James I. Garrels

A sampling of the yeast proteome.

ABSTRACT 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.

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.

Co-expression of vimentin and cytokeratins in parietal endoderm cells of early mouse embryo

Of the five classes of intermediate filaments found in vertebrate tissues, the cytokeratins are considered unique to epithelial tissues, while vimentin is expressed by endothelial and mesenchymal cells1. In neither case is the precise function of the filament system known. Epithelial cells in culture often express vimentin as well as cytokeratins1–3, but co-expression in vivo, as reported for pleomorphic adenomas of the parotid gland4,5 and metastatic carcinoma cells in ascites or pleural fluid6, is still controversial. Here we report the co-expression of cytokeratins and vimentin in situ, in the parietal endoderm of the mouse embryo 8.5–13.5 days old. This population of individual, motile cells seems to be derived from a conventional epithelium by migration and differentiation. Our results support the idea that vimentin expression is specifically related to reduced cell-to-cell contact, and to the independent existence of a cell following detachment from an epithelial sheet.

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 proteins 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 proteins 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

[28] Quantitative two-dimensional gel electrophoresis of proteins

Publisher Summary The fundamental method for two-dimensional separation of complex mixtures of proteins, as first used by O’Farrell, involves the separation of proteins in the first dimension by charge, using isoelectric focusing in high concentrations of urea and nonionic detergents followed by separation in the second dimension by molecular mass using sodium dodecyl sulfate (SDS) electrophoresis. The method has been improved for resolution, reproducibility, and convenience since the application of the original procedure. Some progress has been made in the methods and equipment for routine production of large numbers of two-dimensional gels and in methods of computerized data analysis. The methods described in this chapter are used in the authors laboratory for the resolution and quantitation of radiolabeled proteins from cultured cells. The gel system has been optimized for resolution and reproducibility to facilitate computer quantitation and eventual collection of highly standardized data. Aspects of quantitation, including sample counting and film calibration, are discussed in the chapter.

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.

Characterization of the mRNAs for α-, β- and γ-actin

Abstract The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10 5 daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10 5 daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.

Changes in protein synthesis during myogenesis in a clonal cell line

Abstract Methods of quantitative two-dimensional gel electrophoresis have been used to study the changes in protein synthesis that occur during myogenic differentiation in the L6 clonal line of rat skeletal muscle cells. Pure populations of myoblasts were obtained by maintaining the cells at subconfluent densities, and virtually pure populations of fused myotubes have been obtained by sedimentation at 1 × gravity through a serum gradient. The gel analysis reveals major qualitative differences between myoblasts and myotubes, as well as numerous quantitative changes. Both the α and the β forms of tropomyosin and the LC2 myosin light chain were increased in rate of synthesis by at least 1000-fold during myogenesis. Other proteins were detectable in myoblasts but were not synthesized at a detectable rate in myotubes. One of these is a form of tropomyosin which comigrates under several electrophoretic conditions with smooth muscle tropomyosin. Another protein, which is repressed in rate of synthesis by at least 1000-fold during myogenesis, appears to be a major form of collagen. Computer analysis has been used to analyze in detail a particular region containing about 300 spots from the two-dimensional patterns representing protein synthesis in L6 myoblasts, L6 myotubes, and a rat nerve cell line. Quantiative comparisons have shown that, with respect to this set of proteins, the L6 myoblasts and myotubes are no more alike at the level of protein synthesis than are L6 myoblasts and the cells of the nerve line. Therefore, these studies show that L6 differentiation involves not only the qualitative switching on and off of major gene products but also the quantitative alteration of synthetic rates of many of the common proteins.

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