The polytene and mitotic chromosomes of D. melanogaster, D. simulans, D. ananassae, and D. virilis were stained with the fluorescent dye, quinacrine mustard (QM). In all these species except D. ananassae, we have detected speciesspecific chromosomal loci which exhibit an extremely brilliant fluorescence. Most, but not all, of the brilliantly fluorescing areas are located in heterochromatic chromosome regions. Cytochemical and chemical methods have been employed to demonstrate that the brilliant fluorescence represents regions of acid labile non-covalent binding between DNA and QM whereas the moderate overall fluorescence is primarily due to covalent bonding (by alkylation) of the QM to DNA. The exact mode of binding of QM in the brilliant areas and the nature of the DNA in these areas are not known. The possible biological significance of the DNA in the brilliant regions is discussed.

DNA isolated from Drosophila melanogaster adults (XX, XY, XXY, XYY) was used in DNA-DNA hybridization experiments on nitrocellulose filters. Filter-bound DNA of "high-heterochromatin" flies (those with one or more Y chromosomes) is more effective in forming hybrid duplexes than is XX DNA. The quantitative difference in hybridization efficiency is due primarily to molecules with a relatively high thermal stability (duplexes that dissociate in the temperature range 8090°). Hybridization experiments with DNA samples that have been fractionated into reiterated and unique portions show that the majority of the hybrid duplexes formed involve reiterated DNA. A small, but highly specific, interaction of unique DNA sequences has also been detected in our experiments. These data indicate that a class of repeated DNA sequences is associated with the constitutive heterochromatin, specifically with the Y chromosome of D. melanogaster. Evidence is presented that this same class of molecules, or very similar ones, is to be found in other portions of the genome, presumably in X-chromosomal or centromeric heterochromatin.Recent reports (1-4) have demonstrated a marked capacity of the centromeric regions of Drosophila chromosomes to form hybrid molecules with complementary RNA (c-RNA) transcribed from the rapidly reassociating, repetitious portion of the genome. All of these reports have used the technique of in situ hybridization, developed within the past few years by workers in several different laboratories. We wish to report the results of a series of in vitro hybridization experiments that indicate that Drosophila melanogaster DNA isolated from "high-heterochromatin flies" (i.e., those with one or more Y chromosomes) contains more of a class of repeated DNA sequences than does DNA isolated from "low-heterochromatin" XX females. We have found that the repeated DNA sequences that form hybrid duplexes in our experiments can be derived from either XY or XX flies, and are, therefore, not exclusively Y chromosomal in origin. Finally, these experiments also show that it is possible to use DNA-DNA hybridization techniques on nitrocellulose filters to distinguish between DNA sequences isolated from the unique portion of the genome of D. melanogaster females (XX) and males (XY).D. melanogaster is uniquely suited for studies of the properties of heterochromatin and euchromatin. In this organism, as in no other, we have a wealth of knowledge about the cy-

HE chromosomes of eukaryotes are generally subdivided by cytological and Toperational criteria into heterochromatic and euchromatic regions. Originally, the term heterochromatin was used by HEITZ (1928) to distinguish those hetero-* denotes unseparated, approximately equal numbers of XX and XY ** KAUFMANN 1934

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirmes earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.