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4 (pH 7.5) and 7% SDS; washing was at 65°C in 1 x saline sodium citrate (SSC) and 0.1% SDS. Eight hundred thirty-two hybridizations yielded interpretable results. Many sequences appeared to contain highly repeated elements common to males and females or failed to detect an unambiguously Y-specific restriction fragment, and these were not pursued. (This eliminated sequences derived from the pseudoautosomal regions or other regions of extremely high X-Y nucleotide similarity.) By contrast, 308 sequences hybridized to at least one prominent fragment that was present in 49,XYYYY but absent in 46,XX, which suggests that these sequences derived from the NRY. Each of these 308 sequences was individually used to screen, by hybridization, about 2 million plaques from a λ phage library of human adult testis cDNA (Clontech).
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Genes were localized on a previously reported NRY deletion map by polymerase chain reaction (PCR) testing of individuals carrying partial Y chromosomes (10). Most genes were localized to a single deletion interval. Some genes could not be unambiguously placed, because copies exist in multiple locations in the NRY. In such cases, genes were localized by PCR testing of YACs encompassing the euchromatic region (11). Homologs of DBY, TB4Y, EIF1AY, and UTY were mapped to the human X chromosome by PCR testing of a panel of human/rodent somatic hybrid cell lines (Research Genetics, Huntsville, AL). PCR conditions and primer sequences have been deposited at GenBank, where accession numbers are as follows: DBY, G34990; TB4Y, G34981; EIF1AY, G34991; UTY, G34977; DFFRY, G34983; CDY, G34975; BPY1, G34985; BPY2, G34986; XKRY, G34987; PRY, G34984; TTY1, G34978; TTY2, G34980; DBX, G34988; TB4X, G34979; EIF1AX, G34989; UTX, G34976; and DFFRX, G34982. TB4X primers were designed from an unreported intron; all other PCR primers were chosen from cDNA sequences.
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For each of the previously untested X-linked genes DBX, TB4X, and EIF1AX, we assayed X inactivation status by two methods: (i) reverse transcription (RT)-PCR on human-rodent hybrids retaining inactive human X chromosomes and (ii) CpG methylation studies in which male and female genomic DNAs digested with methylation-sensitive restriction endonucleases were used as templates for PCR [S.-W. Luoh et al., Genomics 29, 353 (1995)]. The previously untested gene UTX was assayed by the first method only. As judged by these assays, each of the four genes escapes X inactivation (B. Lahn and D. C. Page, unpublished results).
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note
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We thank H. Skaletsky and F. Lewitter for help with sequence analysis; Lawrence Livermore National Laboratory for the flow-sorted Y cosmid library; and P. Bain, A. Bortvin, A. de la Chapelle, G. Fink, K. Jegalian, T. Kawaguchi, E. Lander, H. Lodish, p. Matsudaira, D. Menke, U. RajBhandary, R. Reijo, S. Rozen, A. Schwartz, C. Sun, and C. Tilford for comments on the manuscript. Supported by NIH.
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