Biomedical subjects

Linda E Hyman

Publications and source records attributed to Linda E Hyman.

5 recordsLinked to original sources

PubMed2007

Yeast genomic expression patterns in response to low-shear modeled microgravity.

UNLABELLED: The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs), allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. RESULTS: LSMMG differentially changed the expression of a significant number of genes (1372) when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR) genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome-wide changes. This study documents a considerable response to yeast cell growth in low-shear modeled microgravity that is evident, at least in part, by changes in gene expression. Notably, we identified genes that are involved in cell signaling pathways that allow cells to detect environmental changes, to respond within the cell, and to change accordingly, as well as genes of unknown function that may have a unique transcriptional response to microgravity. We also uncovered significant changes in the expression of many genes involved in cell polarization and bud formation that correlate well with the phenotypic effects observed in yeast cells when grown under similar conditions. These results are noteworthy as they have implications for human space flight.

Cluster Analysis↗

PubMed2004

Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii.

Toxoplasma gondii is an Apicomplexan parasite causing significant morbidity and mortality in immunocompromised hosts. Mitogen activated protein kinases regulate diverse biologic processes including proliferation, differentiation, survival and stress responses. We searched a new T. gondii genomic database to identify a 1.6 kilobase pair (kbp) coding region with features suggesting a mitogen activated protein kinase. This gene is predicted to encode a 58kDa protein with a threonine, aspartic acid, tyrosine (TDY) activation loop, similar to parasite and plant mitogen activated protein kinases, but distinct from mammalian mitogen activated protein kinases (with threonine, glycine, tyrosine (TGY) motifs). The predicted protein shares 45% amino acid identity with human stress-activated p38alpha mitogen activated protein kinase. Expression of the cloned gene in Escherichia coli produced a protein with an apparent molecular weight of 63kDa and which exhibited kinase activity. Following osmotic stress, the abundance of the mRNA encoding this T. gondii mitogen activated protein kinase, which we name TgMAPK-1, increased in tachyzoites. Its expression rescued hog1-deficient yeast grown under osmotic stress. These data confirm that the gene product is a stress-response mitogen activated protein kinase. Upon conversion of T. gondii tachyzoites to the latent bradyzoite form in vitro, tgMAPK-1 transcript accumulation increased, suggesting a role in parasite proliferation or stage differentiation. We previously demonstrated that pyridinylimidazole p38 mitogen activated protein kinase inhibitors block T. gondii replication. These inhibitors also blocked TgMAPK-1 autophosphorylation, suggesting that TgMAPK-1, or other parasite mitogen activated protein kinases are novel drug development targets.

Amino Acid Sequence↗

PubMed2003

GRS1, a yeast tRNA synthetase with a role in mRNA 3' end formation.

Transcription termination and 3' end formation are essential processes necessary for gene expression. However, the specific mechanisms responsible for these events remain elusive. A screen designed to identify trans-acting factors involved in these mechanisms in Saccharomyces cerevisiae identified a temperature-sensitive mutant that displayed phenotypes consistent with a role in transcription termination. The complementing gene was identified as GRS1, which encodes the S. cerevisiae glycyl-tRNA synthetase. This result, although unusual, is not unprecedented given that the involvement of tRNA synthetases in a variety of cellular processes other than translation has been well established. A direct role for the synthetase in transcription termination was determined through several in vitro assays using purified wild type and mutant enzyme. First, binding to two well characterized yeast mRNA 3' ends was demonstrated by cross-linking studies. In addition, it was found that all three substrates compete with each other for binding to GlyRS enzyme. Next, the affinity of the synthetase for the two mRNA 3' ends was found to be similar to that of its "natural" substrate, glycine tRNA in a nitrocellulose filter binding assay. The effect of the grs1-1 mutation was also examined and found to significantly reduce the affinity of the enzyme for the three RNA substrates. Taken together, these data indicate that not only does this synthetase interact with several different RNA substrates, but also that these substrates bind to the same site. These results establish a direct role for GRS1 in mRNA 3' end formation.

Adenosine Triphosphate↗

PubMed2002

Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture.

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

Blotting, Northern↗

PubMed2002

Binding to Elongin C inhibits degradation of interacting proteins in yeast.

Elongin C is a highly conserved, low molecular weight protein found in a variety of multiprotein complexes in human, rat, fly, worm, and yeast cells. Among the best characterized of these complexes is a mammalian E3 ligase that targets proteins for ubiquitination and subsequent degradation by the 26 S proteasome. Despite its crucial role as a component of such E3 ligases and other complexes, the specific function of Elongin C is unknown. In yeast, Elongin C is a non-essential gene and there is no obvious phenotype as associated with its absence. We previously reported that in Saccharomyces cerevisiae Elongin C (Elc1) interacts specifically and strongly with a class of proteins loosely defined as stress response proteins. In the present study, we examined the role of yeast Elc1 in the turnover of two of these binding partners, Snf4 and Pcl6. Deletion of Elc1 resulted in decreased steady-state levels of Snf4 and Pcl6 as indicated by Western blot analysis. Northern blot analysis of mRNA prepared from elc1 null and wild type strains revealed no difference in mRNA levels for Snf4 and Pcl6 establishing that the effects of Elc1 are not transcriptionally mediated. Reintroduction of either yeast or human Elongin C into Elc1 null strains abrogated this effect. Taken together, these data document that the levels of Snf4 and Pcl6 are dependent on the presence of Elc1 and that binding to Elc1 inhibits the degradation of these proteins. The results suggest a new function for yeast Elongin C that is distinct from a direct role in targeting proteins for ubiquitination and subsequent proteolysis.

Animals↗