DOE Human Genome Program Contractor-Grantee
54. Sequence-Ready Characterization of the Pericentromeric Region of 19p12: A Strategy for the Analysis of Complex Regions of the Human Genome
Evan E. Eichler1, Anthony P. Popkie1, Laurie A. Gordon2, and Anne S. Olsen2
1Case Western Reserve University, Cleveland, OH, 44106 and 2DOE Joint Genome Institute, Walnut Creek, CA 94598
The pericentromeric region of 19p12 represents one of the most poorly mapped and sequenced regions of chromosome 19. This is due, in large part, to the virtual absence of unique sequence identifiers within this region. The proximal portion of 19p12 possesses sequence attributes consistent with both euchromatic and heterochromatic DNA including a large cluster of ZNF (zinc-finger) genes, an overabundance of human endogenous retroviral elements and an atypical higher-order (~10-30 kb) beta-satellite repeat structure. Analysis of ~425 kb of seed sequence from 19p12 has revealed that less than 15% of the region consists of bonafide unique sequence. This unusual organization has hampered the development of sets of large contiguous clones in this region, resulted in relatively poor clonal coverage (<60%) and has greatly limited the selection of suitable templates for sequencing. To complete mapping and sequencing in this region, we have designed a strategy that takes advantage of the known biological properties of 19p12 repetitive sequences. Our approach has been to distinguish between "generic" and 19p12-specific repeat elements; develop assays to rapidly identify 19p12 clones from different genomic libraries and to confirm the position of these clones at the level of sequence-overlap. Both high-resolution FISH techniques (extended chromatin analysis) and restriction fragment overlap are being implemented as complementary tools to confirm the integrity of the map and to identify potential sites of heteromorphism in the region. To date, a total of 403 19p12 BAC clones have been identified from RPCI-11 and CIT-D libraries. A subset of these have been used to extend clonal coverage into 12 different gap regions of 19p12; four of which are now tentatively closed. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence characterization of other difficult regions of the human genome. The complex organization of this region will be discussed in the context of its unusual biology.
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