George Santangelo - US grants

This award provides funds to update the molecular biology instructional program at the University of Southern Mississippi by establishing an intensive "hands-on" laboratory in molecular biology/biotechnology. The laboratory in molecular biology will give the students practical experience in the fundamental techniques that form the foundation of experimental molecular biology. The major instruments to be obtained include growth chambers, electrophoresis equipment, UV spectrometers and low temperature storage units. The grantee is matching this award with non-Federal sources.

The expression of a very large number of genes (mostly glycolytic genes or translational component genes) that influence the growth rate of the budding yeast Saccharomyces cerevisiae is influenced in turn by the action of only a few regulatory proteins. In a recent publication we presented evidence that two of these regulatory proteins, TUF and GCR, activate transcription interdependently. Our data suggested that GCR is an absolute requirement for activation by a single, isolated TUF binding site (UASrpg element). Here we propose to test the reciprocal idea- that UASrpg is an absolute requirement for activation by GCR. We will try to disprove the idea that GCR-dependent transcriptional activation occurs only through UASrpg, and test for DNA binding by the GCR protein. The successful completion of this research will contribute to our understanding of GCR function, and form the basis for further investigation of the complex roles of TUF, how those roles are balanced in vivo, and the means by which growth rate is controlled in yeast cells. Yeast cells respond to changes in their environment (e.g., availability of nutrients) by changing their rate of growth and division. These changes involve the complex coordination of the action of many different genes in the cell. The mechanism of this coordination is poorly understood, and this is a proposal to study one aspect of this process.

The Organelle Molecular Biology Group at the University of Southern Mississippi is composed of seven energetic young investigators with common research interests and goals. The Group's ultimate research goal is to understand communication between subcellular compartments at the molecular level. This goal encompasses three essential biochemical processes in organelles-- DNA replication, transcription, and translation. We are particularly interested in the regulatory controls exerted by the nucleus on the organelle and vice versa. The two best studied organelles, chloroplasts and mitochondria, are the chosen subjects in a variety of eukaryotes. Funds are solicited for (1) expansion of the present capabilities for centrifugation, radioisotope counting, autoclaving, and dishwashing, and (2) the acquisition of research instrumentation that is not currently available (French pressure cell and fluorescence spectrophotometer).

biomedical equipment purchase;

Cells respond to changes in the nutritional environment by regulating gene expression, thereby controlling and coordinating metabolism and cell division. The focus of this project is on Rap1p and Gcr1p, which are DNA-binding regulatory proteins found in the nucleus of the model eukaryote, Saccharomyces cerevisiae. They mediate expression of genes that encode two important classes of metabolic proteins: translational components and glycolytic enzymes. Rap1p recruits Gcr1p to specific DNA binding sites upstream from the target genes. Preliminary results have demonstrated that both proteins are phosphorylated. These studies will be expanded to elucidate both the significance of these post-translational modifications, and other signaling components involved that might convey signals from the sensory mechanisms at the cell surface. Two potential connections between phosphorylation and Rap1p/Gcr1p function are of particular interest. First, Rap1p is phosphorylated within a single tryptic phosphopeptide, and is degraded upon nutrient starvation in a Gcr1p-dependent fashion. Second, although Gcr1p contains many tryptic phosphopeptides, a single species disappears in the absence of Gcr2p, an ancillary factor that is required for Rap1p/Gcr1p transcriptional activation of glycolytic genes only. Importantly, the Gcr2p-dependent phosphopeptide reappears in a Dgcr2 suppressor strain in which normal growth and glycolytic enzyme levels are restored. These and other potential regulatory roles of phosphorylation in this system will be investigated. The results should contribute to an understanding of the important relationship between environmental sensing and the control of cellular metabolism in eukaryotes.

DESCRIPTION (provided by applicant): Modern biological research is moving towards the examination of large-scale systems. Many advances can be cited to exemplify this trend, including high-throughput DNA sequencing, analysis of gene expression patterns, and drug discovery strategies. Other high-throughput applications involve direct analysis of proteins to characterize their structural modification, cellular localization, or effect on cell morphology. These new technologies are described by the umbrella term "Functional Genomics." Without the networked, high-throughput instrumentation that permits accumulation and bioinformatics-driven analysis of the associated large-scale data sets, competitive biomedical research in the State of Mississippi will soon become difficult, if not impossible. More importantly, the capacity to train talented undergraduate and graduate students for productive careers in biomedical research will decline precipitously. Therefore, it is proposed to establish a BRIN in Mississippi, which will generate an invaluable training pipeline of investigators needed for the modern biomedical research workforce. A critical facet of this Network involves strengthening the basic science departments of undergraduate institutions, most notably the HBCUs in Mississippi. This will include research and training opportunities for HBCU faculty and students to gain hands-on experience with the equipment and methodologies that will dominate the biomedical sciences for years to come. The new Genomics, Cellomics, Imaging, Proteomics, and Pharmacogenetics Facilities will thus help to establish a modern workforce of biomedical investigators helping to correct the dramatic under-representation of minorities in health-related fields of research.

Abstract Not Provided.

[unreadable] DESCRIPTION (provided by applicant): The long term objective of this project is to understand the relationship between the regulation of gene expression and nuclear substructure. Recent reports indicate that, soon after they are transcriptionally induced, genes in the model eukaryote Saccharomyces cerevisiae move to the nuclear periphery and associate with nuclear pore complexes. Specific Aim 1 is to use a single cell assay for regulated gene expression to determine the relationship between perinuclear gene anchoring and the ON and OFF transcriptional states. Specific Aim 2 is to explore the possibility that movement to the nuclear periphery is a universal property of transcriptionally induced genes. If not it will be important to determine whether transcriptionally active genes not anchored at the nuclear periphery become anchored elsewhere in the nucleus, for example within the nucleolus. Understanding these and other related aspects of gene regulation is key to the development of effective therapeutics for human diseases, since improperly regulated transcription is often responsible for congenital disorders and cancer. For example, the neurodegenerative disease spinocerebellar ataxia 7 results from a trinucleotide expansion in the SCA7 subunit of the chromatin modifying TFTC complex. These studies will take advantage of the integrated approach, including genetic, cell biological, and biochemical analyses, that is possible in the S. cerevisiae model system. The ease with which this organism can be grown and manipulated also makes it an ideal choice in providing research opportunities for undergraduate students. PUBLIC HEALTH RELEVANCE: The long-term objective of this project is to understand the relationship between the regulation of gene expression and nuclear substructure. Understanding these and related aspects of gene regulation is key to the development of effective therapeutics for human diseases, since improperly regulated transcription is often responsible for congenital disorders and cancer. [unreadable] [unreadable] [unreadable]