This application proposes to continue and extend the research on heterochromatin and the eu-heterochromatin boundary that he started as a Postdoctoral Fellow in Allan Spradling's laboratory. The investigator is using the Dp(1;f)1187 minichromosome system developed in the Spradling lab over the last few years. This 1.3 Mb minichromosome displays all of the characteristics of a typical higher eukaryotic chromosome, but it affords unusual experimental opportunities. In work done as a postdoctoral fellow the PI found that DNA sequences at the euchromatic-heterochromatic boundary have unusual features and in this application he describes his plans to further characterize these sequences and heterochromatin in general. The proposal contains three specific aims. In the first the PI will continue his characterization of heterochromatic restriction fragments of Dp1187 in a number of different tissues. In previous experiments the PI found that there were differences between, for example, salivary gland and ovary. The PI will carry out mapping experiments to further characterize the structure of these chromosomes and restriction fragments. In the experiments on ovary DNA he will focus on understanding the two reasons that Dp1187 fragments are under represented on Southern blots. The PI has found that these fragments do not transfer with typical efficiency and as well that there are shortened Dp1187 molecules in this sample. The PI will also examine salivary gland Dp1187. In this tissue there is a true under representation of Dp1187 sequences that is it is not due to transfer problems. The nature of the molecules that give rise to the under representation will be characterized by 2D hybridization analysis. The PI also proposes to examine Dp1187 in diploid cells (in embryos and in imaginal discs/brains). The PI already has evidence that in embryo DNA Dp1187 sequences are under represented on Southern blots due to poor transfer and that Dp1187 fragments of increased size are found in embryo DNA. He has been developing techniques for the isolation of DNA from haploid sperm and he will examine the structure of Dp1187 in these cells as well. The second specific aim is to examine the generality of the results on Dp1187 by examining other eu-heterochromatin boundaries. One of those that he will examine is the sc8 inversion which is the parental chromosome for Dp1187. In other experiments he will examine eu- heterochromatic boundaries that result from the insertion of a P element into autosomal heterochromatin. The third specific aim is to identify and characterize the physical property of Dp1187 heterochromatin that leads to selective transfer inhibition. He has already isolated transfer resistant DNA in solution and he will treat this DNA in a variety of ways, such as alkaline denaturation and then examine the effects in a variety of ways such as by examining its sedimentation in sucrose gradients to look for altered conformation.
