Fluorescence activated sorting of chromosomes from 49,XXXXY human lymphoblasts has been used to obtain DNA enriched for the human X. This DNA was cloned in lambda phage Charon 21A to obtain a library of approximately 60,000 pfu. Phage inserts free of human highly repeated DNA sequences are localized to different regions of the human X by two independent hybridization analyses. The first utilized comparative hybridization to rodent-human hybrid cell DNA samples containing all or known portions of the human X, while the second was based on hybridization dosage to DNA samples from human cell lines differing in the number of X chromosomes or X chromosome segments. Of five unique sequence inserts tested, three were X chromosome specific and were localized to regions Xpter leads to Xcen, Xql leads to Xq22 and Xq24 leads to Xqter, respectively. The library presented here represents a highly enriched source of human X chromosome-specific DNA sequences.

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X chromosome, by dosage analysis, and by in situ hybridization to mouse chromosomes. In mouse strain C57BL/10BK, one fragment appeared to be located only on the X chromosome, while the other fragment had homologous sequences on chromosome 11 in addition to the X chromosome. The latter fragment showed DNA variants between mouse strains, which are potentially useful for mapping. Both fragments cross-hybridized to another mouse species: Mus caroli. In this species, each fragment appeared to be located on the X chromosome, indicating that some X-chromosome repetitive sequences are partially conserved. In addition, one fragment cross-hybridized to human DNA.

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short arm of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.INTRODUCTION:

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.

A number of DNA-binding dyes, with spectral properties making them suitable as components of energy donor-acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity of the donor (e.g,, 33258 Hoechst, quinacrine or chromomycin A3), contrast in donor fluorescence can be enhanced, leading to patterns selectively highlighting standard or reverse chromosome bands or particular polymorphic regions. Such results presumably reflect chromosomal regions enriched in 10-20 base pair clusters to which the donor binds and fluoresces but to which the acceptor cannot bind. For other pairs, involving counterstains such as netropsin or echinomycin, which are not suitable as energy acceptors, specific changes observed in polymorphic region fluorescence are most likely due to binding competition between dyes. Dye pairs producing contrast by either method can be used to differentiate between homologous chromosomes or to facilitate detection of specific chromosomal rearrangements. Preliminary data indicate that contrast enhancement generated in fixed metaphase chromosomes spread on microscopic slides can also be observed in suspensions of unfixed metaphase chromosomes, reinforcing the expectation that the methodology described will be of use in flow cytometry.Key terms: Energy transfer, fluorescence, binding competition, chromosome banding patterns, polymorphisms, rearrangements, flow cytometry.Staining metaphase chromosomes simultaneously with pairs of different dyes can lead t o new fluorescence patterns. In addition to the signals produced when the dyes are used independently, information can be generated by interactions between these dyes. For example, two dyes can compete for binding to certain chromosomal sites, which can thereby be stained differentially (15,41) Alternatively, indirect effects of one dye on another can be mediated via conformational changes induced in DNA (46). In addition, because of a coincidence between the range of separations possible between ' Supported by Grant GM21121 from the National Institutes of Health. S.A.L. is the recipient of a Research Career Development Award GM00122 from the National Institute of General Medical Sciences and E.S. is supported in part by a grant from the Whitaker Foundation.* Presented in part at Automated Cytology VII, Asilomar, California, November 25-30, 1979. dye molecules bound to DNA (20) and the effective distance over which dye electronic transition dipoles can interact (typically 5 50 A) (9, 42), the transfer of electronic excitation energy from one dye to another in doubly stained chromatin can be appreciable (14,16,17,36).We have recently shown that the enhanced contrast in metaphase chromosome staining produced by certain dye pairs is due largely to energy transfer, while that produced by other pairs, which fail to satisfy spectral overlap criteria, is due primarily to binding competition (37)....