Showing papers on "Gel electrophoresis published in 2007"

Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.

Abstract: The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

Quantitative analysis of chromosome conformation capture assays (3C-qPCR)

Abstract: Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days.

Separation of nanoparticles by gel electrophoresis according to size and shape.

Abstract: We demonstrate the separation of gold and silver nanoparticles according to their size and shape by agarose gel electrophoresis after coating them with a charged polymer layer. The separation is monitored optically using the size- and shape-dependent plasmon resonance of noble metal particles and confirmed by transmission electron microscopy (TEM). Electrophoretic mobilities are quantitatively explained by a model based on the Henry formula, providing a theoretical framework for predicting gel mobilities of polymer coated nanoparticles.

Complete and Integrated Pyrene Degradation Pathway in Mycobacterium vanbaalenii PYR-1 Based on Systems Biology

Abstract: Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the -ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.

Proteomic analysis of membrane-associated proteins from rat liver autophagosomes.

Abstract: Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pI 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function.

Proteomic analysis of RBC membrane protein degradation during blood storage.

Abstract: Two-dimensional gel electrophoresis and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. New spots with lower molecular masses, ranging between 7 and 15 kDa were observed during the first 7 days storage, while over time, further fragments and high-molecular-mass aggregates appeared, seen as a smearing in the upper part of the gel. Some of the protein changes turned out to be shifts in isoelectric point, as a consequence of chemical oxidations. All these new spots were generated as a result of protein attack by reactive oxygen species (ROS). Protein identification revealed that most of the modified proteins are located in the cytoskeleton. During the first 7 days of storage, oxidative degradation was observed prevalently in band 4.2, to a minor extent in bands 4.1 and 3, and in spectrin. After 14 days, there were new fragments from β-actin, glyceraldehyde-3-phosphate dehy...

Pulsed-field gel electrophoresis.

Abstract: This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to ∼50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploits the fact that very large DNA molecules unravel and “snake” through a gel matrix, and such electrophoretic trajectories are perturbed in a size-dependent manner by carefully oriented electrical pulses. PFGE has enabled the rapid genomic analysis of microbes and mammalian cells, and motivated development of large-insert cloning systems such as bacterial and yeast artificial chromosomes. As such, this protocol includes descriptions of two types of PFGE instrumentation (not commercially available), along with detailed instructions for their operation. Additionally, this protocol provides basic instructions for the preparation of intact chromosomal DNA from several types of organisms. PFGE takes 2–3 days, excluding sample preparation.

Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines.

Abstract: Chemoresistance is a major therapeutic obstacle in cancer patients, and the mechanisms of drug resistance are not fully understood. In the present study, we established platinum-resistant human ovarian cancer cell lines and identified differentially expressed proteins related to platinum resistance. The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP) human ovarian cancer cell lines were isolated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In total, 57 differential protein spots were identified, and five proteins, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1 (GSTO1-1), and cytosolic NADP+-dependent isocitrate dehydrogenase (IDHc), were found to be co-instantaneous significance compared with their parental cells. The expression of the five proteins was validated by quantitative PCR and western blot, and the western blot results showed complete consistency with proteomic techniques. The five proteins are hopeful to become candidates for platinum resistance. These may be useful for further study of resistance mechanisms and screening of resistant biomarkers.

Proteomic Analysis of Wheat Flour Allergens

Abstract: Wheat can cause severe IgE-mediated systematic reactions, but knowledge on relevant wheat allergens at the molecular level is scanty. The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy to wheat using proteomic strategies, referred to as “allergenomics”. Whole flour proteins were separated by two-dimensional gel electrophoresis with isoelectric focusing and lithium dodecyl sulfate−polyacrylamide gel electrophoresis. Then, IgE-binding proteins were detected by immunoblotting with sera of patients with a food allergy to wheat. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. In this study, we identified four previously reported wheat allergens or their sequentially homologous proteins [serpin, α-amylase inhibitor, γ-gliadin, and low molecular weight (LMW) glutenin] by a database search. As a result of the...

Expression of glycolytic enzymes is increased in pancreatic cancerous tissues as evidenced by proteomic profiling by two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry.

Abstract: The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. We tried to identify proteins that might be available for early diagnosis and effective therapies by proteomic profiling of pancreatic cancer tissues. Pancreatic cancerous and paired non-cancerous tissues obtained from surgical resections or autopsies of 10 patients were analyzed by two-dimensional gel electrophoresis. The differential display showed 11 spots whose expression was increased in cancerous tissues compared with the paired non-cancerous tissues. The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system identified the spots as alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, transgelin, calmodulin, superoxide dismutase(Mn) mitochondrial precursor, glutathione S-transferase P, cyclophilin A, protein disulfide isomerase A3 precursor, and apolipoprotein A-I precursor. Two of the 11 spots were detected as GAPDH. We noticed that 4 of 11 spots were enzymes involved in glycolytic pathway. Increased glycolysis in cancer cells has been regarded as the effect of intratumoral hypoxia and is possibly associated with tumor invasion, metastasis or resistance to therapies. These glycolytic proteins and transgelin, were confirmed by Western blotting and immunohistochemistry.

Morphine inhibits erythrocyte carbonic anhydrase in vitro and in vivo.

Abstract: Morphine is implicated in diverse functions, from development to immune modulation in the central and pe- ripheral nervous systems. At the present time, morphine is one of the most effective antinociceptive agents used to manage pain. It has been used extensively in the clinical management of pain due to its potent analgesic effect. In this study, the in vitro and in vivo inhibitory effects of morphine on erythrocyte carbonic anhydrase (CA) were investigated. Human erythrocyte isoenzymes, HCA-I and HCA-II, were purified by Sepharose-4B affinity chro- matography column with a yield of 66.95 and 62.82%, a specific activity of 3892.3 and 11663.2 EU/mg proteins with 745.1 and 2232.6-fold purification of each isoenzyme, respectively. To determine enzyme purity, sodium do- decyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. In vitro inhibition of erythrocyte HCA-I and HCA-II by morphine using the CO2-hydratase enzyme gave IC50 values 4.50� 10 � 5 M (r 2 : 0.954) and 9.23� 10 � 5 M (r 2 : 0.996), respectively. CA activity was significantly attenuated in vivo in Spraque-Dawley rats for up to 3 h (p� 0.001) following intraperitoneal administration of morphine. In conclusion, morphine inhibited CA activity both in vitro and in vivo.

Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte

Abstract: An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.

Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.

Abstract: The present work was started to explore whether a correlation could be detected among proteomic expression, protamine content and DNA integrity in human sperm cells. Towards this goal, we extracted the proteins present in the sperm cells from 47 sperm samples from infertile patients and from ten semen donors, analysed each sample by 2-D gel electrophoresis, and quantified the expression of 101 spots identified by MALDI-TOF analysis. Additionally, the protamine content and DNA integrity were also determined. Several interesting proteins such as transcription factors, prohibitin, heat shock and proteasome proteins have been identified. We have found that the expression of an important number of proteins (58 different 2-D spots) is correlated in independent sperm samples at high statistical significance (p 0.5). Additionally, eight proteins have also been found to correlate with DNA integrity and seven with protamine content (p<0.05). To our knowledge, this is the first report describing the correlation between proteomics, DNA integrity and protamine content. It also sheds new light into the fundamental aspects of the human sperm and points to new potential proteins involved in male infertility.

Gel electrophoresis of gold-DNA nanoconjugates

Abstract: Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effective diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.

Two-dimensional electrophoresis of membrane proteins

Abstract: One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.

Free-flow electrophoresis for purification of plant mitochondria by surface charge.

Abstract: Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.

Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers.

Abstract: Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC−MS/MS), and MALDI-TOF−MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially ...

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays.

Abstract: Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD50 value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.

Comprehensive Proteomic Analysis of the Human Amniotic Fluid Proteome: Gestational Age-Dependent Changes

Abstract: Amniotic fluid (AF) is a significant contributor to fetal health and constitutes a potential rich source of biomarkers for diagnosis of maternal and fetal disorders. In this study, we performed a comprehensive survey of the proteins expressed in AF, combining gel and liquid-based fractionation approaches coupled with LC−MS/MS analysis. Two-dimensional Liquid Chromatography (2D-LC) analysis identified 118 nonredundant proteins with high confidence. One- and two-dimensional gel electrophoresis and in-gel digestion identified 101 proteins. Combining both sets resulted in 219 proteins, of which 96 are unique to AF; 70, 18, and 35 proteins are present in serum, cervico-vaginal fluid, and all three fluids, respectively. Fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE) comparison of first-, second-, and third-trimester AF samples revealed that maximal differences in the relative abundance of AF proteins occur between the first and second trimesters. A systematic analysis of proteins pre...

Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum

Abstract: Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.

The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida). Involvement of acidic polysaccharides from glycoproteins in nacre mineralization.

Abstract: Among molluscs, the shell biomineralization process is controlled by a set of extracellular macromolecular components secreted by the calcifying mantle. In spite of several studies, these components are mainly known in bivalves from only few members of pteriomorph groups. In the present case, we investigated the biochemical properties of the aragonitic shell of the freshwater bivalve Unio pictorum (Paleoheterodonta, Unionoida). Analysis of the amino acid composition reveals a high amount of glycine, aspartate and alanine in the acid-soluble extract, whereas the acid-insoluble one is rich in alanine and glycine. Monosaccharidic analysis indicates that the insoluble matrix comprises a high amount of glucosamine. Furthermore, a high ratio of the carbohydrates of the soluble matrix is sulfated. Electrophoretic analysis of the acid-soluble matrix revealed discrete bands. Stains-All, Alcian Blue, periodic acid/Schiff and autoradiography with (45)Ca after electrophoretic separation revealed three major polyanionic calcium-binding glycoproteins, which exhibit an apparent molecular mass of 95, 50 and 29 kDa, respectively. Two-dimensional gel electrophoresis shows that these bands, provisionally named P95, P50 and P29, are composed of numerous isoforms, the majority of which have acidic isoelectric points. Chemical deglycosylation of the matrix with trifluoromethanesulfonic acid induces a drastic shift of both the apparent molecular mass and the isoelectric point of these matrix components. This treatment induces also a modification of the shape of CaCO(3) crystals grown in vitro and a loss of the calcium-binding ability of two of the main matrix proteins (P95 and P50). Our findings strongly suggest that post-translational modifications display important functions in mollusc shell calcification.

Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels.

Abstract: SUMMARY Erwinia amylovora is a plant pathogenic enterobacterium that causes fire blight disease of apple, pear and other rosaceous plants. A type III (T3) secretion system, encoded by clustered, chromosomal hrp genes (hypersensitive response and pathogenicity), is essential for infection, but only a few proteins are known that are secreted through this pathway (the T3 ‘secretome’). We developed an efficient protocol for purification and concentration of extracellular proteins and used it to characterize the T3 secretome of E. amylovora Ea273 by comparing preparations from the wild-type strain with those from mutants defective in hrp secretion, regulation, or in genes encoding putative T3-secreted proteins. Proteins were resolved by gel electrophoresis and identified using mass spectrometry and a draft sequence of the E. amylovora genome. Twelve T3-secreted proteins were identified, including homologues of known effector and helper proteins, and HrpJ, a homologue of YopN of Yersinia pestis. Several previously uncharacterized T3-secreted proteins were designated as Eops for Erwinia outer proteins. Analysis of the secretome of a non-polar hrpJ mutant demonstrated that HrpJ is required for accumulation of wild-type levels of secreted harpins. HrpJ was found to be essential for pathogenesis, and to play a major role in elicitation of the hypersensitive reaction in tobacco.