a detailed article discussing the differential display methodology and how we have utilized it to identify potential genes involved in cardiomyocyte proliferation. Furthermore, we have provided a list of materials and supplied examples of data obtained, in an effort to allow the reader to perform the technique with success in their own laboratory.     

运用差异展示分离特异性表达的基因

在高等生物中含有约100000个不同的基因,其中仅有15％的基因在任何个体细胞中均表达．因此分离特异的目的基因便显得十分重要.差异展示是通过部分扩增mRNA的逆转录产物、经测序胶电泳,分离到差异性表达的基因.它与消减杂交相比是分离特异表达基因的更有效的手段．虽然这种方法在实际运用中存在着这样或那样的困难,但随着对这种技术的不断改进,它将会有越来越广泛的用途．     

一种快速mRNA差异显示技术及用于分离辐射诱导转录子

介绍一种从不同类型细胞或不同生长状态细胞中分离差异表达基因的快速ｍＲＮＡ差异显示技术．其特点是不用同位素标记,操作简便,在普通琼脂糖凝胶电泳中就能分辨差异显示的ｃＤＮＡ带,便于ＤＮＡ回收和进一步重组克隆．用此方法成功地分离到电离辐射诱导转录子．     

筛选差异表达基因和蛋白质的方法进展

分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries.

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.     

Cloning arbuscule-related genes from mycorrhizas

Until recently little was known about the identity of the genes expressed in the arbuscules of mycorrhizas, due in part to problems associated with cloning genes from the tissues of an obligate symbiont. However, the combination of advanced molecular techniques, innovative use of the materials available and fortuitous cloning has resulted in the recent identification of a number of arbuscule-related genes. This article provides a brief summary of the genes involved in arbuscule development, function and regulation, and the techniques used to study them. Molecular techniques include differential screening, differential display and screening with heterologous probes, and can involve the use of mycorrhizal plant mutants. New technologies such as proteome analysis are also discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.     

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations.Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.     

基因差异表达研究方法的探讨--从DD-PCR到cDNA RDA

ＤＤ－ＰＣＲ和ｃＤＮＡ ＲＤＡ都是近年发展起来的新技术,它们被应用于生物及医学领域,在基因差异表达和筛选差异基因的研究中有着广泛的前景。本文论述了ＤＤ－ＰＣＲ及ｃＤＮＡ ＲＤＡ的基本技术策略,并对两者进行了比较分析。     

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.     

Heterogeneous expression of mRNA in rat liver lobules as detected by differential display

Although liver hepatocytes appear to be uniform histologically, they are considerably heterogeneous with respect to their individual physiological capacities. In order to find still unknown genes that are heterogeneously expressed and with the aim of evaluating the usefulness of the differential display technique for this purpose, we performed differential displays with mRNA isolated from hepatocytes from the periportal and pericentral zone of the rat liver. In this way we identified at least two mRNAs exclusively expressed in the pericentral fraction. Sequence analysis revealed that the corresponding genes encode proteins with proline-glutamate dipeptide repeats similar to ones previously identified in rat pheochromocytoma and brain. In situ hybridization confirmed the heterogeneous distribution of the mRNA. Only one to two cell lines surrounding the terminal hepatic venules were positive, strongly resembling the heterogeneous expression of the enzyme glutamine synthetase. Our work demonstrates that the differential display method is a useful tool for the identification of genes that are differentially expressed in individual parenchymal cells. In fact, our results prove that differential display technology can be used for the identification of cellular markers for distinct subpopulations of cells in a given tissue.     

Isolation of genes expressed in specific tissues of Arabidopsis thaliana by differential screening of a genomic library

The small genome size of Arabidopsis thaliana allows the isolation of genes expressed in specific tissues and under controlled conditions by the differential screening of a genomic library, as has been shown previously for yeast and Drosophila. cDNA probes, based on poly(A)+ mRNA isolated from different Arabidopsis organs, were used in colony hybridizations with 1145 randomly chosen genomic clones, representing 27,000 kb of Arabidopsis DNA. Twenty percent of the clones containing low-copy-number sequences hybridized with one or more of the cDNA probes that were synthesized from mRNA isolated from leaves, stems, seed pods, inflorescences, callus tissue, and light-grown and dark-grown plants. Comparison of the colony hybridizations led to the identification of a large variety of clones which contain differentially expressed genes. The pattern of expression was confirmed by Northern analysis. The advantage of the described method is that it yields directly genomic sequences that contain specifically expressed or induced genes. In particular, it circumvents the construction and differential screening of cDNA libraries for every tissue or environmental parameter to be analyzed.     

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.     

Cloning oncogenic ras-regulated genes by differential display

The coordinated regulation of gene expression is a key cellular function that specifies cell characteristics as well as controls normal physiological processes of the organism. Deregulation of this gene expression leads to a variety of abnormal conditions such as cancer. The ras oncogene is one of the most frequently found mutations in various types of human cancer. The mutated Ras protein constitutively elicits multiple mitogenic signals to the nucleus to alter gene expression of target genes that are involved in a broad range of normal cellular functions. Thus the identification of these genes may provide an important tool toward the understanding of these pathogenic processes. As a first step to reveal these processes at the molecular level and to dissect the key pathway employed by oncogenic Ras protein, we have looked for its target genes in rodent model cell lines using the differential display method. Our initial screening has isolated a number of genes either up- or downregulated by oncogenic ras activation. Although the functional analyses of these genes in terms of ras-mediated cell transformation will be the major challenge, differential display has come to be a very efficient tool that helped us move to the next step. In this short report, we focus primarily on the technical aspects of differential display and experimental designs used in this study.