The long-term objective of this study is to characterize heterochromatin-specific nonhistone chromosomal proteins in Drosophila, with special attention to the role of such proteins in mediating heterochromatic position effects. Towards this end, four lines of investigation are proposed: (1) The molecular basis of mutations in the heterochromatic protein gene C1A9 will be determined by sequence analysis of DNA clones. DNA from the mutant locus will be obtained through the use of the """"""""polymerase chain reaction"""""""". (2) Structure-funcrion relationships in the heterochromatin-specific protein """"""""C1A9"""""""" will be explored through the use of C1A9-LacZ gene fusions. Fusions involving both wild- type and mutant C1A9 sequences will be expressed ty transfection into Drosophila tissue culture cells. Beta-gal fusion peptides will be localized in situ by indirect immunofluorescence using anti-beta-gal serum in bioassays to identify domains of the C1A9 protein important to nuclear localization and chromatin binding. (3) The chromatin srructure of an inducible gene (HsP 82) will be examined in a euchromatic site (where the gene is expressed normally) and a hererochromaric site (where the gene is transcriptionally inactive). This comparison will provide a direct assessment of the structural alterations associated with heterochromatic position effect. (4) A screen will be conducted for transposon-induced dominant suppressors of position effect varigation, many of which are expected to represent mutations in genes encoding heterochromatin-specific proteins. The proposed scheme makes use of the indicible transposition system """"""""jumpstarter"""""""" in order to obtain simple insertion mutations. The mutations recovered by this strategy will be cloned by transposon tagging, and the wild-type alleles will then be cloned and characterized. The identification and characterization of heterochromatin- associated chromosomal proteins and their genes will advance our understanding of eukaryotic chromosome structure. Heterochromatic position effects are frequently observed in association with chromosomal rearrangements, as such, they provide a useful paradigm for studying the genetic consequences of genomic rearrangements in metazoans, including man. Elucidation of the molecular basis for heterochromatic position effects should increase our understanding of mechanisms by which chromosome rearrangements alter gene expression. Such understanding may help explain the pathogenesis of birth defects associated with chromosome rearrangements, and the implications of multiple recurrent chromosome rearrangements associated with a majority of non-Hopkins lymphomas and malignant solid tumors.
