A tandemly repeated DNA sequence organized predominantly, if not entirely, in a specific manner on the human X chromosome has been cloned in pBR322 and characterized. The sequence was detected as a 2-kilobase band in ethidium bromidestained agarose gels ofBamHI-digested total human nuclear DNA. Although in situ hybridization of the cloned sequence to human metaphase chromosomes showed a single major site of hybridization at the centromere region ofthe X chromosome and minor sites of hybridization at several autosomal centromeres, Southern blot analysis of restricted total human DNA indicated that the cloned probe is related to other repeated DNAs, particularly the human alphoid DNAs. Restriction enzyme analysis ofthe cloned fragment revealed an internal repeat structure based upon multiples of 170 base pairs, confirming this relatedness. All available data, however, suggest that the 2-kilobase spacing ofBamHI sites within the repeat may be specific to the X chromosome.The human X chromosome is a particularly interesting system for the molecular investigation of mammalian gene structure expression. A large number ofmedically significant genetic loci have been mapped to it; and one X chromosome in female eutherian mammals exhibits the phenomenon of inactivation, apparently to accomplish dosage compensation of X-linked gene functions (1). Thus, with appropriate DNA probes, this system provides the opportunity to analyze molecular properties ofthe same gene in the active as well as the inactive state in the same cell.Recent advances in DNA technology in conjunction with modem genetic methods have permitted the identification or isolation of X-chromosome specific human DNA sequences. Beauchamp et al. (2) used Southern blot analysis to identify a 2-kilobase-pair (kb) fragment generated by BamHI digestion of rodent-human somatic cell hybrid DNA present in hybrids containing the human X chromosome and which bears homology to human satellite III DNA. Recently, several single-copy sequences from mammalian X chromosomes have been cloned. These include cDNA of the human glucose-6-phosphate dehydrogenase gene (3), a partial cDNA clone of the mouse hypoxanthine phosphoribosyltransferase gene (4), an unidentified human single-copy DNA sequence (5), and two A libraries constructed from flow-sorted chromosomes enriched in human X (6, 7). To date, however, no cloned repetitive sequences that are located predominantly or entirely on the human X chromosome have been reported.We report the cloning and characterization of a 2-kb fragment of human DNA which exists in tandem arrays and is concentrated at or near the centromere region ofthe human X chromosome. The spacing of BamHI sites argues that it is homologous to the sequence noted by Beauchamp et al. (2). Restriction enzyme analysis of the DNA provides data to support the hypothesis that the organization of this sequence is predominantly, if not entirely, specific to the X chromosome. In addition, the DNA displays homology to the human EcoRI dimer and similarities to othe...

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A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. This bifunctional plasmid (pJRSL100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418-resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2-acetylaminofluorene (2-AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b-4, exhibited an increased cytotoxic response to 2-AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.

A (G + C)-rich density satellite DNA (rho = 1.713 gm/cc) has been purified from splenic DNA of Przewalski's horse, Equus przewalskii, by successive equilibrium density gradient centrifugations. The purified satellite, which may comprise as much as 29% of the total DNA, renatures rapidly; however, analyses of native, single-stranded, and reassociated molecules by analytical ultracentrifugation and melting properties suggest that some sequence heterogeniety exists in the 1.713 gm/cc satellite. Complementary RNA (cRNA) transcribed from satellite DNA has been utilized for in situ hybridization studies with E. przewalskii metaphase chromosomes previously identified by quinacrine-banding. These studies establish that sequences complementary to the 1.713 g/cc satellite are greatly enriched in the centromeres of some, but not all, chromosomes. The differential distribution of satellite DNA sequences over heterochromatic regions allows discrimination of three classes of heterochromatin and serves to define three types of pericentromeric regions in the karyotype of this endangered equine species. Additionally, apparent polymorphism in concentrations of satellite DNA sequences between homologs in the same karyotype is noted.