High-resolution physical mapping of a 250-kb region of human chromosome 11q24 by genomic sequence sampling (GSS).
A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling. Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation 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 using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm. The relative orientation and spacing of cosmid contigs with respect to the chromosome was determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping. Each cosmid clone in the contig was subjected to "one-pass" end sequencing, and the resulting ordered sequence fragments represent approximately 5% of the complete DNA sequence, making the entire region accessible by PCR amplification. The sequence samples were analyzed for putative exons, repetitive DNAs, and simple 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 chromosome 11q24. This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps and provides a tool for further structural and functional analysis of the genome.
Selleri
L
L
Molecular Genetics Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Smith
M W
MW
Holmsen
A L
AL
Romo
A J
AJ
Thomas
S D
SD
Paternotte
C
C
Romberg
L C
LC
Wei
Y H
YH
Evans
G A
GA
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D002880:Chromosomes, Human, Pair 11; D003360:Cosmids; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides