Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucoselimited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.

Background: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking.

A physiological and molecular biological study was made of the control of methylenomycin biosynthesis by Streptomyces coelicolor A3(2). A simple and reliable assay for this antibiotic was developed. Conditions that permit the synthesis of methylenomycin by S. coelicolor cultures grown in defined medium were elucidated: a readily assimilated carbon and nitrogen source is required. Under these conditions methylenomycin is produced late in the growth phase, at the time of transition from exponential to linear growth. Provided that the phosphate concentration in the medium is kept high, there is synthesis of methylenomycin but not of the other secondary metabolites that this strain can produce. These conditions were used to study the transcription of the methylenomycin gene cluster during the transition from primary to secondary metabolism. The biosynthetic genes of at least one of the mmy transcription units appear to be transcribed before the mmr resistance determinant. The possibility that methylenomycin induces the transcription of mmr is discussed.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2⌬ mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of ϳ11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway. constitutive activation of PKA by PDE2 deletion; Ras/cAMP pathway; cell wall integrity THE RAS/CAMP PATHWAY is a highly conserved signal transduction pathway operating via the second messenger, cAMP (6). In Saccharomyces cerevisiae, it controls cell-cycle progression, cell growth and proliferation (3, 81), reprogramming of transcription at the diauxic transition (8), mating (1), pseudohyphal morphogenesis (27, 55), metabolism (9), and stress responses. The synthesis of cAMP is catalyzed by adenylate cyclase (40), which is regulated by Ras proteins (18,24,80), the G protein ␣-subunit homolog, Gpa2 (48), and the adenylate-cyclase-associated protein, Cap1p (23,25). The only known biochemical role of cAMP is to activate protein kinase A (PKA) (12,78,79). High activity of PKA in yeast leads to low levels of the storage carbohydrates trehalose and glycogen, low stress resistance due to reduced expression of STRE ("stress response element")-controlled genes, aberrant G 0 arrest, poor growth on nonfermentable and weakly fermentable carbon sources, and failure of sporulation in diploid cells. Low activity yields de-repression of STRE-controlled genes leading to high stress resistance, constitutive expression of heat-shock genes, and sporulation of diploid cells in rich media (for reviews, see Refs. 10,64,[74][75][76]. Two trans-acting factors (Msn2p and Msn4p), negatively regulated by PKA, have been shown to be involved in STRE-mediated gene expression (47,67).Intracellular levels of cAMP, and hence the state of the Ras/cAMP pathway, are also contr...