YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information.

The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease.

The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information.

The Yeast Proteome Database (YPDtrade mark) has been for several years a resource for organized and accessible information about the proteins of Saccharomyces cerevisiae. We have now extended the YPD format to create a database containing complete proteome information about the model organism Caenorhabditis elegans (WormPDtrade mark). YPD and WormPD are designed for use not only by their respective research communities but also by the broader scientific community. In both databases, information gleaned from the literature is presented in a consistent, user-friendly Protein Report format: a single Web page presenting all available knowledge about a particular protein. Each Protein Report begins with a Title Line, a concise description of the function of that protein that is continually updated as curators review new literature. Properties and functions of the protein are presented in tabular form in the upper part of the Report, and free-text annotations organized by topic are presented in the lower part. Each Protein Report ends with a comprehensive reference list whose entries are linked to their MEDLINE s. YPD and WormPD are seamlessly integrated, with extensive links between the species. They are freely accessible to academic users on the WWW at http://www. proteome.com/databases/index.html, and are available by subscription to corporate users.

A sampling of the yeast proteome.

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.

The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data.

The Yeast Proteome Database (YPD) is a model for the organization and presentation of comprehensive protein information. Based on the detailed curation of the scientific literature for the yeast Saccharomyces cerevisiae, YPD contains more than 50 000 annotations lines derived from the review of 8500 research publications. The information concerning each of the approximately 6100 yeast proteins is structured around a convenient one-page format, the Yeast Protein Report, with additional information provided as pop-up windows. Protein classification schema have been revised this year, defining each protein's cellular role, function and pathway, and adding a Functional to the Yeast Protein Report. These changes provide the user with a succinct summary of the protein's function and its place in the biology of the cell, and they enhance the power of YPD Search functions. Precalculated sequence alignments have been added, to provide a crossover point for comparative genomics. The first transcript profiling data has been integrated into the YPD Protein Reports, providing the framework for the presentation of genome-wide functional data. The Yeast Proteome Database can be accessed on the Web at http://www.proteome.com/YPDhome.html

Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination.

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.

The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a curated database for the proteome of Saccharomyces cerevisiae . It consists of approximately 6000 Yeast Protein Reports, one for each of the known or predicted yeast proteins. Each Yeast Protein Report is a one-page presentation of protein properties, annotation lines that summarize findings from the literature, and references. In the past year, the number of annotation lines has grown from 25 000 to approximately 35 000, and the number of articles curated has grown from approximately 3500 to >5000. Recently, new data types have been included in YPD: protein-protein interactions, genetic interactions, and regulators of gene expression. Finally, a new layer of information, the YPD Protein Minireviews, has recently been introduced. The Yeast Protein Database can be found on the Web at http://www.proteome.com/YPDhome. html

Proteome studies of Saccharomyces cerevisiae: identification and characterization of abundant proteins.

Two-dimensional (2-D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2-D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence-derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2-D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2-D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2-D gel patterns. Using the 2-D gel map and the information available in YDP, 2-D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature-sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post-translational modification (N-terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.

Yeast Protein database (YPD): a database for the complete proteome of Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a database for the proteins of the budding yeast,Saccharomyces cerevisiae. YPD is the first annotated database for the complete proteome of any organism. Now that the complete genome sequence of yeast is available, YPD contains entries for each of the characterized proteins and for each of the uncharacterized proteins predicted from the sequence. Contained in YPD are the calculated properties of each protein such as molecular weight and isoelectric point, experimentally determined properties such as subcellular localization and post-translational modifications, and extensive annotations from the yeast literature. YPD contains 25 000 lines of textual annotation that describe the known functions, mutant phenotypes, interactions, and other properties for the approximately 6000 proteins in the yeast proteome. The information in YPD is updated daily, and it is available on the World Wide Web at http://www.proteome.com/YPDhome.html .

YPD-A database for the proteins of Saccharomyces cerevisiae.

YPD is a database for the proteins of the budding yeast, Saccharomyces cerevisiae. YPD has two formats: (i) a spreadsheet which tabulates many of the physical and functional properties of yeast proteins, and (ii) the YPD Protein Reports which are formatted pages containing the protein properties, annotations gathered from the literature, and references with titles. YPD is available through the World-Wide Web, through an Email server, and by anonymous FTP. New releases of the YPD spreadsheet are produced every two to four months, and the on-line information is updated daily.

A Saccharomyces cerevisiae Internet protein resource now available.

The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World-Wide Web (WWW) server (URL http:@siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other.

Protein identifications for a Saccharomyces cerevisiae protein database.

The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two-dimensional (2-D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2-D gels. We are using protein sequencing, overexpression of genes on high-copy number plasmids, and amino acid analysis to identify the proteins from 2-D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins on our yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2-D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.

REF52 on Global Gel Navigator: an internet-accessible two-dimensional gel electrophoresis database.

Until recently, the REF52 2-D gel database of experiments with rat cell lines was accessible only with special software. This database has now been made available to all investigators with access to the Internet, using the World Wide Web (WWW) technology. The package which delivers the database through the WWW has been named the Global Gel Navigator and can be used to explore the data by several methods, including the direct selection of proteins in the displayed gel using the mouse.

Alterations in protein synthesis following transplantation of mouse 8-cell stage nuclei to enucleated 1-cell embryos.

Enucleated mouse 1-cell embryos arrest development at the 2-cell stage following transplantation of cleavage stage nuclei. Earlier studies employing one-dimensional protein gel electrophoresis failed to reveal obvious differences in gene expression in the manipulated embryos that might account for this block. We report here the results of a quantitative, two-dimensional gel electrophoretic analysis that reveals at least 50 alterations in protein synthesis in the 8-->1-cell nuclear transplant embryos. Approximately half of these alterations involve proteins that normally decrease in synthesis between the 2-cell and 8-cell stages and half involve proteins that are synthesized constitutively between these two stages. These results are the first to reveal significant biochemical alterations that accompany the morphological and cytological differences previously described and indicate that the 8-cell stage nucleus is unable to completely recapitulate the normal progression of changes in protein synthesis pattern that occur during the 2-cell stage. Our results indicate that developmental arrest may result from a combination of (1) failure to re-activate genes that normally become repressed between the 2-cell and 8-cell stages and (2) failure to up-regulate genes that become repressed during reprogramming of the 8-cell nucleus by the 1-cell cytoplasm. The failure to up-regulate the synthesis of proteins expressed at both the 2-cell and the 8-cell stages may indicate that, for some genes, a transcriptionally inactive state, possibly related to a particular chromatin configuration, may serve a protective function by restricting access of factors that can permanently reduce the ability of a gene to be expressed. This may partially account for the need to delay the widespread activation of zygotic gene transcription until the 2-cell stage, when much of the nuclear remodeling that occurs postfertilization is complete.

A distributed system for two-dimensional gel analysis.

The Quest II system is a new two-dimensional (2D) gel analysis software system for the construction and analysis of 2D gel protein databases. A new architectural approach to 2D gel software systems has been utilized. This architecture is based on a tightly coupled client/server model. There are three layers to the system architecture: (i) a database layer consisting of three database servers, (ii) a compute layer consisting of three compute servers and (iii) an extensible user interface layer currently consisting of user interface tools for linearization and merging of scanned images, the segmentation and detection of protein spots on the images, matching, editing, and analysis of gels. The ability to store and retrieve the large volume of spot data inherent in 2D gel analysis while utilizing database technology is demonstrated.

Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells.

IL-2 and IL-4 are T cell growth factors that are produced by different T cell subsets and have distinct roles in lymphocyte biology. Despite their importance in the immune system, little is known about the genes that these lymphokines may specifically control and the interaction of these lymphokines in regulating the expression of their target genes. In this paper, we use the factor-dependent murine T cell line (CT.4R) to investigate the interaction of IL-2 and IL-4 in regulating gene expression. We report that the intermediate filament protein vimentin is differentially regulated by these lymphokines. Cells grown in IL-2 typically express 10- to 20-fold more vimentin and vimentin RNA than those grown in IL-4, but express similar levels of other cytoskeletal proteins including actin and tubulin. Vimentin was specifically induced by IL-2 and apparently suppressed by IL-4 in normal lymph node T cells, suggesting that its differential regulation by these lymphokines is physiologically relevant. We investigated the synergy between IL-2 and IL-4 in regulating the expression of vimentin RNA and compared it to that of two other lymphokine-responsive genes, pancreatic lipase and the IL-2R alpha subunit. Complex regulatory interactions were revealed: IL-4 suppressed the ability of IL-2 to induce vimentin RNA but not IL-2R alpha RNA, whereas IL-2 inhibited the ability of IL-4 to induce lipase RNA. These results indicate that IL-2 and IL-4 can cross-regulate lymphokine-responsive genes and can simultaneously exert both positive and negative regulation of different genes within the same cell.

Quantitative analysis of protein synthesis in mouse embryos. II: Differentiation of endoderm, mesoderm, and ectoderm.

The changes in protein synthesis that occur during differentiation of the primitive germ layers were examined by high-resolution, two-dimensional gel electrophoresis of proteins synthesized in 6.5 and 7.5 days postcoitum (d.p.c.) mouse embryos. For 6.5 d.p.c. embryos, protein synthesis patterns were compared between whole extraembryonic and embryonic regions and between embryonic visceral endoderm and embryonic ectoderm. For 7.5 d.p.c. embryos, comparisons were made between extraembryonic and embryonic regions and between isolated embryonic endoderm, mesoderm, and ectoderm. Each of the isolated 7.5 d.p.c. germ layers was divided into anterior and posterior fragments in order to evaluate possible regional differences in gene expression along the anterior-posterior axis. Comparisons of protein synthesis patterns revealed the greatest difference between isolated endoderm and ectoderm, indicating that by as early as 6.5 d.p.c. patterns of gene expression differ significantly between these tissues. The greatest similarities were found between ectoderm and whole embryonic regions and between endoderm and whole extraembryonic regions, which most likely reflects the overall cellular compositions of the embryonic and extraembryonic regions. Based on their patterns of synthesis, four groups of proteins were identified that were preferentially synthesized in either endoderm or ectoderm. These provide useful markers for studying differentiation in these tissues. One other protein, migrating at the position expected for vimentin, was synthesized at an elevated rate in isolated mesoderm. We also observed differences in rates of synthesis of alpha-tubulin and tropomyosin-5 indicative of potential differences in cytoskeletal composition among the germ layers beyond those previously described. The difference in overall protein synthesis patterns between anterior and posterior regions was greatest in the embryonic endoderm, indicating that differentiation along the anterior-posterior axis may be initiated sooner or may proceed more rapidly in the endoderm than in the other germ layers. These data provide the first quantitative evaluation of the degree to which differentiation of the three primitive germ layers affects protein synthesis patterns and reveal potentially useful markers of endoderm and ectoderm differentiation.

Workshop on two-dimensional gel protein databases.

A workshop on two-dimensional gel electrophoresis (2-DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2-DE analysis--namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2-DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or "relational" 2-DE gel protein database. This intermediary database, which would catalogue pertinent information on 2-DE resolved proteins (experimental source, 2-DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2-DE protein databases where primary and more specialized 2-DE data would be housed.

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus.

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.

Analysis of embryonic mouse development: construction of a high-resolution, two-dimensional gel protein database.

Numerous studies have revealed stage specific alterations in protein synthesis that occur in mouse embryos. A thorough analysis of these changes has been hampered by limitations in the ability to resolve individual proteins, the ability to accurately quantify and manage data from two-dimensional gel images, and by variation in the staging of the embryos used. To learn more of the changes in protein synthesis that occur during early development, we constructed a protein database for the mouse embryo using the QUEST system of high-resolution, two-dimensional gel electrophoresis and computerized gel image analysis (Garrels, 1989, J. Biol. Chem., 264:526). Synchronous cohorts of embryos were labeled at 3 h intervals throughout the entire preimplantation period from fertilization to blastocyst stage in order to characterize in detail the changes in protein synthesis pattern that occur during normal preimplantation development. Additional samples were prepared from early post-implantation embryos, isolated inner cell mass and trophoblast cells, and cultured embryonic stem cells. These provide the means for identifying cell and tissue specific proteins and for characterizing changes in protein synthesis that accompany early cellular differentiation in the embryo. We present here a description of the mouse embryo database and discuss its potential usefulness to the study of mammalian embryogenesis.