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This article describes the information contained within World Wide Web of potential uses for molecular biologists. The aim is to provide a basic description of the server provided services of bioinformatics, major program coordinator activities, and current contents of genome nucleic acid and protein databases.
Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Fractionation of opossum (Didelphis virginiana) serum with (NH4)2SO4, followed by chromatography on DEAE-Sepharose, phenyl-Sepharose, and Mono Q HR 5/5, has resulted in the isolation in homogeneous condition of a metalloproteinase inhibitor designated oprin (opossum proteinase inhibitor). Oprin is a single-chain glycoprotein (26% carbohydrate) with an estimated Mr = 52,000, pI = 3.5, and E(1%/1 cm) = 11. Oprin inhibited snake venom metalloproteinases, but showed no activity on venom serine proteinases or on bacterial metalloproteinases. Incubation of Crotalus atrox alpha-proteinase (EC 3.4.24.1) with oprin, and analysis of the reaction products by chromatography on Mono Q HR 5/5 and by electrophoresis under nondenaturing conditions, indicated formation of an inactive enzyme/inhibitor complex. The complex dissociated during SDS/polyacrylamide gel electrophoresis. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for part of the open reading frame for oprin were isolated. The cDNA inserts contained nucleotide sequences corresponding to two internal amino acid sequences of oprin which had been separately determined by protein sequence analysis. Protein database screening using a 211 amino acid sequence deduced from one of the cDNA inserts showed no significant homology to known proteinase inhibitors. There was, however, a 36% identity with human alpha 1B-glycoprotein, a plasma protein of unknown function related to the immunoglobulin supergene family. In addition, the amino-terminal sequence of oprin showed 46% identity with human alpha 1B-glycoprotein in a 26 amino acid residue overlap.(ABSTRACT TRUNCATED AT 250 WORDS)
We present an improved stochastic model of genes in DNA, and describe a method for integrating database homology into the probabilistic framework. A generalized hidden Markov model (GHMM) describes the grammar of a legal parse of a DNA sequence. Probabilities are estimated for gene features by using dynamic programming to combine information from multiple sensors. We show how matches to homologous sequences from a database can be integrated into the probability estimation by interpreting the likelihood of a sequence in terms of the bit-cost to encode a sequence given a homology match. We also demonstrate how homology matches in protein databases can be exploited to help identify splice sites. Our experiments show significant improvements in the sensitivity and specificity of gene structure identification when these new features are added to our gene-finding system, Genie. Experimental results in tests using a standard set of annotated genes showed that Genie identified 95% of coding nucleotides correctly with a specificity of 91%, and 77% of exons were identified exactly.
A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.
Electrospray ionization with an ultralow flow rate (</=4 nanoliters per minute) was used to directly couple capillary electrophoresis with tandem mass spectrometry for the analysis and identification of biomolecules in mixtures. A Fourier transform mass spectrometer provided full spectra (>30 kilodaltons) at a resolving power of approximately 60,000 for injections of 0.7 x 10(-18) to 3 x 10(-18) mole of 8- to 29-kilodalton proteins with errors of <1 dalton in molecular mass. Using a crude isolate from human blood, a value of 28,780.6 daltons (calculated, 28,780.4 daltons) was measured for carbonic anhydrase, representing 1 percent by weight of the protein in a single red blood cell. Dissociation of molecular ions from 9 x 10(-18) mole of carbonic anhydrase gave nine sequence-specific fragment ions, more data than required for unique retrieval of this enzyme from the protein database.
Position-weight matrices (or profiles) are simple mathematical objects traditionally used to capture the information about local sequence patterns (or motifs) characteristic of a given structure or function. Although weight matrices can lead to fast database scanning algorithms their usage has been limited, due to the lack of a reliable method to assess the statistical significance of the matching scores. In this article I first review 3 different computation scheme for designing weight matrices from a block-alignment of any (small or large) number of sequences. I then show that, for patterns spanning 10 positions or more, the best scores expected from matching random sequences are distributed according to the extreme value (Gumbel) distribution. The threshold of statistical significance assessed from this distribution perfectly delineate the range of scores characterizing "true positive" sequences (biological significant matches). This result allows weight matrices to be used to scan an entire protein database for patterns in a highly sensitive way. MODEST (MOtif DEsign and Search Tools), a suite of programs in Unix/C, implements these statistical improvements and is available upon E-mail request (jmc@ncbi.nlm.nih.gov).
The fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, efficient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one- or two-dimensional gels. Each step of the standard protocol of Aebersold et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 6970-6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations of in situ microdigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S-alkylation of gel-separated proteins and accurate identification of tryptophan-containing peptides were introduced to insure overall higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two-dimensional gel of Escherichia coli total protein (120 micrograms), allowed unambiguous identification of the spots but pre-gel enrichment will be required for analysis of most (90-95%) other spots. Integration of comprehensive two-dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging vast amounts of data.
An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios.
The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.
A phosphorylcholine-reactive protein was isolated from serum of Atlantic salmon (Salmo salar L.) by affinity chromatography on a phosphorylcholine-conjugated Sepharose column followed by elution with phosphorylcholine. Based on the method used we describe the isolated protein as salmon phosphorylcholine-reactive protein (salmon PRP). Salmon PRP has calcium-independent binding to phosphorylcholine. The protein exists in a monomeric and dimeric form with molecular weight of approximately 80 and 160 kD, respectively. Separation of the protein preparation on SDS-PAGE under reducing conditions resulted in disappearance of the 80 and 160 kD bands and appearance of a major protein band of approximately 100 kD. The N-terminal amino acid sequences of the non-reduced 80 and 160 kD bands and the reduced 100 kD band were identical. Apart from the dimeric form, the molecular weight of salmon PRP and its appearance on SDS-PAGE is similar to human plasminogen. Comparison of the sequence in a protein database resulted in approximately 50% identity with human and bovine plasminogen. In addition, cross-reactivity between antibodies to human plasminogen and salmon PRP was demonstrated. Thus, salmon PRP appears to be different from other phosphorylcholine-reactive proteins which are mostly reported to be CRP-like proteins with calcium-dependent binding to phosphorylcholine, pentameric ring-structure and sequence homology between species. Whether salmon PRP is a new type of phosphorylcholine-binding protein with an unknown function or a plasminogen-like protein with binding specificity for phosphorylcholine calls for further investigation.
Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.
Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.
BACKGROUND: House dust mites of the Dermatophagoides genus are the most important cause of perennial allergic disease in both humans and companion animals. Although the major mite allergens for humans are proteins of relatively low molecular weight, this is not the case for dogs. Western blotting shows that canine anti-mite IgE responses are directed primarily toward proteins in the molecular weight range of 50 to 120 kd. OBJECTIVE: The objectives of this study were to characterize a D farinae allergen with a molecular weight of approximately 60 kd and to isolate the cDNA coding for this allergen. METHODS: A protein of apparent molecular weight of 60 kd was identified by Western blotting by using canine serum IgE from house dust mite-sensitized atopic dogs. The protein was purified from homogenized D farinae mite bodies by ammonium sulfate precipitation, followed by gel filtration and cation exchange HPLC. The presence of IgE directed to the 60-kd protein in sera from humans and dogs with dust mite allergy was measured by FcepsilonRIalpha-based ELISA. A cDNA encoding a full-length 60-kd protein was isolated from a D farinae cDNA library by a combination of both PCR amplification and hybridization screening. A panel of mAbs specific for the 60-kd protein was generated and used to localize the protein in whole body sections of D farinae mites. RESULTS: ELISA showed that the purified protein bound IgE in 54% of the sera from patients with D farinae allergy. In addition, the 60-kd protein was able to bind IgE in 57% to 77% of D farinae -sensitized dogs. A cDNA was isolated that encoded a protein of 462 amino acids, consisting of a 25 amino acid signal sequence and a 437 amino acid mature protein. The calculated molecular weight of the mature protein is 50 kd, and the amino acid sequence contains a single N-glycosylation site. A protein database search showed homology with multiple chitinases. A mAb specific for the 60-kd chitinase recognized the allergen in the mite digestive system, but fecal pellets did not stain positively for this allergen. CONCLUSIONS: A 60-kd D farinae protein (Der f 18), with homology to chitinase, is a major allergen for humans and dogs sensitive to house dust mites.
Maxicell labelling and two-dimensional gel electrophoresis (2-D PAGE) have identified the proteins encoded by sspA and sspB (SspA, SspB) as proteins D27.1 and A25.8, respectively, in the Escherichia coli gene-protein database. SspA expression increases with decreasing growth rate and is induced by glucose, nitrogen, phosphate or amino acid starvation. The promoter, Pssp, is similar to gearbox promoters. Inactivation of SspA (sspA::neo) blocks sspB expression. [35S]-methionine-labelled proteins synthesized during growth and during stationary phase are different in delta sspA strains compared to sspA+ strains. This difference is enhanced during extended stationary phase (24-72 h). Long-term (10 d) viability of arginine-starved isogenic strains shows that sspA+ cultures remain viable significantly longer than delta sspA mutants. 2-D PAGE of proteins expressed during exponential growth shows that expression of at least 11 proteins is altered in delta sspA strains. A functional relA gene is required for sspA to affect protein synthesis.
Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.
Molecular biology has a communication problem. There are many databases using their own labels and categories for storing data objects and some using identical labels and categories but with a different meaning. Conversely, one concept is often found under different names. Prominent examples are the concepts "gene" and "protein sequence" which are used with different semantics by major international genomic and protein databases thereby making database integration difficult and strenuous. This situation can only be improved by either defining individual semantic interfaces between each pair of databases (complexity of order n2) or by implementing one agreeable, transparent and computationally tractable semantic repository and linking each database to it (complexity of order n). Ontologies are one means to provide such semantic repository by explicitly specifying the meaning of and relation between the fundamental concepts in an application domain. Here, heuristics for building an ontology and the upper level and a database branch of a prospective Ontology for Molecular Biology are presented and compared to other ontologies with respect to suitability for molecular biology (http:/(/)igd.rz-berlin.mpg.de/www/oe/mbo.html).
Muskelin was identified in vertebrates as a novel, intracellular, kelch repeat protein that is needed in cell-spreading responses to the matrix adhesion molecule, thrombospondin-1. The identification and characterization of an orthologue of muskelin in Drosophila melanogaster is now reported. The Drosophila muskelin gene, located on chromosome 2R, is encoded in ten exons. Drosophila muskelin is expressed in embryos, larvae and adult flies. The protein has 45% sequence identity to vertebrate muskelins, with highest sequence identity in an amino-terminal domain and the six kelch repeats that form a beta-propeller structure. Multiple sequence alignment of human, mouse, rat and Drosophila muskelins and protein database searches revealed a novel highly conserved motif within the amino-terminal domain, lissencephaly homology motif (LisH) and C-terminal to LisH motifs in the central region of the molecule, and several conserved consensus motifs for phosphorylation by protein kinase C and casein kinase II. These findings provide new information on the modular structure of muskelin and indicate potential for conserved mechanisms of function.