通过cDNARDA法分离和识别盐藻(Dunaliella salina)盐胁迫相关基因

采用cDNA代表性差异分析 (RDA)技术 ,对盐藻在盐胁迫时差异表达的基因进行了分离鉴定 .在分离到的 10个基因中 ,有 5个与已知基因同源 (包括叶绿素a b结合蛋白基因、蛋白磷酸酶I催化亚基基因和 3个核糖体蛋白基因 ) ,还有 5个未知功能基因则是首次在盐藻中被分离 .值得注意的是 ,所有这 5个已知基因的功能都与细胞分裂或盐胁迫有关 .结果表明 :取样时盐藻细胞仍处于恢复阶段 ,所分离到的基因对于盐藻耐盐可能具有重要意义 ;蛋白磷酸酶I的下调表达可能是盐藻调节离子平衡的一个重要过程和细胞分裂受阻的原因所在 ;盐藻减缓细胞分裂速度可能是为了减少能量消耗 ,以留出足够的能量来应对盐胁迫 ;其它 5个未知基因可能也与盐藻适应盐胁迫机制有关 .     

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'

BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. CONCLUSIONS: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization.

The identification of differential gene expressionbetween cells is a frequent goal in modern biological research. Here we demonstrate the coupling of representational difference analysis (RDA) of cDNA with microarray analysis of the output for high throughput screening. Two primary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was shotgun cloned into a plasmid vector. Inserts from individual colonies from the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hybridized with differentially fluorescently labeled starting amplicons from the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlate well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) were appropriately detected as being >2-fold differentially expressed. Fifty unique, differentially expressed clones were identified. Therefore, the use of RDA essentially provides an enriched library of differentially expressed genes, while analysis of this library with microarrays allows rapid and reproducible screening of thousands of DNA molecules simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.     

A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.     

Monitoring of the subtraction process in solid-phase representational difference analysis: characterization of a candidate drug.

In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.     

Monitoring of representational difference analysis subtraction procedures by global microarrays

Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.     

cDNA representational difference analysis of ileal Peyer's patches in lambs after oral inoculation with scrapie

cDNA representational difference analysis (RDA) was used to study gene expression profiles in the ileal Peyer's patch of a lamb 1 week after oral inoculation with the scrapie agent. Twenty-five differentially expressed cDNA fragments were identified and cloned. Sequence analysis indicated seven novel gene sequences. Other clones shared sequence homology with genes encoding ribosomal and mitochondrial proteins, the translation initiation factor EIF4GII and the bovine pancreatic thread protein. Reverse Northern was used to confirm the differential expression in another four lambs inoculated with scrapie and the tissue distribution of the novel genes was examined using Northern blot analysis.     

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

利用cDNA-RDA技术研究BXSB狼疮小鼠差异表达的基因

利用近年来发展起来的代表性差异分析cDNA-RDA(cDNA-representational difference analysis)技术筛选BXSB红斑狼疮小鼠发病相关基因.发现了3个新的表达序列标签(EST)片段,在GenBank中的登录号分别为AF060113, AF060111, AF060110,同时发现了一些已知与自身免疫病相关的基因如逆转录病毒衣壳蛋白、Line-1逆转录酶等.通过RDA技术可能发现系统性红斑狼疮发病相关新基因,为自身免疫病的理论研究提供新的思路和方法.     

开放的差异基因表达技术研究进展

自 90年代早期发展以来 ,差异基因表达 (DGE)技术在许多领域得到了应用 .“开放”结构系统的DGE技术不需原始的生物学或序列信息 ,而且可应用于任何种群 .主要介绍 6项开放的DGE技术 :cDNA代表性差示分析 (cDNA RDA)、基因表达系统分析 (SAGE)、表达序列标签串联排列连接(TALEST) ,和早期的DGE技术差异显示 (DD)、随机引物聚合酶链反应 (AP PCR) ,以及一项受专利保护的技术———GeneCalling .通过几项重要的参数对这些技术进行了比较 ,认为DD虽然有其致命的弱点 ,但在目前仍然应用得非常广泛 .cDNA RDA能有效富增特异片段 ,扣除共有序列 ,如能和SAGE结合 ,将能进一步促进其发展 .TALEST和GeneCalling操作较简便 ,一次试验能获得大量的数据 ,但是分析这些数据比较麻烦 ,须借助另外的分析软件 .最后介绍了应用DGE技术取得的最新成果 .     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.