Techniques for Determining the Physical Structure 

 of Entire Human Chromosomes 



Cassandra L. Smith. W. Michels, J. S. Cheng. H. Fang. J. Gingrich. D. Wang, 

 and Y. Wu 



Human Genome Center. Lawrence Berkeley Laboratory. Berkeley. CA 94720 

 (415) 486-6800. FTS 4.'^ 1-6800 



Large-fragment DNA methods are being used to construct a macro-restriction map of 

 the smallest human chromosome. Isolation of a human telomere yeast artificial 

 chromosome clone enabled the ends of the map to be defined. About 30 single-copy 

 DNA probes with previously assigned genetic map locations along the length of the 

 chromosome are being employed as anchor points. These probes were used to identify 

 corresponding large Not 1 and Mlii I DNA fragments by hybridization to pulsed-field gel 

 fractionated DNA restriction digests. The map between the anchor points is being 

 reconstructed by combining several approaches: assigning other bands by using single- 

 copy probes with known regional locations; assigning neighboring bands using the 15 

 thus-far-isolated chromosome-specific Not I linking probes: and interpolating between 

 anchor points using partial digests phased either by Smith-Bimstiel type approaches or 

 by using sites that are polymorphic, when different cell lines are compared, as signa- 

 tures of particular regions. A series of clones of repeated DN As are being used to 

 identify all the chromosome-2I restriction fragments present in these hybrid rodent cell 

 lines. These approaches have allowed us to identify about 40 Mb that come from 

 chromosome 21 and to link up Not I fragments of at least 8 Mb near the (/ telomere and 

 additional significant regions along the cj arm. Additionally, these strategies have 

 allowed us to determine that D21S13, the locus most closely linked to the Alzheimer's 

 disease gene on chromosome 21, is in fact located on the same 1.6-Mb fragment as 

 locus D21S16. Although the map is not yet complete, it reveals interesting features such 

 as the uneven distribution of putative genes along the chromosome and a greater-than- 

 expected gradient of enhanced recombination near the (/ telomere. 



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