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Biomedical subjects

Jeffrey M Becker

Publications and source records attributed to Jeffrey M Becker.

At least 19 recordsLinked to original sources

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37 degrees C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Delta mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell-daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2 Delta strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25 degrees C and was not viable at 37 degrees C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

Animals↗

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors.

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.

Animals↗

Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.

Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids.

Amino Acids↗

Nutrient regulation of oligopeptide transport in Saccharomyces cerevisiae.

Small peptides (2-5 amino acid residues) are transported into Saccharomyces cerevisiae via two transport systems: PTR (Peptide TRansport) for di-/tripeptides and OPT (OligoPeptide Transport) for oligopeptides of 4-5 amino acids in length. Although regulation of the PTR system has been studied in some detail, neither the regulation of the OPT family nor the environmental conditions under which family members are normally expressed have been well studied in S. cerevisiae. Using a lacZ reporter gene construct fused to 1 kb DNA from upstream of the genes OPT1 and OPT2, which encode the two S. cerevisiae oligopeptide transporters, the relative expression levels of these genes were measured in a variety of environmental conditions. Uptake assays were also conducted to measure functional protein levels at the plasma membrane. It was found that OPT1 was up-regulated in sulfur-free medium, and that Ptr3p and Ssy1p, proteins involved in regulating the di-/tripeptide transporter encoding gene PTR2 via amino acid sensing, were required for OPT1 expression in a sulfur-free environment. In contrast, as measured by response to toxic tetrapeptide and by real-time PCR, OPT1 was not regulated through Cup9p, which is a repressor for PTR2 expression, although Cup9p did repress OPT2 expression. In addition, all of the 20 naturally occurring amino acids, except the sulfur-containing amino acids methionine and cysteine, up-regulated OPT1, with the greatest change in expression observed when cells were grown in sulfur-free medium. These data demonstrate that regulation of the OPT system has both similarities and differences to regulation of the PTR system, allowing the yeast cell to adapt its utilization of small peptides to various environmental conditions.

Carrier Proteins↗

Harnessing natural diversity to probe metabolic pathways.

Analyses of cellular processes in the yeast Saccharomyces cerevisiae rely primarily upon a small number of highly domesticated laboratory strains, leaving the extensive natural genetic diversity of the model organism largely unexplored and unexploited. We asked if this diversity could be used to enrich our understanding of basic biological processes. As a test case, we examined a simple trait: the utilization of di/tripeptides as nitrogen sources. The capacity to import small peptides is likely to be under opposing selective pressures (nutrient utilization versus toxin vulnerability) and may therefore be sculpted by diverse pathways and strategies. Hitherto, dipeptide utilization in S. cerevisiae was solely ascribed to the activity of a single protein, the Ptr2p transporter. Using high-throughput phenotyping and several genetically diverse strains, we identified previously unknown cellular activities that contribute to this trait. We find that the Dal5p allantoate/ureidosuccinate permease is also capable of facilitating di/tripeptide transport. Moreover, even in the absence of Dal5p and Ptr2p, an additional activity--almost certainly the periplasmic asparaginase II Asp3p--facilitates the utilization of dipeptides with C-terminal asparagine residues by a different strategy. Another, as-yet-unidentified activity enables the utilization of dipeptides with C-terminal arginine residues. The relative contributions of these activities to the utilization of di/tripeptides vary among the strains analyzed, as does the vulnerability of these strains to a toxic dipeptide. Only by sampling the genetic diversity of multiple strains were we able to uncover several previously unrecognized layers of complexity in this metabolic pathway. High-throughput phenotyping facilitates the rapid exploration of the molecular basis of biological complexity, allowing for future detailed investigation of the selective pressures that drive microbial evolution.

Dipeptides↗

Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae.

Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.

Canavanine↗

Transmembrane domain prediction and consensus sequence identification of the oligopeptide transport family.

Few polytopic membrane proteins have had their topology determined experimentally. Often, researchers turn to an algorithm to predict where the transmembrane domains might lie. Here we use a consensus method, using six different transmembrane domain prediction algorithms on six members of the oligopeptide transport family, all of which have been experimentally characterized. PSI-BLAST results indicate that the six chosen oligopeptide transport family members are distributed throughout most branches of the phylogram, suggesting that these members represent a broad view of the oligopeptide transport family. We combined the prediction algorithms with a multiple sequence alignment, and consensus transmembrane domains were assigned not only based on algorithmic output, but also based on conserved familial motifs found by analysis of the PSI-BLAST results. The consensus method combined with the "charge-difference rule" yields a model topology for the family containing 12 transmembrane domains with the N- and C-termini facing extracellular.

Algorithms↗

Interacting residues in an activated state of a G protein-coupled receptor.

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.

Alanine↗

Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.

The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis.

Amino Acid Sequence↗

Synthetic peptides as probes for conformational preferences of domains of membrane receptors.

Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s. A pioneer in this field was Dr. Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides. This article reviews some of Dr. Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae. Peptides containing 30-40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography. CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles. Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues.

Amino Acid Sequence↗

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary adenocarcinomas in Syrian golden hamsters contain beta 2-adrenergic receptor single-nucleotide polymorphisms.

Cigarette smoking contributes to the development of lung cancer throughout the world, with cases of pulmonary adenocarcinoma (PAC) the most numerous. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is formed from nicotine, has been demonstrated to cause mutations in genes that affect cell regulation and proliferation. Moreover, NNK has been shown to interact directly with and stimulate beta adrenergic receptor (ADRB) signal transduction pathways. Our goal was to determine whether single-nucleotide polymorphisms (SNPs) in the Adrb2 from PAC tumors were induced in golden hamsters by the injection of NNK. Here we report the cloning and sequencing of Adrb2 clones from either dissected lung tumors from NNK-injected animals or whole-lung tissue from water-injected controls. Both sets of animals contained SNPs; however, we found significantly more SNPs in the Adrb2 from NNK-injected animals than in the controls. The majority of these SNPs were novel, nonsynonymous mutations found in regions of the Adrb2 known to be involved in ligand binding, G-protein coupling, and desensitization/down-regulation. Our data verified the mutagenic effects of NNK as well as demonstrated that this animal model provides an outstanding way of identifying mutations not only in the Adrb2, but also in other genes that may play essential roles in the regulation and growth of pulmonary adenocarcinomas.

Adenocarcinoma↗

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae.

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.

Amino Acid Sequence↗

A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast.

The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.

4-Chloro-7-nitrobenzofurazan↗

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.

Binding, Competitive↗

Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry.

Protein surfaces are important in most biological processes, including protein-protein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues' chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry. The specific sites of the stable modification were then identified by reverse-phase liquid chromatography coupled to quadropole ion trap tandem mass spectrometry. The solvent accessibility of the residue was shown to directly affect the rate of oxidation by this method (with the exception of methionine), supporting its use as a rapid measure of the solvent accessibility of specific residues, and in some cases, individual atoms.

Amino Acid Sequence↗

Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure.

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.

Amino Acid Sequence↗

The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.

Mating in Saccharomyces cerevisiae is initiated by the secretion of diffusible peptide pheromones that are recognized by G protein-coupled receptors (GPCR). This review summarizes the use of the alpha-factor (WHWLQLKPGQPMY)--GPCR (Ste2p) interaction as a paradigm to understand the recognition between medium-sized peptide hormones and their cognate receptors. Studies over the past 15 years have indicated that the alpha-factor is bent around the center of the pheromone and that residues near the amine terminus play a central role in triggering signal transduction. The bend in the center appears not to be rigid and this flexibility is likely necessary for conformational changes that occur as the receptor switches from the inactive to active state. The results of synthetic, biological, biochemical, molecular biological, and biophysical analyses have led to a preliminary model for the structure of the peptide bound to its receptor. Antagonists for Ste2p have changes near the N-terminus of alpha-factor, and mutated forms of Ste2p were discovered that appear to favor binding of these antagonists relative to agonists. Many features of this yeast recognition system are relevant to and have counterparts in mammalian cells.

Alanine↗

Genome-scale gene function prediction using multiple sources of high-throughput data in yeast Saccharomyces cerevisiae.

Characterizing gene function is one of the major challenging tasks in the post-genomic era. To address this challenge, we have developed GeneFAS (Gene Function Annotation System), a new integrated probabilistic method for cellular function prediction by combining information from protein-protein interactions, protein complexes, microarray gene expression profiles, and annotations of known proteins through an integrative statistical model. Our approach is based on a novel assessment for the relationship between (1) the interaction/correlation of two proteins' high-throughput data and (2) their functional relationship in terms of their Gene Ontology (GO) hierarchy. We have developed a Web server for the predictions. We have applied our method to yeast Saccharomyces cerevisiae and predicted functions for 1548 out of 2472 unannotated proteins.

Automation↗