- United States
M Washburne
University of New Mexico, Biology, Faculty Member
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Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase... more
Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion...
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Enzymatic biofuel cells employ enzymes as the catalysts for either anodic or cathodic processes, and use biofuels that are already available in nature such as sugars and alcohols [1]. In order to fully oxidize complex fuels like sugars in... more
Enzymatic biofuel cells employ enzymes as the catalysts for either anodic or cathodic processes, and use biofuels that are already available in nature such as sugars and alcohols [1]. In order to fully oxidize complex fuels like sugars in the anode, there is a need to ...
We are interested in characterizing the process of entry into and the maintenance of the stationary phase. To identify proteins that are induced during growth to stationary phase, we examined protein synthesis in long-term... more
We are interested in characterizing the process of entry into and the maintenance of the stationary phase. To identify proteins that are induced during growth to stationary phase, we examined protein synthesis in long-term stationary-phase cultures using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Although the total rate of protein synthesis declined when growth ceased after the postdiauxic phase, the pattern of proteins synthesized remained similar throughout the experimental period (28 days), except at the diauxic shift. At the diauxic shift most proteins detectable by 2D-PAGE undergo a transient reduction in their relative rate of synthesis that ends when cells resume growth during the postdiauxic phase. We conclude from this that the transient repression of protein synthesis at the diauxic shift is not directly associated with stationary-phase arrest. A number of proteins that are synthesized after exponential phase have been identified by 2D-PAGE. These protei...
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We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly... more
We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog....
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Members of the Joint Working Group on Improving Underrepresented Minorities (URMs) Persistence in Science, Technology, Engineering, and Mathematics (STEM)—convened by the National Institute of General Medical Sciences and the Howard... more
Members of the Joint Working Group on Improving Underrepresented Minorities (URMs) Persistence in Science, Technology, Engineering, and Mathematics (STEM)—convened by the National Institute of General Medical Sciences and the Howard Hughes Medical Institute—review current data and propose deliberation about why the academic “pathways” leak more for URM than white or Asian STEM students. They suggest expanding to include a stronger focus on the institutional barriers that need to be removed and the types of interventions that “lift” students’ interests, commitment, and ability to persist in STEM fields. Using Kurt Lewin’s planned approach to change, the committee describes five recommendations to increase URM persistence in STEM at the undergraduate level. These recommendations capitalize on known successes, recognize the need for accountability, and are framed to facilitate greater progress in the future. The impact of these recommendations rests upon enacting the first recommendati...
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Page 1. HYPERSPECTRAL IMAGING OF BIOLOGICAL TARGETS: THE DIFFERENCE A HIGH RESOLUTION SPECTRAL DIMENSION AND MULTIVARIATE ANALYSIS CAN MAKE Jerilyn A. Timlin a , Michael B. Sinclair a , David M. Haaland a , M. Juanita Martinez b , Monica... more
Page 1. HYPERSPECTRAL IMAGING OF BIOLOGICAL TARGETS: THE DIFFERENCE A HIGH RESOLUTION SPECTRAL DIMENSION AND MULTIVARIATE ANALYSIS CAN MAKE Jerilyn A. Timlin a , Michael B. Sinclair a , David M. Haaland a , M. Juanita Martinez b , Monica ...
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The regulatory subunit of cAMP-dependent protein kinase in yeast, encoded by the BCY1 gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have... more
The regulatory subunit of cAMP-dependent protein kinase in yeast, encoded by the BCY1 gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have identified novel isoforms of Bcy1 in cells under these conditions. The isoforms are distinguishable by their migration on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional nonequilibrium pH gradient gel electrophoresis. The isoforms observed by one-dimensional SDS-PAGE bind cAMP, as determined by [32P]8-azido-cAMP labeling (diagnostic of Bcy1 protein). Proteins isolated from cells grown to stationary phase in rich medium exhibit five antibody-reactive bands, by one-dimensional SDS-PAGE immunoblot analysis, with apparent molecular masses of 50, 52, 55, 59 and 61 kDa. Total Bcy1 protein increases at least 8-fold between exponential and stationary phase. Analysis of proteins from a variety of yeast mutants i...
Research Interests: Biological Chemistry, Western blotting, Biological Sciences, Saccharomyces cerevisiae, Protein Kinases, and 9 moreMutation, cyclic AMP, Phosphorylation, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Two-Dimensional Gel Electrophoresis, Two dimensional Gel Electrophoresis, and Medical and Health Sciences
SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly... more
SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream. The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved. This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants. An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated. SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced. In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced. Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil a...
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The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces... more
The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringenc...
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For cells of the yeast Saccharomyces cerevisiae, heat shock causes a transient inhibition of the cell cycle-regulatory step START. We have determined that this heat-induced START inhibition is accompanied by decreased CLN1 and CLN2... more
For cells of the yeast Saccharomyces cerevisiae, heat shock causes a transient inhibition of the cell cycle-regulatory step START. We have determined that this heat-induced START inhibition is accompanied by decreased CLN1 and CLN2 transcript abundance and by possible posttranscriptional changes to CLN3 (WHI1/DAF1) cyclin activity. Persistent CLN2 expression from a heterologous promoter or the CLN2-1 or CLN3-1 alleles that are thought to encode cyclin proteins with increased stability eliminated heat-induced START inhibition but did not affect other aspects of the heat shock response. Heat-induced START inhibition was shown to be independent of functions that regulate cyclin activity under other conditions and of transcriptional regulation of SWI4, an activator of cyclin transcription. Cells lacking Bcy1 function and thus without cyclic AMP control of A kinase activity were inhibited for START by heat shock as long as A kinase activity was attenuated by mutation. We suggest that hea...
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The yeast AAP1 gene, encoding a putative amino-peptidase, was isolated based on its ability to suppress the temperature-sensitive growth on nonfermentable carbon sources of spr5, a stationary phase regulatory mutant. AAP1 was physically... more
The yeast AAP1 gene, encoding a putative amino-peptidase, was isolated based on its ability to suppress the temperature-sensitive growth on nonfermentable carbon sources of spr5, a stationary phase regulatory mutant. AAP1 was physically mapped to chromosome VIII between PUT2 and CUP1. Sequence analysis of the AAP1 gene showed a 1581-nucleotide open reading frame capable of encoding a 59-kilodalton protein. The protein encoded by this open reading frame exhibits approximately 40% sequence identity to human, rat, and mouse aminopeptidases. In limited regions, sequence identity between Aap1 and the mammalian aminopeptidases ranges from 53% to 93%. Insertional inactivation of the AAP1 gene resulted in a decrease in glycogen accumulation and the loss of the major band of arginine/alanine aminopeptidase activity. Strains carrying the AAP1 gene on a high copy plasmid show an increase in the major arginine/alanine aminopeptidase activity, a dramatic increase in glycogen accumulation, and an...
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Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate... more
Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.
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Saccharomyces cerevisiae contains a large family of genes related to hsp70, the major heat shock-inducible gene of Drosophila melanogaster. One subfamily, identified by sequence homology, contains four genes, SSA1, SSA2, SSA3, and SSA4... more
Saccharomyces cerevisiae contains a large family of genes related to hsp70, the major heat shock-inducible gene of Drosophila melanogaster. One subfamily, identified by sequence homology, contains four genes, SSA1, SSA2, SSA3, and SSA4 (formerly YG100, YG102, YG106, and YG107, respectively). Previous studies showed that strains containing mutations in SSA1 and SSA2 are temperature sensitive for growth. SSA4, which is normally heat inducible and not expressed during vegetative growth, is expressed at high levels in ssa1 ssa2 strains at 23 degrees C. We constructed mutations in SSA3 and SSA4 and analyzed strains carrying mutations in the four genes. Strains carrying mutations in SSA3 SSA4 or SSA3 and SSA4 were indistinguishable from the wild type. However, ssa1 ssa2 ssa4 strains were inviable. SSA3, like SSA4, is a heat-inducible gene that is not normally expressed at 23 degrees C. Nevertheless, an intact copy of SSA3 regulated by the constitutive SSA2 promoter was capable of rescuing...
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SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton... more
SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.
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Yeast Hsp70 genes constitute a multigene family in which at least five of the nine members are heat inducible. Hsp70 RNA levels also vary dramatically during stationary arrest and sporulation. During growth to stationary phase, SSB1-SSB2... more
Yeast Hsp70 genes constitute a multigene family in which at least five of the nine members are heat inducible. Hsp70 RNA levels also vary dramatically during stationary arrest and sporulation. During growth to stationary phase, SSB1-SSB2 and SSC1 RNAs decreased in abundance as cell density increased. In contrast, SSA1-SSA2 RNA levels increased before the diauxic shift and then decreased as cells approach stationary phase. SSA3 RNA was detected only after the diauxic shift and accumulated to high levels as cells entered stationary phase. This accumulation was reversed by addition of glucose. Studies with cyr1 mutants indicated that SSA3 RNA accumulation is stimulated by decreasing intracellular cyclic AMP concentrations. When cells were incubated in sporulation medium, most Hsp70 RNAs, with the exception of SSA1-SSA2 RNA, decreased in abundance. This finding contrasted with the SSA1-SSA2 pattern observed during growth to stationary phase. SSA3 RNA was not detected during growth in ac...
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In this chapter, we argue that with careful attention to cell types in stationary-phase cultures of the yeast, S. cerevisiae provide an excellent model system for aging studies and hold much promise in pinpointing the set of causal genes... more
In this chapter, we argue that with careful attention to cell types in stationary-phase cultures of the yeast, S. cerevisiae provide an excellent model system for aging studies and hold much promise in pinpointing the set of causal genes and mechanisms driving aging. Importantly, a more detailed understanding of aging in this single celled organism will also shed light on aging in tissue-complex model organisms such as C. elegans and D. melanogaster. We feel strongly that the relationship between aging in yeast and tissue-complex organisms has been obscured by failure to notice the heterogeneity of stationary-phase cultures and the processes by which distinct cell types arise in these cultures. Although several studies have used yeast stationary-phase cultures for chronological aging, the majority of these studies have assumed that cultures in stationary phase are homogeneously composed of a single cell type. However, genome-scale analyses of yeast stationary-phase cultures have identified two major cell fractions: quiescent and non-quiescent, which we discuss in detail in this chapter. We review evidence that cell populations isolated from these cultures exhibit population-specific phenotypes spanning a range of metabolic and physiological processes including reproductive capacity, apoptosis, differences in metabolic activities, genetic hyper-mutability, and stress responses. The identification, in S. cerevisiae, of multiple sub-populations having differentiated physiological attributes relevant to aging offers an unprecedented opportunity. This opportunity to deeply understand yeast cellular (and population) aging programs will, also, give insight into genomic and metabolic processes in tissue-complex organism, as well as stem cell biology and the origins of differentiation.
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... Molecular and Cellular Biology, 2002. 16. MJ Martinez, S. Roy, AB Archuletta, PD Wentzell, SS Anna-Arriola, AL Rodriguez, AD Aragon, GA Quiones, C. Allen, and M. Werner-Washburne. ... 18. A. Niemistö, T. Aho, H. Thesleff, M. Tiainen,... more
... Molecular and Cellular Biology, 2002. 16. MJ Martinez, S. Roy, AB Archuletta, PD Wentzell, SS Anna-Arriola, AL Rodriguez, AD Aragon, GA Quiones, C. Allen, and M. Werner-Washburne. ... 18. A. Niemistö, T. Aho, H. Thesleff, M. Tiainen, K. Marjanen, M. Linne, and O. Yli-Harja. ...
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Neurospora crassa was the central organism in the development of biochemical genetics, providing a model system that established the relationship between genes and enzymes; it remains the best-studied filamentous fungus. This review... more
Neurospora crassa was the central organism in the development of biochemical genetics, providing a model system that established the relationship between genes and enzymes; it remains the best-studied filamentous fungus. This review focuses on the impact that the recent publication of a high-quality draft sequence of the N. crassa genome will have upon efforts to understand the biology of the
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Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant... more
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental change...
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Research Interests: Stress, Mitochondria, Multidisciplinary, Nature, Cell Culture, and 14 moreTranscription, Saccharomyces cerevisiae, Nucleic acid hybridization, Endoplasmic Reticulum, Peptides, Proteins, Biological evolution, Translocation, Heat Shock Proteins, Heat Shock Protein, Affinity, Gene Family, Secretion, and Thallophyta
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Research Interests: Computational Biology, Cell Cycle, Gene expression, Biological Sciences, Saccharomyces cerevisiae, and 15 moreVisual Analysis, Structured data, Three Dimensional Imaging, Cluster Analysis, Comparative Analysis, Ribosomal proteins, Genome, Physiological Response, Protein Interaction, Fungal genome size, Ribosomal Protein, Gene expression profiling, Gene Expression Data, False Positive, and Medical and Health Sciences
Research Interests: Genetics, Microbiology, Plant Biology, Gene expression, Protein synthesis, and 14 moreCell Division, Neurospora crassa, Cell Signalling, Genome Analysis, New Mexico, Life Cycle, Fungal Genetics and Biology, Fungal genome size, Amino Acid Sequence, Base Sequence, Fungal Genetics, Pilot project, Molecular Sequence Data, and cDNA cloning
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The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that... more
The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.
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ABSTRACT With the increasing availability of genome scale data, a plethora of algo-rithms are being developed to infer genome scale regulatory networks. How-ever, identifying the appropriate algorithm for a particular inference task is a... more
ABSTRACT With the increasing availability of genome scale data, a plethora of algo-rithms are being developed to infer genome scale regulatory networks. How-ever, identifying the appropriate algorithm for a particular inference task is a critical and difficult modeling question. This is because for most real-world data, the true network is rarely known and different algorithms produce dif-ferent networks, which may be due to assumptions inherrent to the algorithm or the objective the algorithm is designed to optimize. One approach to ad-dress this issue is to build artificial, but biologically realistic, gene networks for which the true topology is known. These networks can be sampled to gen-erate data, which can then serve as a testbed to compare a suite of network inference algorithms. Existing simulation systems either require highly detailed descriptions of network components making the generation of large-scale networks (> 100 nodes) infeasible, or, make simplifications that render the simulated networks very far from true models of gene regulation. We have developed a simula-tion system that builds a network of genes and proteins and models the rate of transcriptional activity as a non-linear function of a set of transciption factor complexes. Our simulator uses a system of differential equations and interfaces with Copasi, a differential equation solver, to produce steady-state and time-series expression datasets. Overall, our simulator has the following advantages: (i) it can produce expression datasets in an efficient and high-throughput manner, (ii) it is biologically more realistic since it models tran-scription rate as a function of transcription factor levels rather than gene ex-pression levels, (iii) it incorporates the combinatorial control among different transcription factors, and (iii) it incorporates gene and protein expressions, thus allowing the comparison of algorithms that use different combinations of these data sources.