The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.

We report herein the isolation of ccf, a new gene located in region 82E and essential for Drosophila development. This gene, expressed throughout development, encodes a novel product of 68 kDa which is found in the nucleus during interphase and labels, in a novel pattern, centrosomes and chromosome arms during mitosis. Mutations in ccf give rise to late larvae with small imaginal discs and to adults showing appendages of reduced size, consistent with CCF involvement in cell proliferation. Neuroblast squash analyses show that CCF is required for proper condensation of mitotic chromosomes and, therefore, for progression through mitosis. Furthermore, we observe that adult ccf mutants as well as animals overexpressing CCF during larval stages exhibit homeotic transformations. We also find that mutations in the Pc-G genes Polycomb, polyhomeotic and Enhancer of zeste are enhanced by ccf mutations. Finally, we show that the CCF protein binds to specific sites on polytene chromosomes, many of which are shared with the Posterior sex combs Pc-G protein. Together, these results suggest a role for the CCF protein in the maintenance of chromosome structure during mitosis and interphase.

The supragenic loop organization of the Drosophila genome was investigated on a 800 kilobase (kb) DNA continuum from the 14B-15B first chromosome region. Nuclear scaffolds from 0-18 hr embryos were prepared with Laemmli's low-salt, detergent procedure and digested with restriction enzymes. Scaffold-associated regions (SARs) were mapped by probing Southern transfers of total, scaffold-associated and free DNA with a set of 70 recombinant phages overlapping the investigated genomic region. In all, 85 restriction fragments showed association to scaffolds. 12 of them were present in the majority of scaffolds. They bore strong SARs organizing the DNA molecule as consecutive loops with sizes ranging from 15 to 115 kb. 44 were present in only a fraction of scaffolds. They contained weak SARs subdividing the basic loops into smaller ones. 29 additional restriction fragments were present in a very small fraction of scaffolds. The position of SARs with respect to transcribed regions was investigated. Strong SARs appeared to be located on untranscribed DNA and to frame transcription units. In contrast, at least some weak SARs were shown to comap with transcribed regions or to reside within characterized transcription units. Statistical analyses established that strong and weak SARs were periodically positioned on the DNA continuum and that there was a potential contact point between scaffolds and the DNA continuum every 11 kb, or multiples thereof. Implications for SAR role(s) are discussed.