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BRIEF PROPOSAL LATE REPLICATION-DEPENDENT DENSITY ENRICHMENT OF MAMMALIAN X CHROMOSOME: AN AID TO ITS PURIFICATION NARENDRA G. MEHTA* Of the mammalian chromosomal complement, the X chromosome is unique in that it occurs in two copies in the female and in only one copy in the male. One of the two X chromosomes in the female, and any supernumerary X's occurring in abnormal conditions, get randomly inactivated during embryonic development. The inactivation represents a special form of en bloc transcriptional regulation, although expression of a few loci is apparendy required for maintenance of fertility (reviewed in [I]). Many inherited disorders occur owing to mutations on the X chromosome. It is therefore not surprising that efforts have been in progress for the purification of this chromosome. Centrifugation methods taking advantage ofthe size ofchromosomes have not yielded significant results [2]. Success (approximately 90 percent purity) has recently been achieved by flow cytometry and sorting [3]. The mediod, however, requires specialized equipment , and the quantities purified are radier small (400—500 ng DNA equivalent [3]). Some success has also been achieved in the isolation of X chromosomal DNA by the removal of mouse-specific sequences from the DNA of mousehuman hybrid cell that contains X as the only human chromosome [4]. By this method a preparation of DNA is obtained that is about 50 percent pure with respect to human X chromosomal DNA. Some fragments of DNA have also been isolated that hybridize with the X chromosome [5, 6]. A characteristic feature of the inactive X chromosome is that its DNA is late replicating [I]. The replication of die inactive X-DNA is not only initiated much later than the rest of DNA but is also the last to be completed. For example, in some human cells in culture, no synthesis of the inactive X-DNA takes place in the first half of the S phase [7], and much of it occurs after the synthesis ofbulk DNA is complete [8]. One often sees autoradiographs in the literature where the X chromosome shows much heavier labeling with 3H-thymidine compared with other chromosomes, thus earning the nickname of"hot X" [9]. It is thus possible in practice to achieve much greater labeling of the DNA of the inactive X chromosome than of other chromosomes. ?Biological Chemistry Division, Cancer Research Institute, Parel, Bombay 400 012, India. Permission to reprint'this brief proposal may be obtained only from the author. 486 I Narendra G. Mehta ¦ BriefProposal I wish to suggest that the property above can be the basis for an inexpensive procedure for large-scale purification of the X chromosome. If during die period of its preferential synthesis, nucleotides containing heavy isotopes of C, N, and H are made available, the incorporated heavy nucleotides would impart a higher buoyant density to its DNA and consequently to the chromosome. Thus if a synchronized population of cells growing on normal medium is shifted to a medium containing nucleotides substituted with 15N, 13C, and 2H late in the S phase, die subsequently replicating DNA of die inactive X chromosome would be labeled heavy. The precise time of the medium shift would be determined by the knowledge of kinetics of the inactive X chromosomal DNA replication in the cells used for isolation, as well as of the kinetics of nucleotide phosphorylation and die levels of intracellular nucleotide pools. Following the completion of metaphase, the harvest ofchromosomes and their separation by density gradient centrifugation should yield pure X chromosomes. Bromodeoxyuridine could also be used in place of the heavy isotope-substituted bases for density labeling. The cell lines possessing supernumerary X's would increase the yield manyfold. Reproducible density labeling of DNA with bromodeoxyuridine at specific times in the S phase has recently been demonstrated [10]. Some investigators (e.g., [11, 12]) have reported that the Y chromosome is also late replicating. The approach above could also be used for the isolation of diis chromosome. REFERENCES 1.Gordon, J. W., and Ruddle, F. H. Mammalian gonadal determination and gametogenesis. Science 211:1265-1271, 1981. 2.SALZMan, N. P., and Mendelsohn, J. Isolation and fractionation of metaphase chromosomes. Methods Cell Physiol. 3:277-292, 1968. 3.Davies, K. E.; Young, B...


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