HUMAN DISTAL XQ DNA--ANALYSIS WITH YACS

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The long-range objective is to make a yeast artificial chromosome (YAC)-based map of Xq24-q28 human DNA (50 Mb; about 1.5% of the genome). A start has been made with an overlapping YAC/probe map of nearly all of the region, and mapping will now be continued to more refined structural and functional levels. Structural analysis will focus on sequences placed at the ends of chromosomes, including the Xq28 telomere, and the Fragile X and common fragile site in Xq27.2-27.3, which will be analyzed in detail. In addition, the distribution of defined sequence elements win be determined in YACs and YAC contigs across cytogenetic bands. The experiments are designed to test conjectures about the distribution of overall GC content; highly repetitive sequences (Alu, LI); selected loci for the moderately repetitive sequence pTR5; and some simple sequence repeats [including poly(dGdC).(dA-dT), and the (CCG)n motif at the Fragile X site]. Functional analyses will compare several techniques for mapping transcription units in the vicinity of glucose 6-phosphate dehydrogenase (G6PD); in portions of an 8 Mb contig across Xq26; and in the search for a gene in which lesions are responsible for a particular X-linked disease. G6PD-containing YACs will be transfected to determine if, either as such or fitted with appropriate selective markers, they can recombine at homologous sites in the genome. Comparable experiments will test whether fragility in the Fragile X region can be localized purely to the (CCG)n repeat sequence or requires other structural features. Finally, one approach will extend structural and functional studies to an evolutionary perspective. The human G6PD gene has been sequenced. Attempts will now be made to identify important regions of the gene and observe features of its evolution by comparing sequences to corresponding portions amplified by PCR from other primates, and also to the complete sequence of the cloned mouse gene. Conserved areas of putative functional importance will thus be inferred; and finally, YACs fitted with selective markers will be modified in those regions, and the YACs will be transfected to test predictions.