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Biomedical subjects

R A Young

Publications and source records attributed to R A Young.

At least 19 recordsLinked to original sources
PubMed2001

Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins.

DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

Binding Sites↗
PubMed2001

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

Carrier Proteins↗
PubMed2001

The plasticity of dendritic cell responses to pathogens and their components.

Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to Escherichia coli, Candida albicans, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses.

Antigen Presentation↗
PubMed2001

Serial regulation of transcriptional regulators in the yeast cell cycle.

Genome-wide location analysis was used to determine how the yeast cell cycle gene expression program is regulated by each of the nine known cell cycle transcriptional activators. We found that cell cycle transcriptional activators that function during one stage of the cell cycle regulate transcriptional activators that function during the next stage. This serial regulation of transcriptional activators forms a connected regulatory network that is itself a cycle. Our results also reveal how the nine transcriptional regulators coordinately regulate global gene expression and diverse stage-specific functions to produce a continuous cycle of cellular events. This information forms the foundation for a complete map of the transcriptional regulatory network that controls the cell cycle.

Cell Cycle↗
PubMed2001

Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse.

An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.

Animals↗
PubMed2001

Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase.

The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCF(Cdc4)-dependent manner in vivo. Upon fractionation of yeast extracts to identify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosphorylates Gcn4 and thereby marks it for recognition by SCF(Cdc4) ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Gcn4 is almost completely stabilized in srb10Delta pho85Delta cells, or upon mutation of all Srb10 phosphorylation sites within Gcn4, suggesting that the Pho85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumulation of Gcn4. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. We propose that Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors. Simultaneous down-regulation of both transcriptional regulatory proteins and RNA polymerase may enhance the potency and specificity of transcriptional inhibition by Srb10.

Amino Acids↗
PubMed2001

An unusual combination of diaphragmatic hernias in a patient presenting with the clinical features of restrictive pulmonary disease: report of a case.

The combination of a Morgagni hernia and a paraesophageal hernia in adults is very rarely encountered in clinical practice. In fact, to our knowledge, only three cases of this condition, which is probably a coincidental occurrence, have been reported in the medical literature. We discuss the management of a 74-year-old man found to have combined Morgagni and paraesophageal hernia who presented with clinical features of a restrictive pulmonary disease.

Aged↗
PubMed2001

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes.

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.

Ascomycota↗
PubMed2001

Remodeling of yeast genome expression in response to environmental changes.

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves approximately 10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

Adaptation, Physiological↗
PubMed2001

Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo.

NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

DNA-Binding Proteins↗
PubMed2001

Using graphical models and genomic expression data to statistically validate models of genetic regulatory networks.

We propose a model-driven approach for analyzing genomic expression data that permits genetic regulatory networks to be represented in a biologically interpretable computational form. Our models permit latent variables capturing unobserved factors, describe arbitrarily complex (more than pair-wise) relationships at varying levels of refinement, and can be scored rigorously against observational data. The models that we use are based on Bayesian networks and their extensions. As a demonstration of this approach, we utilize 52 genomes worth of Affymetrix GeneChip expression data to correctly differentiate between alternative hypotheses of the galactose regulatory network in S. cerevisiae. When we extend the graph semantics to permit annotated edges, we are able to score models describing relationships at a finer degree of specification.

Bayes Theorem↗
PubMed2001

The Gaussian derivative model for spatial-temporal vision: I. Cortical model.

How do we see the motion of objects as well as their shapes? The Gaussian Derivative (GD) spatial model is extended to time to help answer this question. The GD spatio-temporal model requires only two numbers to describe the complete three-dimensional space-time shapes of individual receptive fields in primate visual cortex. These two numbers are the derivative numbers along the respective spatial and temporal principal axes of a given receptive field. Nine transformation parameters allow for a standard geometric association of these intrinsic axes with the extrinsic environment. The GD spatio-temporal model describes in one framework the following properties of primate simple cell fields: motion properties, number of lobes in space-time, spatial orientation. location, and size. A discrete difference-of-offset-Gaussians (DOOG) model provides a plausible physiological mechanism to form GD-like model fields in both space and time. The GD model hypothesizes that receptive fields at the first stage of processing in the visual cortex approximate 'derivative analyzers' that estimate local spatial and temporal derivatives of the intensity profile in the visual environment. The receptive fields as modeled provide operators that can allow later stages of processing in either a biological or machine vision system to estimate the motion as well as the shapes of objects in the environment.

Animals↗
PubMed2001

The Gaussian derivative model for spatial-temporal vision: II. Cortical data.

Receptive fields of simple cells in the primate visual cortex were well fit in the space and time domains by the Gaussian Derivative (GD) model for spatio-temporal vision. All 23 fields in the data sample could be fit by one equation. varying only a single shape number and nine geometric transformation parameters. A difference-of-offset-Gaussians (DOOG) mechanism for the GD model also fit the data well. Other models tested did not fit the data as well as or as succinctly, or failed to converge on a unique solution, indicating over-parameterization. An efficient computational algorithm was found for the GD model which produced robust estimates of the direction and speed of moving objects in real scenes.

Animals↗
PubMed2001

Joint actions of parents and adolescents in health conversations.

Based on an action theory approach, 32 videotaped parent-adolescent conversations about health in two Canadian ethno-cultural groups were analyzed to identify and describe the joint action of parents and adolescents in the health domain. The data include manifest behavior (the conversation), internal processes (recalled thoughts and feelings on viewing the videotape of the conversation), and social meaning. These data were analyzed using the goals, functions, and elements of the conversations. Five categories of joint actions emerged from these data: sharing and exploring information, values, and beliefs about health; negotiating and struggling about aspects of the adolescent's independence; providing and receiving guidance; adjusting to family and home issues; and fostering joint understanding by giving voice to the adolescent.

Adolescent↗
PubMed2000

Genome-wide location and function of DNA binding proteins.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.

Binding Sites↗
PubMed2000

Insulin regulation of beta-cell function involves a feedback loop on SERCA gene expression, Ca(2+) homeostasis, and insulin expression and secretion.

The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.

Animals↗
PubMed2000

Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells.

Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.

Animals↗