Concentration and detection of Salmonella in mung bean sprout spent irrigation water by use of tangential flow filtration coupled with an amperometric flowthrough enzyme-linked immunosorbent assay.

The development of a culture-free method for Salmonella screening of spent irrigation water derived from sprouting mung bean beds is described. The system used tangential flow filtration (TFF) to nonspecifically concentrate cells from large (2- to 10-liter) sample volumes. The retentate (100 ml) from the TFF was then flowed over an anti-Salmonella antibody-modified cellulose acetate membrane. The captured Salmonella was detected by reacting with a secondary anti-Salmonella and goat anti-rabbit biotin labeled antibody, followed by avidin-tagged glucose oxidase. The hydrogen peroxide generated from the enzymic oxidation of glucose was amperometrically detected at an underlying platinum electrode. It was found that 10 liters of Salmonella suspensions of 2 log CFU/ml could be concentrated to 4 log CFU/ml with 60% recovery regardless of the flow rate (112 to 511 ml/min) or transmembrane pressure (0 to 20 lb/in2) applied. The solids content of spent irrigation water negatively affected the filtration rate of TFE. This was most evident in spent irrigation water collected in the initial 24 h of the sprouting period, where the solids content was high (4,170 mg/liter) compared with samples collected at 96 h (560 mg/ liter). Trials were performed using mung bean beds inoculated with different Salmonella levels (1.3 to 3.3 log CFU/g). By using the optimized TFF and flowthrough immunoassay it was possible to detect Salmonella in spent irrigation water at levels of 2.43 log CFU/ml within 4 h. The integrated concentration and detection system will provide a useful tool for sprout producers to perform in-house pathogen screening of spent irrigation water.

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Two dimensions and two States in DNA nanotechnology.

Abstract The construction of periodic matter and nanomechanical devices are central goals of DNA nanotechnology. The minimal requirements for components of designed crystals are [1] programmable interactions, [2] predictable local intermolecular structures and [3] rigidity. The sticky-ended association of DNA molecules fulfills the first two criteria, because it is specific and diverse, and it results in the formation of B-DNA. Stable branched DNA molecules permit the formation of networks, but individual single branches are too flexible. Antiparallel DNA double crossover (DX) molecules can provide the necessary rigidity, so we use these components to tile the plane. It is possible to include DNA hairpins that act as topographic labels for this 2-D crystalline array, because they protrude from its plane. By altering sticky ends, it is possible to change the topographic features formed by these hairpins, and to detect these changes by means of AFM. We can modify arrays by restricting hairpins or by adding them to sticking ends protruding from the array. Although individual branched junctions are unsuitable for use as crystalline components, parallelograms of four 4-arm junction molecules are sufficiently rigid that they can be used to produce 2D arrays. The arrays contain cavities whose dimensions are readily tuned by changing the edges of their parallelogram components. We have used these arrays to measure directly the angle between the helices of the Holliday junction. The rigidity of the DX motif can also be exploited to produce a nanomechanical device predicated on the B-Z transition. Two DNA double crossover molecules have been joined by a segment of DNAcapable of undergoing the B-Z transition. In the B-conformation, the unconnected helices of the two molecules are on the same side of the connecting helix, whereas in the Z conformation they are on opposite sides, leading to movements of as much as 60Å. This effect is shown by fluorescence resonance energy transfer, because dyes attached to the unconnected helices have different separations in the two states.

Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription.

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.

Abortive initiation of transcription at a hybrid promoter. An analysis of the sliding clamp activator of bacteriophage T4 late transcription, and a comparison of the sigma70 and T4 gp55 promoter recognition proteins.

Bacteriophage T4 late promoters are transcribed by an RNA polymerase holoenzyme comprising the Escherichia coli core, E, the phage gene 55-encoded promoter recognition subunit, gp55, and the gene 33-encoded co-activator, gp33. Transcriptional initiation is activated by the T4 gene 45-encoded sliding clamp, which is loaded on to DNA at enhancer-like sites by its clamp-loader. Correct initiation of transcription at late promoters in basal mode requires only RNA polymerase core and gp55 (E.gp55). Dinucleotide-primed abortive initiation of basal and activated T4 late transcription has been compared. Only the trinucleotide non-productive transcript is made at a high rate; all other short transcripts are made at rates of less than one molecule per productive transcript. Gp45 increases abortive trinucleotide synthesis along with productive transcription, although the proportion of productive transcripts is also elevated. Nevertheless, this increase accounts for only a small part of the activation of T4 late transcription that is generated by its activator and co-activator. The pattern of production of short transcripts differs subtly between basal and enhanced transcription, indicating that linking the RNA polymerase with its sliding clamp activator only generates minor changes in the transition from abortive to productive RNA chain elongation. The T4 late promoter is converted to a strong sigma70 promoter by inserting an appropriate -35 promoter element. A direct comparison at such a hybrid promoter shows sigma70 and gp55 generating qualitatively and quantitative different patterns of abortive initiation at the same start site.

A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence.

Bacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription. In the latter context, DNA tracking is an essential feature of the unique mechanism of action. T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small sigma-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core. A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways. (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking. (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNA-tracking state of gp45 is short-lived, but is stabilized by gp55. The ability of this topologically linked DNA-tracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.

Dynamics of DNA-tracking by two sliding-clamp proteins.

Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA. gp45 also activates T4 late transcription. DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer-template junctions is assisted by single-stranded DNA-binding proteins. The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites for proteins that block gp45 tracking. DNA loading of gp45 can also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S. At saturation, DNA is very closely packed with gp45 or beta. When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking state of beta is much more stable. The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol. We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.

Cleavage of double-crossover molecules by T4 endonuclease VII.

DNA double-crossover molecules containing two Holliday junctions have been prepared and treated with endonuclease VII, the resolvase from bacteriophage T4. One molecule contains antiparallel double-helical domains, and the other molecule contains parallel domains. The parallel double-crossover model system has been made tractable by closing the free ends of the molecule, to convert it to a catenane. The products resulting from the two substrates differ substantially. The molecule containing antiparallel helical domains is cleaved three nucleotides 3' to the crossover points, in a fashion similar to single Holliday junction analogs. The molecule containing parallel helical domains is cleaved, but the major points of scission are five nucleotides 5' to a branch point on the crossover strands and six nucleotides 3' to the same branch point on the non-crossover strands. The major sites of scission reflect features of molecular symmetry in each case, suggesting that the resolvase recognizes structural features. The cleavage results suggest that the antiparallel structure is the natural substrate, if the Holliday junction is unconstrained within the cell. It is straightforward to reconcile antiparallel Holliday junctions with the conventional parallel paradigm of recombination. Nevertheless, the cleavage of the parallel molecule shows that a parallel substrate could also be cleaved symmetrically by endonuclease VII (but with different products) if the molecule were constrained to assume that conformation within the cell.

Holliday junction crossover topology.

The Holliday junction is a key intermediate in genetic recombination. It consists of four strands of DNA that associate to form four double-helical arms. Studies over the past decade with asymmetric analogs of Holliday junctions have shown that the four arms stack to generate two stacking domains. This arrangement of arms results in two strands with a roughly helical structure, and two that contain a crossover structure connecting the domains. Both parallel and antiparallel orientations of the helical strands are possible, although the antiparallel orientation is favored. In principle, there are two possible isomers of the Holliday junction, depending on which pairs of strands contain the crossover structure; isomerization between these two structures is key to many molecular models of recombination. Isomerization of parallel domain structures entails large end-to-end helical rotations if braiding of the crossover strands is to be avoided. We have examined the ability of the crossover strands to braid. This has been done by employing a double crossover molecule, whose termini are all hairpin loops. Such a molecule is a catenane of two single strands of DNA, whose linking number is a function of the sign of the node at the crossover. We have prepared linking standards by means of topological protection techniques, and have compared them to the catenane formed by the double crossover molecule. We find no evidence for braiding of the strands. Furthermore, no braided structures can be detected when the double crossover molecule is treated with Escherichia coli DNA topoisomerase I in the presence of varying amounts of Mg2+ cations.

Symmetric immobile DNA branched junctions.

Branch migration is an isomerization of Holliday recombination intermediates that arises from their homologous (2-fold) sequence symmetry. This isomerization relocates the branch point in an apparently random fashion and thereby complicates the study of the physical and structural properties of these structures. For the past decade, these properties have been studied in low-symmetry immobile junctions, whose sequence asymmetry eliminates branch migration. The asymmetric findings of many of these studies suggest the need for a system combining both immobility and symmetry. Double-crossover DNA molecules have been used to create molecules with both these properties. Immobility is achieved by flanking one crossover with a symmetric junction and the other crossover with an asymmetric junction. Close torsional coupling between the two junctions renders the symmetric junction immobile. These molecules will enable the characterization of thermodynamic, structural, dynamic, liganding, and substrate properties of symmetric branched DNA molecules in a sequence-specific fashion.

DNA double-crossover molecules.

DNA molecules containing two crossover sites between helical domains have been suggested as intermediates in recombination processes involving double-strand breaks. We have modeled these double-crossover structures in an oligonucleotide system. Whereas the relative orientations of the helical domains must be specified in designing these molecules, there are two broad classes of the molecules, the parallel, DP, and antiparallel, DA, molecules. The distance between crossover points must be specified as multiples of half-turns, in order to avoid torsional stress in this system; hence, there are two further subdivisions, those double-crossover molecules separated by odd, O, and even, E, numbers of half-turns. In addition, the parallel molecules with odd numbers of half-turns between crossovers must be divided into those with an excess major or wide-groove separation, W, or those with an excess minor- or narrow-groove separation, N. We have constructed models of all five of these classes, DAE, DAO, DPE, DPOW, and DPON. DPE molecules containing 1 and 2 helical turns between crossovers have been constructed; the DAE molecule contains 1 turn between crossovers, and the DAO, DPOW, and DPON molecules contain 1.5 helical turns between crossovers. None of the parallel molecules is well-behaved; the molecules either dissociate or form multimers when visualized on native polyacrylamide gels. In contrast, antiparallel molecules form single bands when assayed in this fashion. Hydroxyl radical autofootprinting analysis of these molecules reveals protection at expected sites of crossover and of occlusion, suggesting that all the complexes contain linear helix axes that are roughly coplanar between crossovers. However, the DPOW molecule and the DPE molecule with 2 turns between crossovers show decreased protection in the portion between crossovers, suggesting that their helices may bow in response to charge repulsion. We conclude that the helices between parallel double crossovers must be shielded from each other or distorted from linearity if they are to participate in recombination. We have analyzed the possibilities of branch migration and crossover isomerization in double-crossover molecules. Parallel molecules need no sequence symmetry beyond homology to branch migrate, but the sequence symmetry requirements for antiparallel molecules restrict migration to directly repetitive segments that iterate the sequence between crossovers. Crossover isomerization appears to be a very complex process in parallel double-crossover molecules, suggesting that it may be catalyzed by topoisomerases if it occurs within the cell.