Identifying differences in mRNA expression by representational difference analysis of cDNA.

Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.     

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.     

用表达性差异显示分析技术分离人胎肝组织选择性表达基因

目的：建立4月龄人胎肝组织选择性表达基因EST库,为研究胚胎肝组织特异表达基因提供有力的工具。方法：采用表达性差异显示分析（representational difference analysis,RDA) 技术建立4月龄人胎肝组织选择性表达基因EST库,并测定部分克隆核苷酸序列,以竞争PCR检测代表性基因的差异表达。结果与结论：以珠蛋白家族基因为标志,所建差减cDNA文库中α－珠蛋白家族基因的表达频率较未差减cDNA文库增高7倍,同时该文库也含有多种与肝生长密切相关的基因,表明RDA技术确是研究差异表达基因的有效手段,所建EST库富集胎肝特异表达基因。部分克隆序列分析获得2种未报道序列,其中一株含有丝／苏氨酸激酶结构域,表明可望从此库中分离具有重要未知功能的新基因。     

Isolation of YAC insert sequences by representational difference analysis.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.     

Mapping of porcine genetic markers generated by representational difference analysis

Representational difference analysis (RDA) was performed using pig genomic DNA from a Landrace non-selected control population and a Landrace population selected for increased loin muscle area (LMA) for five generations. Pigs used for the analysis differed phenotypically for various carcass traits and were divergent in genotype at the skeletal muscle ryanodine receptor 1 locus. Two RDA experiments were performed using BamHI and BglII. Fourteen BamHI and 37 BglII difference products were cloned and sequenced. Oligonucleotide primers were designed to amplify RDA difference products and sequence-tagged sites (STS) were developed for 16 RDA fragments (two BamHI and 14 BglII). These 16 STS were mapped using the INRA-Minnesota porcine Radiation Hybrid panel. Polymorphisms identified in nine of the STS were used to place these markers on the PiGMaP genetic linkage map. Sequence-tagged sites were localized to 11 different chromosomes including three markers on chromosome 11 and four markers on chromosome 14. Development of RDA markers increases the resolution of the pig genome maps and markers located within putative quantitative trait locus (QTL) regions can be used to refine QTL positions.     

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.     

Targeting of marker loci to chicken chromosome 16 by representational difference analysis

Representational difference analysis (RDA) was initially used to identify differences between two inbred lines of chickens, line N and line 15I, on which the Compton mapping reference population is based. RDA was subsequently used to identify marker loci targeted specifically to chicken chromosome 16. Chromosome 16 contains the major histocompatibility complex (MHC), nucleolar organiser region (NOR) and Rfp-Y complex. To generate markers specific for this chromosome a bird was selected from the Compton mapping reference population which had inherited N line alleles for the MHC, NOR and Rfp-Y regions on this chromosome. DNA from this bird was compared with pooled DNA from 16 of its siblings, all of which had inherited line 15I alleles for the MHC, NOR and Rfp-Y regions. Initially amplicons were derived from Bam HI digested samples, RDA products were cloned after the first round of hybridisation and 113 clones were investigated: 45 of these identified Bam HI polymorphisms in this population. Of the 45 polymorphic clones, 17 have been mapped in the reference population so far, and these have identified seven new loci on chromosome 16. Interestingly a group of 16 other loci were linked on chromosome 4. The same birds were also compared by RDA following digestion with Taq I. Again large numbers of clones were generated of which 65 were investigated. Of these 17 clones were polymorphic and of five clones mapped so far three lie on chromosome 16. Two of the loci mapped to chromosome 16 have been used to identify yeast artificial chromosome (YAC) clones (GenBank accession numbers: AF057302, AF057303, AF057304, AF063218, AF06347, AF06348, AF06349, AF06350, AF063#51, AF06353, AF06354, AF06355, AF06356).     

Targeted mapping of rye chromatin in wheat by representational difference analysis.

A targeted mapping strategy using representational difference analysis (RDA) was employed to isolate new restriction fragment length polymorphism probes for the long arm of chromosome 6 in rye (6RL), which carries a gene for resistance to Hessian fly larvae. Fragments from the 6RL arm were specifically isolated using a 'Chinese Spring' (CS) wheat - rye ditelosomic addition line (CSDT6RL) as tester, and CS and (or) CS4R as the driver for the genomic subtraction. Three RDA experiments were performed using BamHI amplicons, two of which were successful in producing low-copy clones. All low-copy clones were confirmed to have originated from 6RL, indicating substantial enrichment for target sequences. Two mapping populations, both of which are derived from a cross between two similar wheat-rye translocation lines, were used to map five RDA probes as well as five wheat probes. One of the populations was prescreened for recombinants by C-banding analysis. Fifteen loci, including seven new RDA markers, were placed on a map of the distal half of 6RL. The Hessian fly resistance gene was localized by mapping and C-banding analysis to approximately the terminal 1% of the arm. The utility of RDA as a method of targeted mapping in cereals and prospects for map-based cloning of the resistance gene are discussed.     

cDNA representational difference analysis of the deltamelthrin-resistant and -susceptible populations in diamondback moth (Plutella xylostella L.)

Abstract:  The improved cDNA representational difference analysis was preliminarily used to analyse the genetic differences between the deltamethrin-susceptible and -resistant strain of the diamondback moth. The driver amplicon is the cDNA from the susceptible strain while the tester amplicon is from the resistant strain. After four rounds of subtractive hybridization, we obtained one different product, the size of which was about 200 bp. Comparisons between our sequencing results and data in GenBank show that there is one sequence which has a relatively high homology to the ubiquitin gene, but there are no reports indicating that its upregulated expression is correlated with insecticide resistance. Hence further studies are warranted.     

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis. To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B. hyodysenteriae strain B204. Both fragments were present in a single copy and mapped to different positions on the genome of B. hyodysenteriae B78(T). Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed. Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis.     

Antioxidative activity of flavonoids in stimulated human neutrophils

The release and production of oxidative products generated by the respiratory burst under the influence of natural flavonoids: quercetin, kaempferol and isorhamnetin derivatives have been studied in the polymorphonuclear neutrophils (PMNs) from healthy human donors. Flavonoids were tested in vitro at concentration range 1-100 microM. The antioxidative potential of flavonoids was compared to the activity of a food preservative, butylated hydroxyanisole. Two methods were applied to the measurement of the PMNs respiratory burst: flow cytometry using dichlorofluorescein diacetate and luminol-dependent chemiluminescence. It was found that the studied products decreased the neutrophil hydrogen peroxide production in concentration-dependent manner. The highest degree of inhibition was registered for concentration of 100 microM, although in the chemiluminescence method the metabolic activity inhibition was more prominent. Antioxidative activity of flavonoids depended on the number of hydroxyl groups. These results provide useful data for establishing methods used to assess the respiratory burst in phagocytic cells.     

Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation

Leukocyte chemoattractants were inactivated when exposed to human neutrophils and either ingestible particles or phorbol esters. Loss of biologic activity was time- and temperature-dependent, required physiologic concentrations of viable neutrophils and a halide, and was inhibited by azide or catalase. Neutrophils from patients with either hereditary myeloperoxidase deficiency or chronic granulomatous disease failed to inactivate the chemoattractants unless purified myeloperoxidase or H2O2, respectively, was added. Susceptibility to inactivation by neutrophils correlated with the presence of methionine in the attractant. Loss of chemotactic activity was blocked by low concentrations of methionine and by higher concentrations of other reducing agents, but was unaffected by oxidized methionine. Paper chromatography demonstrated that exposure of a formyl-methionyl peptide chemotactic factor to either the cellfree myeloperoxidase system or stimulated neutrophils resulted in its conversion to a molecular species whose location in the chromatographs was identical to that of the peptide containing oxidized methionine. Thus, stimulated human neutrophils inactivate peptide chemoattractants by secretion of myeloperoxidase and H2O2, which combine with halides to form oxidants that react with a critical methionine residue. We suggest that myeloperoxidase-catalyzed oxidation of thioethers may constitute an inflammatory control mechanism as well as a general means of modifying the functional properties of biologic mediators.