1986 — 1988 |
Tsubota, Stuart I |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Position Effects and the Control of Gene Expression
The goal is to understand how changes in the local chromosomal environment of particular genes influences their transcriptional activity. This phenomenon which has been called "position effect" is observed when the DNA sequence organization of particular genes is altered, either by insertion of foreign DNA sequences or by large scale chromosomal rearrangments. These changes in sequence organization can cause dramatic alterations in gene expression, and may either suppress or activate transcription. Moreover, position dependent alterations in gene activity have been directly implicated in oncogenesis. To study this phenomenon, we have chosen two different genes in the fruit fly Drosophila melanogaster, rudimentary and Bar, which exhibit position dependent changes in gene activity. Position effect mutations at both of these loci have been identified and these are associated either with the insertion of foreign DNA sequences or with chromosomal rearrangements. We will use a combination of genetic and molecular approaches to examine how specific changes in the DNA sequence environment of these genes alters their expression. These studies will include: (1) genetic experiments to generate new "position effect" mutations; (2) the analysis of the transcriptional activity of the wild type and mutant loci; (3) a detailed characterization of the DNA sequence organization of the wild type and mutant loci; and (4) an examination of how the chromatin structure of each locus is altered by the chromosomal rearrangements.
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0.951 |
2002 |
Tsubota, Stuart I |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
The Enhancer of Rudimentary Gene
DESCRIPTION (provided by applicant): Pyrimidine biosynthesis is regulated in a cell-cycle specific manner to ensure sufficient pools for DNA synthesis. At the core of this regulation is the gene CAD in mammals and its homologue, rudimentary (r) in Drosophila melanogaster. These genes encode the first three enzymes in the pyrimidine biosynthetic pathway, including the rate-limiting step, carbamyiphosphate synthetase. The enhancer of rudimentary gene, e(r), was isolated as a regulatory gene of r. It encodes a highly conserved protein, whose function within the cell has not been determined. The maternal and embryonic expression patterns of e(r) suggest an embryonic function in the cell cycle. Other clues to the function and regulation of the e(r) protein (ER) are the presence of three casein kinase II sites on the protein and the binding of the protein to the ribosomal protein S3 (RPS3). The long-term goals of this project are to determine the function of e(r) in growth and development and to elucidate its mode of action in the cell. In this project, the role of the conserved casein kinase II phosphorylation sites in the regulation of the activity of ER will be determined by mutating the sites to either glutamic acid to mimic phosphorylation or to alanine to mimic the unphosphorylated residue. Each mutant gene will then be assayed for activity by its ability to rescue the mutation e(r)pl. Second, the regions of ER and RPS3 that interact will be determined by testing the ability of fragments of each protein to interact in a yeast two-hybrid assay. In this assay, interacting clones will be seen as LEU2 LACZ+ colonies. Finally, null mutations of e(r) will be isolated using a gene-targeting procedure that will utilize a mutant episomal copy of e(r) to recombine with the resident allele. The expression data of e(r) suggest that mutants may be embryonic lethals. If this is the case, the mutants will be analyzed for cellular defects in the embryo that that will help uncover the normal function of e(r).
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