R Sikorski

Bio: R Sikorski is an academic researcher. The author has contributed to research in topics: Shuttle vector & Saccharomyces cerevisiae. The author has an hindex of 1, co-authored 1 publications receiving 7587 citations.

Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus.

Abstract: The transmembrane kinase Ire1p is required for activation of the unfolded protein response (UPR), the increase in transcription of genes encoding endoplasmic reticulum (ER) resident proteins that occurs in response to the accumulation of unfolded proteins in the ER. Ire1p spans the ER membrane (or the nuclear membrane with which the ER is continuous), with its kinase domain localized in the cytoplasm or in the nucleus. Consistent with this arrangement, it has been proposed that Ire1p senses the accumulation of unfolded proteins in the ER and transmits the signal across the membrane toward the transcription machinery, possibly by phosphorylating downstream components of the UPR pathway. Molecular genetic and biochemical studies described here suggest that, as in the case of growth factor receptors of higher eukaryotic cells, Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER and is phosphorylated in trans by other Ire1p molecules as a result of oligomerization. In addition to its kinase domain, a C-terminal tail domain of Ire1p is required for induction of the UPR. The role of the tail is probably to bind other proteins that transmit the unfolded protein signal to the nucleus.

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

Correlation between Protein and mRNA Abundance in Yeast

Abstract: The description of the state of a biological system by the quantitative measurement of the system constituents is an essential but largely unexplored area of biology. With recent technical advances including the development of differential display-PCR (21), of cDNA microarray and DNA chip technology (20, 27), and of serial analysis of gene expression (SAGE) (34, 35), it is now feasible to establish global and quantitative mRNA expression profiles of cells and tissues in species for which the sequence of all the genes is known. However, there is emerging evidence which suggests that mRNA expression patterns are necessary but are by themselves insufficient for the quantitative description of biological systems. This evidence includes discoveries of posttranscriptional mechanisms controlling the protein translation rate (15), the half-lives of specific proteins or mRNAs (33), and the intracellular location and molecular association of the protein products of expressed genes (32). Proteome analysis, defined as the analysis of the protein complement expressed by a genome (26), has been suggested as an approach to the quantitative description of the state of a biological system by the quantitative analysis of protein expression profiles (36). Proteome analysis is conceptually attractive because of its potential to determine properties of biological systems that are not apparent by DNA or mRNA sequence analysis alone. Such properties include the quantity of protein expression, the subcellular location, the state of modification, and the association with ligands, as well as the rate of change with time of such properties. In contrast to the genomes of a number of microorganisms (for a review, see reference 11) and the transcriptome of Saccharomyces cerevisiae (35), which have been entirely determined, no proteome map has been completed to date. The most common implementation of proteome analysis is the combination of two-dimensional gel electrophoresis (2DE) (isoelectric focusing-sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis) for the separation and quantitation of proteins with analytical methods for their identification. 2DE permits the separation, visualization, and quantitation of thousands of proteins reproducibly on a single gel (18, 24). By itself, 2DE is strictly a descriptive technique. The combination of 2DE with protein analytical techniques has added the possibility of establishing the identities of separated proteins (1, 2) and thus, in combination with quantitative mRNA analysis, of correlating quantitative protein and mRNA expression measurements of selected genes. The recent introduction of mass spectrometric protein analysis techniques has dramatically enhanced the throughput and sensitivity of protein identification to a level which now permits the large-scale analysis of proteins separated by 2DE. The techniques have reached a level of sensitivity that permits the identification of essentially any protein that is detectable in the gels by conventional protein staining (9, 29). Current protein analytical technology is based on the mass spectrometric generation of peptide fragment patterns that are idiotypic for the sequence of a protein. Protein identity is established by correlating such fragment patterns with sequence databases (10, 22, 37). Sophisticated computer software (8) has automated the entire process such that proteins are routinely identified with no human interpretation of peptide fragment patterns. In this study, we have analyzed the mRNA and protein levels of a group of genes expressed in exponentially growing cells of the yeast S. cerevisiae. Protein expression levels were quantified by metabolic labeling of the yeast proteins to a steady state, followed by 2DE and liquid scintillation counting of the selected, separated protein species. Separated proteins were identified by in-gel tryptic digestion of spots with subsequent analysis by microspray liquid chromatography-tandem mass spectrometry (LC-MS/MS) and sequence database searching. The corresponding mRNA transcript levels were calculated from SAGE frequency tables (35). This study, for the first time, explores a quantitative comparison of mRNA transcript and protein expression levels for a relatively large number of genes expressed in the same metabolic state. The resultant correlation is insufficient for prediction of protein levels from mRNA transcript levels. We have also compared the relative amounts of protein and mRNA with the respective codon bias values for the corresponding genes. This comparison indicates that codon bias by itself is insufficient to accurately predict either the mRNA or the protein expression levels of a gene. In addition, the results demonstrate that only highly expressed proteins are detectable by 2DE separation of total cell lysates and that therefore the construction of complete proteome maps with current technology will be very challenging, irrespective of the type of organism.

Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions

Abstract: Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

Abstract: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.