L. Selleri et al., HIGH-RESOLUTION PHYSICAL MAPPING OF A 250-KB REGION OF HUMAN-CHROMOSOME 11Q24 BY GENOMIC SEQUENCE SAMPLING (GSS), Genomics, 26(3), 1995, pp. 489-501
A physical map of the region of human chromosome 11q24 containing the
FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma an
d primitive neuroectodermal tumors, was analyzed by genomic sequence s
ampling. Using a 4- to 5-fold coverage chromosome 11-specific library,
22 region-specific cosmid clones were identified by phenol emulsion r
eassociation hybridization, with a 245-kb yeast artificial chromosome
clone containing the FLI1 gene, and by directed ''walking'' techniques
. Cosmid contigs were constructed by individual clone fingerprinting u
sing restriction enzyme digestion and assembly with the Genome Reconst
ruction and AsseMbly (GRAM) computer algorithm. The relative orientati
on and spacing of cosmid contigs with respect to the chromosome was de
termined by the structural analysis of cosmid clones and by direct vis
ual in situ hybridization mapping. Each cosmid clone in the contig was
subjected to ''one-pass'' end sequencing, and the resulting ordered s
equence fragments represent similar to 5% of the complete DNA sequence
, making the entire region accessible by PCR amplification. The sequen
ce samples were analyzed for putative exons, repetitive DNAs, and simp
le sequence repeats using a variety of computer algorithms. Based upon
the computer predictions, Southern and Northern blot experiments led
to the independent identification and localization of the FLI1 gene as
well as a previously unknown gene located in this region of chromosom
e 11q24. This approach to high-resolution physical analysis of human c
hromosomes allows the assembly of detailed sequence-based maps and pro
vides a tool for further structural and functional analysis of the gen
ome. (C) 1995 Academic Press, Inc.