bluuebunny/crossref_metadata_2025_split huggingface dataset:
Separation of 4‐color DNA sequencing extension products in noncovalently coated capillaries using low viscosity polymer solutions
Authors: Mandabhushi Ramakrishna S. 
Published: May 2005

AbstractA low viscosity (ca. 75cp) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length‐of‐read (LOR) value of approximately 600 bases in about 2 h using four‐color sequencing in 50 μm capillary at 42°C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA‐capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high‐throughput instruments for DNA sequencing.
Capillary electrophoresis of peptides and proteins in fused‐silica capillaries coated with derivatized polystyrene nanoparticles
Authors: Kleindienst Gerald , Huber Christian G. , Gjerde Douglas T. , Yengoyan Leon , Bonn Günther K. 
Published: May 2005

AbstractHigh‐resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 μm ID fused‐silica capillaries with 40‐140 nm polystyrene particles which have been derivatized with α‐ω‐diamines such as ethylenediamine or 1, 10‐diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400 000 plates per meter. A substantial improvement of separation efficiency in particle‐coated capillaries was observed after in‐column derivatization of amino functionalities with 2,3‐epoxy‐1‐propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1 900 000 theoretical plates per meter. Finally, coated capillaries were applied to the high‐resolution analysis of protein glycoforms and bioactive peptides.
Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II)
Authors: Schmid Martin G. , Gübitz Gerald , Kilár Ferenc 
Published: May 2005

AbstractFurther studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron‐free human serum transferrin are described. The substances passed a highly concentrated pseudo‐stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.
Quantitative trace analysis of interleukin‐3, interleukin‐6, and basic model proteins using isotachophoresis‐capillary zone electrophoresis with hydrodynamic counterflow
Authors: Bergmann Jens , Jaehde Ulrich , Schunack Walter 
Published: May 2005

AbstractA quantitative analytical technique to determine trace concentrations of recombinant human interleukin‐3 (rhIL‐3), recombinant human IL‐6 (rhIL‐6), and various basic model proteins is described using isotachophoresis‐capillary zone electrophoresis (ITP‐CZE). Proteins were separated on coated fusedsilica capillaries using a commercial capillary electrophoretic system modified for the application of isotachophoretic preconcentration with hydrodynamic counterflow. The effect of injection time and isotachophoretic focusing time was investigated and compared with predictions from existing mathematical models. Good linearity of the calibration graphs (r > 0.995) was observed for all investigated proteins. The limit of quantification was in the 10−8 M range using UV detection at 200 nm. Within‐day and between‐day precison of peak area ranged between 1 and 6%. Precision was unaffected by isotachophoretic preconcentration. In conclusion, the described method is feasible to quantify trace concentrations of rhIL‐3, rhIL‐6, and basic proteins. Potential applications comprise issues of pharmaceutical quality control.
Capillary affinophoresis of pea lectin with polyliganded affinophores: A model study of divalent‐polyvalent interactions
Authors: Shimura Kiyohito , Kasai Ken‐Ichi 
Published: May 2005

AbstractAffinophoresis is a type of affinity electrophoresis using an affinophore, a soluble ionic carrier bearing affinity ligand(s). It was reported previously that an affinophore, prepared by coupling multiple p‐aminophenyl α‐D‐mannoside ligands to a part of the carboxyl groups of succinylated polylysine, specifically changed the mobility of pea lectin in agarose gel. The affinophoresis of this divalent lectin with the polyliganded affinophore was investigated by using capillary electrophoresis. Analysis of the mobility change of the lectin in the presence of differently modified affinophores showed that the affinity was larger for affinophores having higher ligand density. Analysis of the inhibition of the mobility change by a neutral ligand, with a known affinity constant for the lectin, allowed estimation of the contributions of monovalent and divalent interactions to the binding in the lectin‐affinophore complex. The proportion of divalent complexes was greater for affinophores having higher ligand density. This approach to estimate the contribution of divalency in complex formation should be generally applicable to the analysis of divalent interactions with different techniques other than electrophoresis.
A diagnostic test for scrapie‐infected sheep using a capillary electrophoresis immunoassay with fluorescent‐labeled peptides
Authors: Schmerr Mary Jo , Jenny Allen 
Published: May 2005

AbstractScrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod‐shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease‐resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and β‐mercaptoethanol, a monomer form of ∼27 kDa molecular mass is observed. A competition immunoassay to detect PrPSCfrom scrapie‐infected sheep was developed using free zone capillary electrophoresis with laser‐induced fluorescence (LIF) for detection and flourecein‐labeled sysnthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent‐labeled peptides bound to the antibody were separated from the unbound peptides using 200 mMTricine, pH 8.0, containing 0.1%n‐octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind ∼50% of the fluorescent‐labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, ∼2 fmoles of the peptide could be measured. When PrPSCextracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSCcould be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142–154 and 155–178, clearly differentiated scrapie‐positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopathies in animals and in humans.
Exploitation of a monoclonal antibody for weak affinity‐based separation in capillary gel electrophoresis
Authors: Ljungberg Helén , Ohlson Sten , Nilsson Staffan 
Published: May 2005

AbstractWeak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the α‐ and β‐forms of p‐nitro‐phenyl‐labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.
Genome encyclopedias and their use for comparative analysis of Rhodobacter capsulatus strains
Authors: Fonstein Michael , Nikolskaya Tatiana , Kogan Yakov , Haselkorn Robert 
Published: May 2005

AbstractThis paper consists of two components: the use of gene encyclopedias in genomic studies and Rhodobacter capsulatus genome project. A survey of vectors used for encyclopedia construction includes a brief discussion of their relative advantages and limitations. Projects employing various methods of encyclopedia assembly including the comparison of restriction patterns, restriction maps, linking by hybridization, oligonucleotide fingerprinting, sequence tagged site (STS) fingerprinting and encyclopedias derived from genetic maps are listed and briefly described. The R. capsulatus SB 1003 genome project started with the construction of its cosmid encyclopedia, which comprises 192 cosmids covering the chromosome and the 134 kbp plasmid in strain SB 1003, with the exact map coordinates of each cosmid. In a pilot sequencing study, several cosmids were individually subcloned using the vector M13mp18 and merged into one 189 kbp contig. About 160 open reading frames (ORFs) identified by the CodonUse program were subjected to similarity searches. The biological functions of eighty ORFs could be assigned reliably using the WIT (what is there) genome investigation environment. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Recently, another 1.2 Mbp genome fragment of the Rhodobacter genome was sequenced using a slightly modified approach. These results together with some genome investigation tools, have been placed at our web site (http://capsulapedia.uchicago.edu). The sequence of R. capsulatus is expected to be completed by summer 1998. A project to construct a systematic set of deletion strains of R. capsulatus in order to assign functions to unknown ORFs has been started. Preliminary data demonstrate the extreme convenience of the unique gene transfer agent (GTA) system to perform such work.
Selective generation of chromosomal cosmid libraries within the Trypanosoma cruzi genome project
Authors: Frohme Marcus , Hanke Jens , Åslund Lena , Pettersson Ulf , Hoheisel Jörg D. 
Published: May 2005

AbstractFrom a total genomic cosmid library of the pathogen Trypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed‐field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross‐hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III‐ and IV‐specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross‐hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being diploid pairs.
Hybridization mapping ofTrypanosoma cruzichromosomes III and IV
Authors: Hanke Jens , Frohme Marcus , Laurent Jean‐Pierre , Swindle John , Hoheisel Jörg D. 
Published: May 2005

AbstractAs part of theTrypanosomaGenome Initiative launched by the World Health Organization (WHO), a physical clone map ofTrypanosoma cruzichromosomes III and IV was generated to facilitate both DNA sequence analysis of the parasite's genome and the investigation of chromosome organization. Apart from a few genetic markers, anonymous cosmids were taken from chromosomal sublibraries and individually hybridized to filter arrays of the relevant cosmid library. The probe order was determined from the hybridization fingerprint results and used to define a fitting clone order, with few gaps remaining. The results were independently verified by hybridizations to a bacterial artificial chromosome (BAC) library and, in case of chromosome III, restriction mapping. For gap closure, additional experiments on a total cosmid library were carried out. The possible tiling paths consist of 26 clones for chromosome III (610 kbp) and 28 clones for chromosome IV (680 kbp).
Optimization and automation of fluorescence‐based DNA hybridization for high‐throughput clone mapping
Authors: Scholler Patrik , Heber Steffen , Hoheisel Jörg D. 
Published: May 2005

AbstractLarge‐scale hybridization‐based genome mapping projects, such as the production of sequence‐ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high‐throughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin‐modified at their 5′ ends. Alternatively, digoxigenin‐labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti‐digoxigenin antibody upon the addition of AttoPhosTM substrate. Signals are directly detected with a charge‐coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.
Global analysis of genomic texts: The distribution of AGCT tetranucleotides in the Escherichia coli and Bacillus subtilis genomes predicts translational frameshifting and ribosomal hopping in several genes
Authors: Hénaut Alain , Lisacek Frédérique , Nitschké Patrick , Moszer Ivan , Danchin Antoine 
Published: May 2005

AbstractPresent availability of the genomic text of bacteria allows assignment of biological known functions to many genes (typically, half of the genome's gene content). It is now time to try and predict new unexpected functions, using inductive procedures that allow correlating the content of the genomic text to possible biological functions. We show here that analysis of the genomes of Escherichia coli and Bacillus subtilis for the distribution of AGCT motifs predicts that genes exist for which the mRNA molecule can be translated as several different proteins synthesized after ribosomal frameshifting or hopping. Among these genes we found that several coded for the same function in E. coli and B. subtilis. We analyzed in depth the situation of the infB gene (experimentally known to specify synthesis of several proteins differing in their translation starts), the aceF/pdhC gene, the eno gene, and the rplI gene. In addition, genes specific to E. coli were also studied: ompA, ompF and tolA (predicting epigenetic variation that could help escape infection by phages or colicins).
Structural and functional implications of sequence diversity of Pseudomonas aeruginosa genes oriC, ampC and fliC
Authors: Spangenberg Claudia , Montie Thomas C. , Tümmler Burkhard 
Published: May 2005

AbstractSequence analysis of three representative gene loci, oriC, ampC and fliC, in 19 Pseudomonas aeruginosa strains revealed a low sequence diversity that does not correlate with the extensive diversity of P. aeruginosa habitats. Single point mutations lead to a mean sequence diversity of 0.40%, 0.38% and 0.59% for oriC, ampC and a‐type fliC, respectively, but of only 0.05% for b‐type flagellin genes. The analyzed genes encode highly conserved functions that are subject to strong selective pressure. The detected nucleotide substitutions of oriC, accumulating in a central 95 bp region, affect neither the putative DnaA binding sites nor the 13 bp direct repeats that presumably provide the sites to openoriC duplex DNA. Even in P. aeruginosa strain DSM 1128, which exhibits an unusually high sequence variability in several analyzed genes, the 9 bp and 13 bp motifs are conserved, reflecting their essential functional role in replication initiation. The two flagellin types, differing by 37–38% in their primary structure, exhibit pronounced structural and functional homology, as shown by alignment of flagellin variants by hydrophobicity index, probability of surface exposure, chain flexibility and antigenicity, and by cross‐reactivity between both proteins using specific antisera. Five nonsynonymous nucleotide substitutions of ampC lead to β‐lactamase variants that differ in recognition and turnover of substrate, as deduced from the three‐dimensional structure of the highly homologous Enterobacter cloacae β‐lactamase and confirmed by inhibition kinetics. The identified point mutations in the three genes are classified as selectively equivalent sequence variants indicating neutral genetic drift as a mechanism of molecular evolution in P. aeruginosa, rather than positive selection.
Chromosomal rearrangements in enteric bacteria (minireview)
Authors: Sanderson Kenneth E. , Liu Shu‐Lin 
Published: May 2005

AbstractEarly genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I‐CeuI, which digests the DNA of bacteria in the rrn operon for rRNA (ribosomal RNA), thus establishing the “rrn genomic skeleton” (the size in kbp of the intervals between rRNA operons); (ii) analysis of XbaI and B1nI sites within Tn10 insertions in the chromosome. The order of I‐CeuI fragments, which is ABCDEFG in S. typhimurium LT2 and E. coli K‐12, was found to be conserved in most Salmonella species, most of which grow in many hosts (host‐generalists). However, in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum, species which are host‐specialized, these fragments are rearranged, due to homologous recombination between the rrn operons, resulting in translocations and inversions. Inversions and translocations not involving the rrn operons are seldom detected except for inversions over the TER (termination of replication) region. Additive genetic changes (due to lateral transfer resulting in insertion of nonhomologous DNA) have resulted in “loops” containing blocks of DNA which provide new genes to specific strains, thus driving rapid evolution of new traits.
Leptospira genomics
Authors: Boursaux‐Eude Caroline , Girons Isabelle Saint , Zuerner Richard 
Published: May 2005

AbstractThe bacterial species Leptospira interrogans (sensu stricto) has a complex genome containing two circular chromosomal replicons. Comparative analysis of the larger chromosome reveals a fluid genetic organization with many large rearrangements differentiating two closely related strains. In the present study new genes were identified by partial sequence analysis of randomly cloned fragments of L. interrogans DNA. These genes were localized in regions of the genome by nucleic acid hybridization with DNA fragments separated by pulsed‐field gel electrophoresis. The resulting genetic maps provide improved resolution for each strain and provide evidence for additional chromosomal rearrangements. Insertion elements may be involved in recombination events, as several are near regions of the chromosome that have undergone rearrangement.
European functional analysis network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome (minireview)
Authors: Dujon Bernard 
Published: May 2005

AbstractLess than two yeras after the sequence of its genome was completed, the baker's yeast, Saccharomyces cerevisiae, is a leading organism in the rapidly growing field of functional genomics. Two thousands novel protein coding genes, nearly all of them “orphans”, have already been disrupted by the coordinated efforts of a large consortium of European laboratories, EUROFAN, and other initiatives. The mutants are submitted to many specialized functional assays, and studies are performed in parallel at the transcriptome and the proteome levels. With a central repository of mutant yeast strains, and a centralized database, EUROFAN lays the foundations for the future of genomics with yeast serving both as a model and a tool.
Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in‐line transfer to polyvinylidene difluoride membranes for sequencing
Authors: Masuoka James , Glee Pati M. , Hazen Kevin C. 
Published: May 2005

AbstractHydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two‐dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in‐line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.