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PubMed1997

Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme.

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.

Amino Acid Sequence↗

PubMed1987

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.

Amino Acid Sequence↗

PubMed1997

Mapping of microsatellite markers developed from a flow-sorted swine chromosome 6 library.

Swine Chromosome (Chr) 6-enriched libraries, generated with size-fractionated DNA isolated from chromosomes sorted by flow cytometry, have been used to develop new Chr 6 microsatellite markers. Chromosome isolation procedures were established to reproducibly prepare high quality chromosomes from phytohemagglutinin (PHA)-stimulated swine peripheral blood lymphocytes and to sort individual chromosomes after staining with Hoechst 33258 and chromomycin A3. Chromosome purity was verified by specific staining of swine Chr 6 with fluorescence in situ hybridization (FISH) by use of painting probes generated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of as few as 300 sorted Chr 6. For library construction, DNA was extracted from flow-sorted pools representing Chr 6, amplified, size selected for fragments from 300 to 700 bp, and ligated into pBluescript SK II+ or Lambda ZAP Express. The libraries were then screened with a radiolabeled poly-(dCA) DNA probe. Of 107 (CA)n repeat-containing clones verified by sequencing, 21 were polymorphic and used to genotype the University of Illinois swine reference families. Linkage analysis was then performed with CRIMAP 2.4 (LOD > 3.0), and the results showed that 15 of the microsatellites mapped to swine Chr 6. At least three of these new markers map to locations where there were gaps in the consensus Chr 6 map. Another four markers, because of their PIC values, should provide more informative markers in other areas of the map. Most of the new markers can also be used for automated genotyping with fluorescent labeling. This set of 15 new Chr 6 markers will, therefore, be useful in helping to define QTL associated with swine Chr 6.

Animals↗

PubMed2006

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes.

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure-function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.

Enzymes↗

PubMed2001

Cellular biosensors for drug discovery.

Recent advances in cell biology, fluorescent probe chemistry, miniaturization and automation have allowed the use of mammalian cells in a variety of medical and industrial applications. Here we describe the generation of cell-based biosensors, engineered to optically report specific biological activity. Cellular biosensors are comprised of living cells and can be used in various applications, including screening chemical libraries for drug discovery and environmental sensing. Panels of biosensors may also be useful for elucidating the function of novel genes. Here we describe two examples of the construction and use of engineered cell lines as biosensors for drug discovery.

Animals↗

PubMed1997

Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools.

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.

Amino Acid Sequence↗

PubMed1992

An evaluation of the source selection elements of the prototype UMLS Information Sources Map.

The Information Sources Map (ISM) is a component of the National Library of Medicine's Unified Medical Language System (UMLS) project. The ISM is intended to provide both human-readable and machine-interpretable information about the content, scope, and access conditions for various information sources such as databases, expert systems, and the organizations which make these information sources available. Automated source selection is supported by three types of indexing in the ISM: Medical Subject Heading (MeSH) terms and subheadings; Semantic Types from the UMLS Semantic Network; and Semantic Type Relations, which depict pairs of semantic types joined by a relationship chosen from the Semantic Network. This paper reports a study of the recall and precision of the source selection elements in the prototype version of the ISM.

Abstracting and Indexing↗

PubMed2005

Analysing proteomic data.

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.

Animals↗

PubMed1998

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 up-regulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro)n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photoirreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.

Amino Acid Sequence↗

PubMed1995

Efficient pooling designs for library screening.

We describe efficient methods for screening clone libraries, based on pooling schemes that we call "random k-sets designs." In these designs, the pools in which any clone occurs are equally likely to be any possible selection of k from the v pools. The values of k and v can be chosen to optimize desirable properties. Random k-sets designs have substantial advantages over alternative pooling schemes: they are efficient, flexible, and easy to specify, require fewer pools, and have error-correcting and error-detecting capabilities. In addition, screening can often be achieved in only one pass, thus facilitating automation. For design comparison, we assume a binomial distribution for the number of "positive" clones, with parameters n, the number of clones, and c, the coverage. We propose the expected number of resolved positive clones--clones that are definitely positive based upon the pool assays--as a criterion for the efficiency of a pooling design. We determine the value of k that is optimal, with respect to this criterion, as a function of v, n, and c. We also describe superior k-sets designs called k-sets packing designs. As an illustration, we discuss a robotically implemented design for a 2.5-fold-coverage, human chromosome 16 YAC library of n = 1298 clones. We also estimate the probability that each clone is positive, given the pool-assay data and a model for experimental errors.

Binomial Distribution↗

PubMed2004

Large-scale expression screening by automated whole-mount in situ hybridization.

Gene expression profiling is an important component of functional genomics. We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos. Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development. Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development. cDNA inserts were colony PCR amplified in a 384-format. The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process. Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed. Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" (http://www.embl-heidelberg.de/mepd/), that is publicly accessible.

Animals↗

PubMed1999

Towards the pathogenesis of autoimmune liver disease.

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗

PubMed1994

A neural network system for detection of atrial fibrillation in ambulatory electrocardiograms.

INTRODUCTION: A neural network classifier has been designed, which is able to distinguish atrial fibrillation (AF) from other supraventricular arrhythmias in ambulatory (Holter) ECGs. METHOD AND RESULTS: The classification algorithm uses a rhythm analysis that considers the ECG to be a time series of RR interval durations. This is combined with an analysis of baseline morphology that considers the morphological characteristics of the non-QRS portions of the waveform. A backpropagation-based neural network has been used as part of the classifier implementation. When applied to a library consisting exclusively of 42,970 examples of AF and other supraventricular rhythm disturbances validated by an experienced cardiologist, the algorithm demonstrated a sensitivity of 82.4% for 10-beat runs of paroxysmal atrial fibrillation (PAF) and a specificity of 96.6%. Since this system has been implemented as a postprocessor to a conventional automated Holter system, operating only on segments of ECG that are known to contain supraventricular arrhythmias rather than ventricular arrhythmias or sinus rhythm, it can be added to most existing Holter processing systems without significantly increasing the average time to process a tape. CONCLUSION: A neural network system has been designed, which can potentially provide, for the first time, an accurate, quantitative technique to determine the natural history of PAF and to evaluate potential treatments for PAF.

Atrial Fibrillation↗

PubMed1987

Knowledge-based decision support for general practitioners: an integrated design.

Most decision-support systems (DSSs) in medicine have been developed in hospital environments, for use in hospitals. Only a few are designed for use by general practitioners (GPs) in primary care. The work reported in this paper aims to (a) design DSSs for GPs in primary care, taking into consideration that primary care is the first level in a health care organization, where the reasons for the patients attendance are seen in a social context; and (b) integrate three approaches-Hypertext, Knowledge-Based Systems and Online Library-since any one of these will not suffice for the varying needs of GPs.

Academic Medical Centers↗

PubMed2005

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Carcinoma, Hepatocellular↗

PubMedDate not supplied

Detection and classification of threat agents via high-content assays of mammalian cells.

One property common to all chemical or biological threat agents is that they damage mammalian cells. A threat detection and classification method based on the effects of compounds on cells has been developed. This method employs high-content screening (HCS), a concept in drug discovery that enables those who practice cell-based assays to generate deeper biological information about the compounds they are testing. A commercial image-based cell screening platform comprising fluorescent reagents, automated image acquisition hardware, image analysis algorithms, data management and informatics was used to develop assays and detection/classification methods for threat agents. These assays measure a cell's response to a compound, which may include activation or inhibition of signal transduction pathways, morphological changes or cytotoxic effects. Data on cell responses to a library of compounds was collected and used as a training set. At the EILATox-Oregon Workshop, cellular responses following exposure to unknown samples were measured by conducting assays of p38 MAP kinase, NF-kappaB, extracellular-signal related kinase (ERK) MAP kinase, cyclic AMP-response element binding protein (CREB), cell permeability, lysosomal mass and nuclear morphology. Although the assays appeared to perform well, only four of the nine toxic samples were detected. However the system was specific, because no false positives were detected. Opportunities for improvement to the system were identified during the course of this enlightening workshop. Some of these improvements were applied in subsequent tests in the Cellomics laboratories, resulting in a higher level of detection. Thus, an HCS approach was shown to have potential in detecting threat agents, but additional work is necessary to make this a comprehensive detection and classification system.

Algorithms↗

PubMed2005

Signaling protein inhibitors via the combinatorial modification of peptide scaffolds.

Compounds that selectively interfere with protein-protein interactions are not only invaluable as biological reagents, but may ultimately serve as therapeutically useful drugs for the treatment of a wide variety of disease states. However, unlike active site directed inhibitors that bind to a relatively small, well-defined, hydrophobic pocket, reagents that disrupt protein-protein interactions must contend with a protein surface that is comparatively large, ill defined, and solvent exposed. We have developed a straightforward method for the acquisition of protein-protein interaction inhibitors. The library-based strategy starts with low affinity consensus sequence peptides, which are then transformed in a stepwise fashion into high affinity inhibitors. The approach has been used to create potent ligands for SH2 and SH3 domains, as well as powerful and highly selective inhibitors for protein kinases and phosphatases. The protocol is easily automated and therefore has the potential to be routinely applied, in a high throughput fashion.

Animals↗

PubMed1990

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family.

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.

Amino Acid Sequence↗