Barbara J. Trask, Hillary Massa, Cynthia Friedman, Lee Rowen, Hiroki Yokota, Ger van den Engh, Heather Christy, Leah LaTray, Shawn Iadonato, David Wong, Forrester Johnson, Carolyn Akinbami, John Blankenship, Eric Green, Mark Keating, Antonia Martin-Gallardo, and David Miller
Dept. of Molecular Biotechnology, University of Washington,
Low-copy repeats can present a challenge to mapping efforts. This challenge is severe when the repeated segments are large (>kbp), very similar, or closely spaced in the genome. This challenge must be met because these sequence duplications can have biological significance as members of gene families, mediators of chromosome rearrangements, indicators of steps in chromosomal evolution, or descriptors of human variation. Here we present analyses of three biologically interesting regions whose maps have been complicated by low-copy repeats.
a) A collaborative effort (BT, EG, and MK laboratories) is underway to physically map the Williams region of 7q11.2, to correlate patient deletions at the molecular level with the phenotypic features, and to identify and characterize the genes contained within the deleted intervals. These studies have demonstrated that the region is associated with duplications of sequences within a single cytogenetic band. Some parts of the region are also duplicated on other chromosomes. The results of a combination of FISH analyses of patient and normal chromosomes and STS-content mapping of YACs will be presented.
b) We have used FISH to map the boundaries of the duplication of part of the T-cell receptor locus (chromosome 7) on chromosome 9. This duplication encompasses a trypsinogen gene.
c) Different challenges are presented by the low-copy duplications identified by cosmid 7501. The 35-kbp segment cloned from chromosome 19 is inserted at the ends of chromosomes 3, 15, and 19 in diverse human populations. Fifteen other chromosomes carry this sequence in at least one of 45 individuals analyzed, but the sequence is restricted to one chromosome in chimps and gorillas. The duplicated segment contains three regions with high homology to olfactory receptor genes. The extent of the duplication and the sequence similarity of the different copies has been analyzed by a combination of physical mapping approaches (of YACs, P1s, cosmids), molecular analyses of different chromosomes purified by flow-sorting, and FISH. Preliminary data suggest that parts of the cosmid may be duplicated within short YAC-sized segments of the same chromosome. These analyses may reveal the rearrangements chromosomes have undergone during evolution and a more complete description of human variation.
d) FISH analyses of straightened DNA fibers offers the possibility of detecting and analyzing very closely spaced duplications. We will report on some of the parameters that are critical to obtaining uniformly and reproducibly straightened DNA fibers for FISH.
*Supported by a grant from the Director, Office of Energy Research, OHER, U. S. Department of Energy under contract DE-FG06-93ER61553 and DE-FG06-93ER61662.
 National Center for Human Genome Research, NIH.
 Dept. Human Genetics, University of Utah
 Centro Nacional de Biotecnologia, Madrid.
 Dept. of Electrical Engineering, Pennsylvania State University.
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