Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes.
A collection of 2,000 lambda phage-carrying human single-copy inserts (greater than 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two human-hamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs; 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100-150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.
Laboratory of Immunobiology, National Cancer Institute-Frederick Cancer Research Facility, MD 21701.
M I
MI
Lerman
F
F
Latif
G M
GM
Glenn
L N
LN
Daniel
H
H
Brauch
S
S
Hosoe
K
K
Hampsch
J
J
Delisio
M L
ML
Orcutt
O W
OW
McBride
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D005260:Female; D015723:Gene Library; D016147:Genes, Tumor Suppressor; D006801:Humans; D006822:Hybrid Cells; D008297:Male; D010375:Pedigree; D012150:Polymorphism, Restriction Fragment Length; D014158:Transcription, Genetic; D006623:von Hippel-Lindau Disease / Q000235:genetics
D015342:DNA Probes; D004247:DNA