HIGH RESOLUTION INTERPHASE CHROMOSOME STRUCTURE

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The overall objective of the work proposed here is to determine the complete 3-dimensional interphase chromosome structure of two specific genes, the Notch and engrailed genes of Drosophila melanogaster, at several critical times in their expression programs in diploid embryonic and in larval polytene chromosomes. The purpose of this project is to determine what structural changes occur in the genes during the Drosophila life cycle and how these changes correlate to changes in gene expression. As a result of increasingly detailed molecular and genetic analysis, it has become clear that chromosomal context can have profound effects on gene behavior, probably because of the influence of chromatin structure on expression. A number of observations support this idea: 1) The inactive X chromosome in human females is highly condensed, suggesting a correlation between chromosome decondensation and gene expression. The fragile X syndrome, causing mental retardation and other defects primarily in human males, has been hypothesized to result from incomplete reactivation of a portion of the maternally derived X chromosome. 2) The expression of some genes in Drosophila is altered when homologue pairing is interrupted. 3) Expression of integrated copies of mammalian genes is location sensitive as are P-element mediated gene insertions in Drosophila. High resolution optical microscopy and in situ hybridization, in conjunction with molecular techniques will be used to determine the chromosome structure of the Notch locus and the engrailed gene in diploid interphase cells in embryos, when the genes are silent and after the onset of gene expression. The same techniques will then be used in conjunction with in vitro mutagenesis and P-element mediated germline transformation of Drosophila to determine which DNA sequences are required to maintain the gene structure. In accomplishing these aims, basic questions concerning the state of normal chromosomes will be answered. In addition, new insights will be gained into the mechanism by which certain transposable DNA sequences choose to interrupt genes and induce mutations.