Several new finding imply that a number of previous ideas about the organization and replication of heterochromatin are probably wrong. In particular, heterochromatin is not a boring genetic wasteland of countless megabases of tandemly repeated satellite DNAs interrupted here and there by an occasional functional gene and an occasional defective transposable element in the heterochromatic """"""""graveyard."""""""" Rather, the heterochromatin has a complex and interesting genetic structure consisting of """"""""islands"""""""" of unique sequence, many of which contain coding regions, interspersed with blocks of satellite DNA. Understanding the remarkable genetic properties of heterochromatin will require physical mapping and genomic sequencing. We proposed to study the representation of heterochromatin of the same P1 libraries that have been instrumental in mapping the euchromatin and to investigate whether these clones could provide suitable material for physical mapping and sequencing of the genetically interesting portions of the heterochromatin.
Specific aim 1 : We will carry out fluorescent in situ hybridization (FISH) with prometaphase and metaphase mitotic chromosomes on 388 p1 clones that hybridize with the chromocenter of salivary gland nuclei. These localization will serve to identify the chromosome arm(s) containing the site(s) of major hybridization, with finer localization possible to quartiles of the pericentromeric heterochromatin at the base of each arm. This set of clones covers heterochromatic sequences other than simple satellites with an estimated redundancy of 1.1.
Specific aim 2 : We will isolate p1 clones containing known heterochromatic sequences by screening pools of DNA from p1 clones with oligonucleotide primers specific to known heterochromatic single-copy genes as well as to simple satellites and more complex repetitive sequences present in heterochromatin.
Specific aim 3 : We will determine whether the complex """"""""islands"""""""" of unique sequence present in heterochromatin can probably be covered by p1 contigs. Preliminary data suggest that heterochromatic P1 clones have sufficient complexity to support contig assembly by means of STS mapping with the ends of the cloned inserts. Mapping with pairs of clone ends has been shown to be a highly efficient strategy for the construction of physical maps. We will investigate the possibility of mapping heterochromatin in the manner by sequencing at least 200 P1 clone ends and designing oligonucleotide primers to a sample of the ends to be able to amplify them specifically from genomic DNA.
