title: Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.
abstract: Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.
authors.affiliation: Section of Hematology/Oncology, University of Chicago, IL 60637.
authors.forename: F V
authors.initials: FV
authors.lastname: Rassool
authors.forename: T W
authors.initials: TW
authors.lastname: McKeithan
authors.forename: M E
authors.initials: ME
authors.lastname: Neilly
authors.forename: E
authors.initials: E
authors.lastname: van Melle
authors.forename: R
authors.initials: R
authors.lastname: Espinosa
authors.forename: M M
authors.initials: MM
authors.lastname: Le Beau
mesh_terms: D000818:Animals; D002871:Chromosome Banding; D043283:Chromosome Fragile Sites; D002873:Chromosome Fragility; D002874:Chromosome Mapping; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D005260:Female; D015321:Gene Rearrangement; D005819:Genetic Markers; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology; D007621:Karyotyping; D009693:Nucleic Acid Hybridization; D010920:Placenta / Q000166:cytology / Q000502:physiology; D011247:Pregnancy; D014162:Transfection
chemical_list: D005819:Genetic Markers; D004247:DNA