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Mark Johnston

Publications and source records attributed to Mark Johnston.

At least 19 recordsLinked to original sources
PubMed2006

Two glucose-sensing pathways converge on Rgt1 to regulate expression of glucose transporter genes in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.

Amino Acid Sequence↗
PubMed2006

Linking DNA-binding proteins to their recognition sequences by using protein microarrays.

Analyses of whole-genome sequences and experimental data sets have revealed a large number of DNA sequence motifs that are conserved in many species and may be functional. However, methods of sufficient scale to explore the roles of these elements are lacking. We describe the use of protein arrays to identify proteins that bind to DNA sequences of interest. A microarray of 282 known and potential yeast transcription factors was produced and probed with oligonucleotides of evolutionarily conserved sequences that are potentially functional. Transcription factors that bound to specific DNA sequences were identified. One previously uncharacterized DNA-binding protein, Yjl103, was characterized in detail. We defined the binding site for this protein and identified a number of its target genes, many of which are involved in stress response and oxidative phosphorylation. Protein microarrays offer a high-throughput method for determining DNA-protein interactions.

Base Sequence↗
PubMed2006

Dysplasia and cancer in a large multicenter cohort of patients with Barrett's esophagus.

BACKGROUND & AIMS: The exact incidence of adenocarcinoma in patients with Barrett's esophagus (BE) is not known and is reported to vary from 0.2%-2% per year. Published series of patients with BE have included relatively small numbers of patients with limited duration of follow-up. The goal of this study was to define the prevalence and incidence of dysplasia and cancer and evaluate the paths of progression in a large multicenter cohort of BE patients. METHODS: The BE study is a multicenter clinical and endoscopic outcomes project involving a single large database of patients with BE. Data from each of the participating centers were merged into the main study database. Cancers and HGD occurring within 12 months of the index endoscopy were regarded as prevalent cases. RESULTS: One thousand three hundred seventy-six patients met the study criteria (95% white, 14% women); 91 patients had cancer at the initial endoscopy (prevalent cases, 6.7%; 95% confidence interval [CI], 4.8%-8.7%). Six hundred eighteen patients were followed for a total of 2546 patient-years; mean follow-up was 4.12 years. Twelve patients developed cancer during follow-up, a cancer incidence of 1 in 212 patient-years of follow-up (0.5% per year; 95% CI, 0%-1.1%). The combined incidence of HGD and/or cancer was 1 in 75 patient-years of follow-up or 1.3% per year (95% CI, 0%-2.2%). Of the 34 patients developing HGD and/or cancer, 18 patients (53%) had at least 2 initial consecutive endoscopies with biopsies revealing nondysplastic mucosa. The incidence of LGD was 4.3% per year (95% CI, 2.8%-6.0%). In the 156 patients with LGD, regression to no dysplasia occurred in 66%, persistent LGD in 21%, and progression to HGD/cancer in 13%. The incidence of cancer in patients with LGD was 1 in 156 patient-years of follow-up or 0.6% per year (95% CI, 0%-1.3%). CONCLUSIONS: Preliminary results from this trial define the prevalence and incidence of dysplasia and cancer in a multicenter cohort of patients with BE. At least half the patients who developed HGD and/or cancer had 2 consecutive initial endoscopies with biopsies revealing nondysplastic mucosa. The majority of patients with LGD regressed and had a cancer incidence similar to all BE patients.

Adenocarcinoma↗
PubMed2006

New roles for model genetic organisms in understanding and treating human disease: report from the 2006 Genetics Society of America meeting.

Fundamental biological knowledge and the technology to acquire it have been immeasurably advanced by past efforts to understand and manipulate the genomes of model organisms. Has the utility of bacteria, yeast, worms, flies, mice, plants, and other models now peaked and are humans poised to become the model organism of the future? The Genetics Society of America recently convened its 2006 meeting entitled "Genetic Analysis: Model Organisms to Human Biology" to examine the future role of genetic research. (Because of time limitations, the meeting was unable to cover the substantial contributions and future potential of research on model prokaryotic organisms.) In fact, the potential of model-organism-based studies has grown substantially in recent years. The genomics revolution has revealed an underlying unity between the cells and tissues of eukaryotic organisms from yeast to humans. No uniquely human biological mechanisms have yet come to light. This common evolutionary heritage makes it possible to use genetically tractable organisms to model important aspects of human medical disorders such as cancer, birth defects, neurological dysfunction, reproductive failure, malnutrition, and aging in systems amenable to rapid and powerful experimentation. Applying model systems in this way will allow us to identify common genes, proteins, and processes that underlie human medical conditions. It will allow us to systematically decipher the gene-gene and gene-environment interactions that influence complex multigenic disorders. Above all, disease models have the potential to address a growing gap between our ability to collect human genetic data and to productively interpret and apply it. If model organism research is supported with these goals in mind, we can look forward to diagnosing and treating human disease using information from multiple systems and to a medical science built on the unified history of life on earth.

Animals↗
PubMed2006

A glucose sensor in Candida albicans.

The Hgt4 protein of Candida albicans (orf19.5962) is orthologous to the Snf3 and Rgt2 glucose sensors of Saccharomyces cerevisiae that govern sugar acquisition by regulating the expression of genes encoding hexose transporters. We found that HGT4 is required for glucose induction of the expression of HGT12, HXT10, and HGT7, which encode apparent hexose transporters in C. albicans. An hgt4Delta mutant is defective for growth on fermentable sugars, which is consistent with the idea that Hgt4 is a sensor of glucose and similar sugars. Hgt4 appears to be sensitive to glucose levels similar to those in human serum ( approximately 5 mM). HGT4 expression is repressed by high levels of glucose, which is consistent with the idea that it encodes a high-affinity sugar sensor. Glucose sensing through Hgt4 affects the yeast-to-hyphal morphological switch of C. albicans cells: hgt4Delta mutants are hypofilamented, and a constitutively signaling form of Hgt4 confers hyperfilamentation of cells. The hgt4Delta mutant is less virulent than wild-type cells in a mouse model of disseminated candidiasis. These results suggest that Hgt4 is a high-affinity glucose sensor that contributes to the virulence of C. albicans.

Animals↗
PubMed2006

Integration of transcriptional and posttranslational regulation in a glucose signal transduction pathway in Saccharomyces cerevisiae.

Expression of the HXT genes encoding glucose transporters in the budding yeast Saccharomyces cerevisiae is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to the HXT promoters. It is well established that glucose induces degradation of Mth1, but the fate of its paralogue Std1 has been less clear. We present evidence that glucose-induced degradation of Std1 via the SCF(Grr1) ubiquitin-protein ligase and the 26S proteasome is obscured by feedback regulation of STD1 expression. Disappearance of Std1 in response to glucose is accelerated when glucose induction of STD1 expression due to feedback regulation by Rgt1 is prevented. The consequence of relieving feedback regulation of STD1 expression is that reestablishment of repression of HXT1 expression upon removal of glucose is delayed. In contrast, degradation of Mth1 is reinforced by glucose repression of MTH1 expression: disappearance of Mth1 is slowed when glucose repression of MTH1 expression is prevented, and this results in a delay in induction of HXT3 expression in response to glucose. Thus, the cellular levels of Std1 and Mth1, and, as a consequence, the kinetics of induction and repression of HXT gene expression, are closely regulated by interwoven transcriptional and posttranslational controls mediated by two different glucose-sensing pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

After the duplication: gene loss and adaptation in Saccharomyces genomes.

The ancient duplication of the Saccharomyces cerevisiae genome and subsequent massive loss of duplicated genes is apparent when it is compared to the genomes of related species that diverged before the duplication event. To learn more about the evolutionary effects of the duplication event, we compared the S. cerevisiae genome to other Saccharomyces genomes. We demonstrate that the whole genome duplication occurred before S. castellii diverged from S. cerevisiae. In addition to more accurately dating the duplication event, this finding allowed us to study the effects of the duplication on two separate lineages. Analyses of the duplication regions of the genomes indicate that most of the duplicated genes (approximately 85%) were lost before the speciation. Only a small amount of paralogous gene loss (4-6%) occurred after speciation. On the other hand, S. castellii appears to have lost several hundred genes that were not retained as duplicated paralogs. These losses could be related to genomic rearrangements that reduced the number of chromosomes from 16 to 9. In addition to S. castellii, other Saccharomyces sensu lato species likely diverged from S. cerevisiae after the duplication. A thorough analysis of these species will likely reveal other important outcomes of the whole genome duplication.

Adaptation, Physiological↗
PubMed2005

The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone methylation by COMPASS.

To date, several classes of enzymes have been shown to affect transcription by catalyzing the modifications of nucleosomes via methylation. Employing our global proteomic screen, GPS, we have determined that the loss of Bur2, a component of the Bur1/Bur2 cyclin-dependent protein kinase, results in a decrease in histone H3(K4) methylation catalyzed by COMPASS. Furthermore, Bur1/Bur2 is required for histone H2B monoubiquitination by Rad6/Bre1. The effect on histone monoubiquitination and methylation is the result of defective Bur1/Bur2-mediated phosphorylation of Rad6 on its serine residue 120 and proper recruitment of the Paf1 complex to chromatin. We have also demonstrated that serine 120 of Rad6 is required for histone H2B monoubiquitination and the regulation of gene expression in vivo. Our results identify in vivo substrates for Bur1/Bur2, thus linking its role to transcriptional elongation and demonstrating a potential activation mechanism for histone H2B monoubiquitination by the Rad6/Bre1 complex.

Amino Acid Sequence↗
PubMed2004

How the Rgt1 transcription factor of Saccharomyces cerevisiae is regulated by glucose.

Rgt1 is a transcription factor that regulates expression of HXT genes encoding glucose transporters in the yeast Saccharomyces cerevisiae. Rgt1 represses HXT gene expression in the absence of glucose; high levels of glucose cause Rgt1 to activate expression of HXT1. We identified four functional domains of Rgt1. A domain required for transcriptional repression (amino acids 210-250) is required for interaction of Rgt1 with the Ssn6 corepressor. Another region of Rgt1 (320-380) is required for normal transcriptional activation, and sequences flanking this region (310-320 and 400-410) regulate this function. A central region (520-830) and a short sequence adjacent to the zinc cluster DNA-binding domain (80-90) inhibit transcriptional repression when glucose is present. We found that this middle region of Rgt1 physically interacts with the N-terminal portion of the protein that includes the DNA-binding domain. This interaction is inhibited by the Rgt1 regulator Mth1, which binds to Rgt1. Our results suggest that Mth1 promotes transcriptional repression by Rgt1 by binding to it and preventing the intramolecular interaction, probably by preventing phosphorylation of Rgt1, thereby enabling Rgt1 to bind to DNA. Glucose induces HXT1 gene expression by causing Mth1 degradation, allowing Rgt1 phosphorylation, and leading to the intramolecular interaction that inhibits DNA binding of Rgt1.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I.

The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.

Casein Kinase I↗
PubMed2004

Large-scale screening of yeast mutants for sensitivity to the IMP dehydrogenase inhibitor 6-azauracil.

Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6-azauracil (6AU), an inhibitor of IMP dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase. To gain insight into the basis of the 6AU-sensitive phenotype and discern its specificity, we screened almost 3000 deletion mutants for growth in the presence of drug; 42 (1.5%) were reproducibly sensitive to the drug. The sensitive mutants included several missing known transcription elongation factors, but the majority were in genes involved in other cellular processes. Not all of the 6AU-sensitive strains displayed cross-sensitivity to mycophenolic acid (MPA), another drug that inhibits IMP dehydrogenase and has been employed as a screening agent for elongation mutants, showing that these two drugs are mechanistically distinct. Several of the mutants were tested for the ability to induce transcription of IMP dehydrogenase-encoding genes, in response to 6-AU and MPA treatment. As expected, mutants defective in transcriptional elongation factors were unable to fully induce IMPDH expression. However, most of the 6AU-sensitive strains had normal levels of IMPDH expression. Thus, although 6AU-sensitivity often results from defects in the elongation machinery, mutations that compromise processes other than transcription and induction of IMPDH also lead to sensitivity to this drug.

Blotting, Northern↗
PubMed2004

The promise of functional genomics: completing the encyclopedia of a cell.

Genome sequencing provides complete parts lists of organisms. This presents the obvious challenge of determining how each gene contributes to the life of the organism. This task seems increasingly feasible; however, progress to date suggests that increased interaction between systematic efforts and individual investigators will be critical to completing the encyclopedia of the yeast cell.

Gene Expression Regulation, Fungal↗
PubMed2004

Methods for the differential integrative omic analysis of plasma from a transgenic disease animal model.

Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.

Animals↗
PubMed2004

Regulatory network connecting two glucose signal transduction pathways in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

Base Sequence↗