Slab Gel Research Articles

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

Capillary electrophoretic restriction fragment length polymorphism patterns for the Mycobacterial hsp65 gene.

PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of Mycobacterium species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (hsp65) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12 Mycobacterium species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify Mycobacterium species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of Mycobacterium species that is feasible for automation and routine use without the need for costly probes.

Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing.

A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment.

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver ( Sphyrna zygaena)

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400±20 Fe 3+/SZLF to 1890±20 Fe 3+/SZLF within the protein shell. Nature SZLF (SZLF N) consisting of holoSZLF and SZLF with unsaturated iron (SZLF UI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLF E) not only had 1890±20 Fe 3+/SZLF E but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of ∼21 kDa for SZLF E subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge ( m/ z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLF E were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M +) and its polymers, namely, [M] 2+, [M] +, [2M] +, and [3M] +, respectively. Both SZLF E and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe 3+ within the SZLF E core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.

Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational polymorphism analysis

As genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5-9 have been developed as reference materials for the detection of TP53 mutations. The goal of this study was to validate the reference materials in providing quality assurance for the detection of TP53 mutations in clinical specimens. We studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5-9 for mutation detection. Of the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned. Mutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST) TP53 reference panel in TP53 mutation detection in clinical tissue specimens.

Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals.

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (</= 5 degrees C), but become a gel-like micellar liquid crystal upon warming. Nucleic acids are well separated by electrophoresis in these gels as previously shown by Rill and Liu. Good separations of myoglobin tryptic peptides were accomplished by electrophoresis on slab gels of 24% Pluronic F127 or 15% polyacrylamide using the alkaline Laemmli buffer system (without sodium dodecyl sulfate). Labeling of peptides with the succinimidyl ester of Cascade Yellow (CY) prior to electrophoresis allowed sensitive detection with blacklight illumination at 365 nm. Labeled tryptic peptides were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. An inverse dependence of electrophoretic mobility on mass of peptides with charge Z = -1 was observed in both media. Two-dimensional (2-D) electrophoresis of myoglobin peptides on polyacrylamide, then on Pluronic media, at pH 8.3 indicated that the primary separation mechanism of most peptides was the same in both media. A few off-diagonal spots indicated that some peptides were preferentially retarded in Pluronic gels, perhaps due to hydrophobic effects. The ease of gel preparation and peptide recovery are advantages of Pluronic F127 gels for oligopeptide electrophoresis. The two media can be combined conveniently for 2-D electrophoresis, providing means to facilitate protein identification and peptidomics.

Formation of Molecular Complexes by N-Methyl-d-aspartate Receptor Subunit NR2B and Ryanodine Receptor 2 in Neonatal Rat Myocard

The N-methyl-d-aspartate (NMDA) receptor is a glutamate gated cation channel prevalent in the postsynaptic membranes of central nervous system neurons. The neurotransmitter receptor complex is thought to represent a tetramer where variable NR2 or NR3 polypeptides form heteromeric assemblies with an obligatory NR1 subunit. Recently, we showed that cardiac myocytes from perinatal rats transiently express the NMDA receptor subunit NR2B, the function of which in heart is unknown. To characterize the cardiac NR2B protein, we determined its subcellular distribution and specific molecular interaction partners. By immunostaining of rat heart tissue slices and acutely dissociated cardiac myocytes, the NR2B antigen was localized at the sarcomeric Z-bands. Using immunoprecipitation of detergent-solubilized NR2B protein and subsequent analysis employing matrix-assisted laser desorption/ionization time of flight mass spectrometry, ryanodine receptor 2 was identified as a molecular interaction partner of the cardiac NR2B polypeptide. Differences in antibody recognition indicate that the cardiac NR2B polypeptide carries a structurally altered C terminus as compared with the NR2B variant prevalent in central nervous system. Based on its localization and protein interaction, the function of cardiac NR2B protein may relate to mechanosensitivity or play a role in the regulation of the contractile apparatus of neonatal heart.

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

Quantitation of transgenic Bt event-176 maize using double quantitative competitive polymerase chain reaction and capillary gel electrophoresis laser-induced fluorescence.

In this work, a new procedure useful to quantitatively analyze genetically modified organisms (GMOs) in foods is described and applied to analyze transgenic Bt Event-176 maize. The method developed consists of coamplifications of specific DNA maize sequences with internal standards using quantitative competitive PCR (QC-PCR). The QC-PCR products are quantitatively analyzed using a capillary gel electrophoresis (CGE) with laser-induced fluorescence detection (LIF) method developed at our laboratory that utilizes a physically adsorbed coating. The CGE-LIF procedure allows the use of internal standards differing by only 10 bp from the original target fragments, to our knowledge, the smallest size difference that can be found in the bibliography for QC-PCR of GMOs. A spectrofluorometric procedure using ROX reference dye is proposed to solve calibration problems of input DNA concentration. It is demonstrated that the use of ROX drastically enhances the accuracy of the quantitative analysis by QC-PCR. Reproducibility of analysis times and corrected peak areas (measured as target/competitor PCR products ratio) for the CGE-LIF separations are determined to be better than 0.91 and 1.93% (RSD, n = 15) respectively, for three different days. It is shown that CGE-LIF provides better resolution and a signal/noise ratio improvement of approximately 700-fold compared to slab gel electrophoresis. The good possibilities in terms of quantitative analysis of GMOs provided by this new method are confirmed by determining the Bt Event-176 maize content in certified reference maize powder and food samples of known composition. This procedure opens the possibility for accurate quantitation of multiple GMOs in a single run.

Capillary electrophoresis to characterize ricin and its subunits with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been employed as highly efficient methods to characterize ricin, its subunits, and the chemically deglycosylated forms. As a CE method, sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) was used because of its merit over the conventional slab gel techniques. SDS–CGE showed higher resolution capability over other analytical tools in the analysis of the ricin mixture as well as in each of its purified forms. The high resolution was considered to be a result of the presence of carbohydrates on ricin subunits, and this property was useful for identifying the native ricin or its A chain from their chemically deglycosylated forms. However, this method exhibited an overestimation of the molecular mass due to the carbohydrate moieties on ricin subunits, and the inaccuracies were observed to be dependent on the carbohydrate content of the subunits. The exact molecular masses were measured by MALDI-TOF MS, and the results were almost consistent with the expected values. This study clearly illustrates the usefulness and necessity of complementary use of two powerful analytical techniques to characterize ricin and its subunits in a various research fields such as poisoning and immunotoxin research.

One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection.

Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area. A detection limit of 0.1 microg of total protein was achieved. The operation should be amenable to total automation.

Concentration and separation of low-abundant proteins in human plasma by ammonium sulfate fractionation followed by a three-step electrophoresis technique

In this paper, we describe a technique to concentrate and separate low-abundant proteins in human plasma. A human plasma sample was fractionated by ammonium sulfate and the precipitate obtained at 35% saturated ammonium sulfate was dialyzed and subjected to isoelectric focusing employing an agarose slab gel (55mm×73mm×3mm, 1% agarose). The IEF gel was cut into strips and each strip was transferred on the top of an SDS-containing column gel (5% T, 5% C polyacrylamide gel) prepared in a Pasture pipette. After SDS electrophoresis, each pipette column gel was transferred onto a top of a micro slab gel (38mm×38mm×1mm, 8-17% T, 5% C polyacrylamide gradient gel) and second SDS gel electrophoresis was run. If the step of pipette electrophoresis was deleted, the proteins appeared as horizontal faint bands on the slab gel. By using the three-step electrophoresis, the low-abundant proteins were detected as densely stained spots on the slab gel with 10-25 fold higher concentration of corresponding proteins than in the case of two-step electrophoresis. The spots on the slab gels after the three-step electrophoresis have been subjected to protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The results showed the advantages of the present technique for the structural analysis of low-abundant proteins in human plasma.

The application of LTR retrotransposons as molecular markers in plants.

Retrotransposons are major, dispersed components of most eukaryotic genomes. They replicate by a cycle of transcription, reverse transcription, and integration of new copies, without excising from the genome in the process. Because they represent a major share of the genome, cause easily detectable genetic changes having known ancestral and derived states, and contain conserved regions for which polymerase chain reaction (PCR) primers may be designed, retrotransposon insertions can be exploited as powerful molecular marker systems. Here, we describe the background and strategies, as well as give detailed laboratory protocols, for four key retrotransposon-based methods: SSAP, IRAP, REMAP, and RBIP. The SSAP, IRAP, and REMAP methods are multiplex and generate anonymous marker bands; RBIP scores individual loci, much as microsatellite-based marker systems do. The methods are variously suited to marker detection on agarose and polyacrylamide slab gels, slab and capillary sequencing devices, and arrays on solid supports. The different strengths and weaknesses of these approaches and their performance relative to conventional marker methods are discussed, together with their applicability to marker-assisted breeding, phylogenetic analyses, biodiversity determinations, and evolutionary studies.

Molecular and Biochemical Methods Used for the Identification of Globodera Species in Slovenia (from 6&lt;sup&gt;th&lt;/sup&gt; Conference of European Foundation for Plant Pathology, Prague, Czech Republic, 8-14 September 2002)

Globodera&amp;nbsp;species identification is based on specific morphological characteristics which are included in the majority of keys used for&amp;nbsp;Globodera&amp;nbsp;identification. To confirm and make more precise the morphometrical method, other methods have been developed. At Agricultural Institute of Slovenia, molecular and biochemical methods are applied. With the use of PCR-RFLP method we try to distinguish between&amp;nbsp;G. rostochiensis,&amp;nbsp;G. pallida&amp;nbsp;and&amp;nbsp;G. tabacum. Patterns of nematode DNA digested with restriction endonucleases and subjected to agarose gel electrophoresis are analysed. Differences in DNA sequence result in the number and size of fragments produced (RFLPs). For the differentiation of all three nematodes we have introduced a method for protein electrophoresis. Samples are compared after being separated by polyacrilamide slab gel electrophoresis. Proteins are visualised after silver staining. &amp;nbsp; &amp;nbsp; &amp;nbsp; &amp;nbsp;

Changes in physicochemical properties and microstructure of dried squid during softening treatment

AbstractDried squid were prepared at 4 or 40 °C and softened first in water and then in alkaline solution. The physicochemical and structural changes in the dried squid during the softening treatment were examined. A significantly higher wet weight was observed for the 4 °C‐dried squid during the softening treatment compared with the 40 °C‐dried squid. The rupture stress and rupture energy of the 40 °C‐dried squid were significantly higher than those of the 4 °C‐dried squid during the softening treatment. The sodium dodecyl sulphate polyacrylamide slab gel electrophoresis (SDS‐PAGE) pattern of the 4 °C‐dried squid was almost the same as that of raw squid. The SDS‐PAGE pattern of the 40 °C‐dried squid showed many fragments of lower molecular weight. After soaking in distilled water the SDS‐PAGE pattern of the 40 °C‐dried squid did not change significantly; however, the SDS‐PAGE pattern of the 4 °C‐dried squid became the same as that of the 40 °C‐dried squid. Histological analysis by light microscopy showed the formation of muscle fibre bundles in the 40 °C‐dried squid. A higher water permeation was observed among the muscle fibres of the alkali‐softened 4 °C‐dried squid when compared with the alkali‐softened 40 °C‐dried squid. Copyright © 2003 Society of Chemical Industry

Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection

DNA and RNA amplification, through polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) are essential steps in most procedures of nucleic acid analysis. Microfabricated devices (chips) for continuous-flow PCR and/or RT-PCR provide very rapid thermal energy transfer and cycling compared to conventional PCR systems. However, time-consuming slab gel electrophoresis has been used for analysis of the amplified fragments obtained from the flow-through chips. In order to facilitate high throughput and automation, we have combined the advantages of continuous-flow DNA and RNA amplification chip with an in-house built laser-induced fluorescence (LIF) detection system that allows analysis of amplification products within seconds. Upon exiting from the chip, the products are mixed with the intercalating dye SYBR Green I (SG1) and introduced into the LIF system. The fluorescence is linearly related to the DNA concentration in the range of 0.025–30 mg/l. The CVs of the LIF measurements range from 0.9 to 12.7%. We studied the effect of the number of amplification cycles on the fluorescence. The fluorescence was also studied as a function of the input DNA molecules in the range of 1.56×10 5–4×10 7. The reproducibility of the entire process including continuous-flow amplification on the chip and LIF detection was 18%. No sample carry-over was observed in the chip or the LIF detector.

Penicillin G hydrolysis in an electro-membrane reactor with immobilized penicillin G acylase

Penicillin G hydrolysis in a compartmentalised electro-membrane reactor with penicillin G acylase immobilised in a polyacrylamide gel slab was studied. Penicillin G solution was fed to the substrate compartment stacked to one gel slab surface and reaction products were collected in the product compartment adjacent to the other slab surface. An electric field perpendicular to the slab surface was imposed to the reactor by a couple of platinised titanium electrodes. The enzyme activity of the gel slab was varied from 30 to 150 U cm −3 and density of the electric current passing through the slab (5% polyacrylamide, dimensions 40 mm×40 mm×2.7 mm) was varied from −1250 to +625 A m −2. The current was considered as negative when electrophoretic migration augmented penicillin G transport from the substrate compartment to the product one. The reactor was operated in recirculation (batch) mode. The initial penicillin G concentration was 2% (w/v). Substantial increase of the rate of the penicillin G hydrolysis and of the volumetric slab productivity was observed both at negative and positive electric currents applied to the reactor. Fast replenishment of the consumed substrate was the reason of intra-slab reaction acceleration under the negative currents. The positive currents resulted to a close-to-optimum intra-slab pH value and consequent increase of the reaction rate. The positive currents, however, yielded only very poor separation of the reaction products from the unreacted substrate. Good separation was achieved when negative current of the density of −1250 A m −2 was applied. The volumetric productivity of the slab at this current density was about 240 mol of the penicillin G hydrolysed per litre of the gel per one hour of reaction time. Temperature rise in the reactor due to the Joule heating was acceptable at all current densities.

Shrinking Kinetics of Polyacrylate Gels in Surfactant Solution

Volume transition of covalently cross-linked sodium polyacrylate gels (micrometer-sized) due to the absorption of dodecyltrimethylammonium bromide from bulk aqueous solution is studied by means of light and fluorescence microscopy. The volume transition occurs in a narrow range of surfactant concentrations. In the presence of 0.01 M sodium bromide in the solution, the equilibrium volume is reduced typically 50 times for surfactant concentrations larger that 4 × 10-4 M. During the transition, a collapsed, surfactant-rich, surface phase is formed, enclosing the swollen gel core. Evidence is found for equilibrium between the core and surface phase in the collapsed state of the gel. The relation to surfactant self-assembly in solutions of linear polyion and in centimeter-sized gels is discussed. The kinetics of diffusion-controlled shrinking is analyzed theoretically. The model considers the transport of surfactant from the bulk, through the “stagnant” liquid layer and the surface phase, to the gel core. During shrinking, the osmotic swelling of the gel is assumed to be in quasi-equilibrium with the bulk and calculated using a recent model for equilibrium swelling of phase-separated gels (Hansson et al. J. Phys. Chem. B 2002, 106, 9777). The kinetic model is used to analyze time-resolved experimental shrinking curves. The result suggests that the shrinking is controlled by stagnant layer diffusion of surfactant, and that the relative swelling at intermediate stages during the collapse is the same as for slab gels of the same composition. The lag time measured before shrinking starts is longer than the time expected for the surfactant concentration to exceed the critical aggregation concentration in the gels, suggesting that the gels are arrested for some time in a metastable state before the surface phase starts to form.

First Report of Phytophthora palmivora on Grevillea spp. in Italy.

The genus Grevillea (family Proteaceae) comprises over 300 species and is a popular and widely cultivated group of Australian plants. In the last 3 years, numerous potted grevilleas with symptoms of decline associated with a rot of feeder roots were found in ornamental nurseries in Sicily. Aboveground symptoms were reduced growth, yellowing of foliage, wilt, dieback, and death of the entire plant. The disease was observed on many commercial cultivars and was especially severe on G. alpina (mountain grevillea), G. juniperina (juniper-leaf grevillea), G. lavandulacea (lavender grevillea), and G. rosmarinifolia (rosemary grevillea) as well as the hybrid cultivars Clearview David (G. lavandulacea × rosmarinifolia) and Poorinda Rondeau (G. baueri × lavandulacea), while G. lanigera (woolly grevillea) cv. Mount Tamboritha and G. thelemanniana subsp. obtusifolia appeared resistant. A species of Phytophthora was consistently isolated from rotted roots of symptomatic plants using a selective medium (4), and pure cultures were obtained by single-hypha transfers. The species was identified as P. palmivora (E.I. Butler) E.I. Butler on the basis of morphological and cultural characters. On solid media, all isolates produced elliptical to ovoid, papillate sporangia with a mean length/width ratio of 1.8. Sporangia were caducous with a short pedicel (5 μm) and a conspicuous basal plug. All isolates were heterothallic (mating type A1) and produced oogonia and oospores only when paired with A2 mating type reference isolates of P. nicotianae and P. palmivora. Antheridia were amphyginous. Identification was confirmed by electrophoresis of mycelial proteins in polyacrylamide slab gels (1). The electrophoretic patterns of total soluble proteins and six isozymes (alkaline phosphatase, esterase, fumarase, NAD-glucose dehydrogenase, malate dehydrogenase, and superoxide dismutase) of isolates from grevillea were identical to those of a reference isolate of P. palmivora from Coronilla valentina subsp. glauca (2) but distinct from those of reference strains of eight other papillate species of Phytophthora included in Waterhouse's taxonomic group VI. Koch's postulates were fulfilled using 6-month-old rosemary grevillea plants that were transplanted into pots filled with soil that was artificially infested with chlamydospores (50 per gram of soil) produced in submerged cultures (3) by grevillea isolate IMI 390579. Plants were maintained in a glasshouse at 20 to 28°C and watered to field capacity once a week. One month after transplanting, infected plants showed decline symptoms similar to those of naturally infected plants. Control plants grown in pots containing noninfested soil remained healthy. P. palmivora was reisolated from roots of symptomatic plants. It appears that P. palmivora has become a widespread root pathogen in commercial ornamental nurseries in Italy (2).

Evaluation of denaturing conditions in analysis of DNA variants applied to multi‐capillary electrophoresis instruments

AbstractDenaturant slab gel techniques (DGGE and variants thereof) and denaturing CE (DCE) have been used for the analysis of mutations and single nucleotide polymorphisms by several research groups. Recently, DCE applied to commercially available capillary electrophoresis instruments has been demonstrated to be sensitive, specific, and robust for mutation and SNP analysis. However, instruments currently available on the market are not designed for DCE in their present forms. In both single and multi‐capillary instruments inaccurate temperature control units lead to irreproducible denaturing conditions, both between runs and from capillary to capillary within an electrophoresis run. We have previously suggested that cycling a temperature gradient several times around a theoretically defined optimum for a given DNA fragment results in more reproducible and controllable denaturing conditions compared to a constant denaturant or single sweep temperature gradient. In this paper, we demonstrate that with the temperature differences observed, the common technique of applying a temperature gradient is not sufficient for optimal separation conditions in all capillaries. Furthermore, we demonstrate that cycling of the temperature around a fragment's theoretically calculated melting temperature during electrophoresis leads to reproducible results in all capillaries. These results clearly show the potential of standard commercial capillary instruments in automated cycling temperature CE applications.