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Fouling is a major challenge for low-pressure membrane drinking water treatment systems. Previous research has demonstrated that under the right conditions, biofiltration is an effective method to reduce fouling of low-pressure polymeric membranes. This study provides additional insight into the effect of biofiltration as a pretreatment for fouling reduction by using river water with different raw water quality characteristics than has been examined in previous studies. Two parallel pilot-scale dual media (sand/anthracite) biological filters were operated continuously over a period of 14 months. Liquid chromatography-organic carbon detection analysis confirmed that the parallel biofilters performed similarly with both averaging on 21% biopolymer removal. Raw and treated water biopolymer concentrations were correlated, with increased absolute removals occurring at higher raw water concentrations. Ultrafiltration (UF) membrane fouling experiments showed substantial improvement in performance following biofiltration pretreatment by reducing hydraulically irreversible and reversible fouling rates by 14-68% and 8-55%, respectively. The results also reaffirm the importance of biopolymers at concentrations as low as -0.1 mg/L on irreversible and reversible UF membrane fouling and a minimal impact of humic substances. [ABSTRACT FROM AUTHOR]

The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination. While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described. Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with beta-galactosidase. In electrophoretic mobility shift assays, recombinant Cdp1pDelta1-867 bound Pu-motif triplex DNAs with high affinity (K:(d) approximately 5 nM) and bound Py-motif triplex, duplex and single-stranded DNAs with far lower affinity (0.5-5.0 microM). Genetic analyses revealed that the CDP1 gene product was required for proper chromosome segregation. The possible involvement of triplex DNA in this process is discussed.

The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiae whole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K(d) of 61 pM) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Delta yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1, MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed.

The influence of pH, temperature and catechol concentration on germanium (Ge) accumulation byPseudomonas stutzeri AG259 was investigated. Increasing the incubation temperature or pH of the culture medium markedly enhanced Ge accumulation. High amounts of Ge were accumulated at pH 11 and at 50°C, conditions under whichP. stutzeri cells were non-viable. Ge accumulation was unaffected by treatment with toluene or 2,4-dinitrophenol. These results indicate that Ge was accumulated by an energy-independent process. Ge accumulation increased as the catechol concentration increased. The use of autoclaved catechol solutions consistently increased the amount of Ge accumulated at all concentrations of catechol tested. It is possible that Ge enters the bacterial cells as a Ge-catechol complex and this uptake is enhanced by autoclaved catechol.

The combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) was used to determine the preferred duplex DNA binding sites of the peptide N-methylpyrrolecarboxamide antibiotic distamycin A. After 12 rounds of REPSA, several sequences were identified that bound distamycin with an apparent affinity of 2−20 nM. Among these, the highest-affinity sites averaged 10 bp in length, suggesting that these sites may be occupied by multiple, cooperatively interacting distamycin molecules. Presently, REPSA is the only combinatorial approach that allows the identification of preferred DNA targets for small molecule ligands at physiologically relevant concentrations in solution. As such, it should prove useful in the design and screening of sequence-specific DNA-binding molecules.

An important control point for gene regulation is the frequency of initiations leading to different numbers of RNA polymerases simultaneously transcribing the same gene. To date, the only direct assay for multiple-round transcription by RNA polymerase II in vitro required G-free cassette-containing templates and GTP-free conditions and was thus restricted in application. Here we used instead templates containing a triplex-directed interstrand psoralen-DNA cross-link to block RNA polymerase II elongation at a specific location. Covalently cross-linked templates allowed simultaneous detection of both specific initiation and reinitiation with any combination of promoter and transcribed sequence. In reconstituted systems, identical stacking of RNA polymerases was observed when the first polymerase was halted by GTP deprivation at the end of a G-free cassette or by a covalent cross-link downstream of different transcribed sequences. In contrast to transcription of G-free cassettes, reinitiation was unaffected by the transcription factor SII on sequences containing all four nucleotides. In crude nuclear extracts, transcription of covalently cross-linked templates yielded a reinitiation pattern with a wider spacing than in more purified fractions, indicating that the elongation complexes from nuclear extract contained a different form of RNA polymerase II or a different complement of associated factors.

We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II) cleaves double helical DNA with low sequence specificity. Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs. We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

The human gene-specific upstream stimulatory transcription factor (USF) is required, both in vivo and in vitro, for maximal expression of the major late promoter (MLP) of adenovirus. We report here the complete purification and identification of USF from HeLa cell nuclei. The protein was followed throughout its purification using a quantitative filter binding assay. With a combination of classical purification techniques and fast-flow protein liquid chromatography, USF can be purified to homogeneity starting either with a standard HeLa cell nuclear extract or with a higher salt extract from (lysed) HeLa cell nuclei (nuclear pellet extract). Approximately 20,000-fold purification from the nuclear pellet extract and 80,000-fold from the nuclear extract are necessary to obtain homogeneous preparations of the transcription factor. A maximum of 20,000 molecules of USF appear to be present in HeLa cells. Two major forms of the USF protein can be distinguished both by their slightly different mobilities in sodium dodecyl sulfate gel electrophoresis (apparent molecular weights 44,000 and 43,000, respectively) and by different electrophoretic mobilities of the corresponding protein-DNA complexes. Both forms of USF are heat-stable and interact with the MLP as monomers. Antibodies elicited against purified HeLa USF interact with the transcription factor bound to the MLP upstream element.