The regulation of DNA replication in multicellular organisms is critical for their development. In addition, the improper regulation of DNA replication can lead to disease states such as malignancy or cell death. The experiments in this proposal address two aspects of the developmental regulation of DNA replication. One of the earliest controls in development is the restart of DNA replication in the embryo in response to fertilization. In most organisms the control of DNA replication in altered in some of the cells such that S phase becomes unlinked from mitosis and cell division, leading to an increase in the DNA content of the cell (polyteny or polyploidy). These two aspects of the developmental regulation of DNA replication will be investigated in the fruit fly, Drosophila melanogaster, because mutations can be isolated in which DNA replication is improperly controlled. Two genes have been identified, plutonium (plu) and pan gu (png), that regulate DNA replication at the onset of development. Unfertilized eggs mutant in either of these genes inappropriately undergo extensive DNA replication; moreover, the DNA in all four meiotic products appears to replicate rather than simply the pronucleus. Analysis of plu and png will be key to understanding how a resting oocyte is converted into a developing embryo in response to maturation and fertilization signals. The mechanisms by which plu and png regulate DNA replication in early development will be elucidated by experiments that will determine whether they control entry into S phase or whether they regulate DNA replication within S phase, possibly by blocking reinitiation. The products encoded by the plu and png genes will be identified by cloning the genes, and their cellular location will be determined. The developmental expression of the genes will be addressed, particularly whether the gene products are altered after fertilization. The interaction between plu, png, and four other genes that regulate the earliest cell cycles of development will be investigated by genetic and molecular approaches. Polytenization in Drosophila results from a modified cell cycle, the endo cell cycle, in which S phase alternates with a gap phase, but no mitosis occurs. The endo cell cycle is under developmental control because the onset of polytenization occurs with precise temporal and spatial regulation during the latter half of embryogenesis. Regulatory genes that trigger the onset of polytenization in development or that control the endo cell cycle will be identified and analyzed. Candidate regulatory genes have been isolated by the criteria that they are transcriptionally activated at the onset of polytenization in several tissues in the embryo. The effect of mutation of these genes on polytenization will be determined, and those that are shown to regulate this process will be cloned. A selection will be done for mutants in which polytenization is affected.
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