The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. We have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. A series of five single-copy genomic probes from the 70-kilobase first intron of BCR were used to localize rearrangements in 8 of 10 Philadelphia chromosomepositive ALLs. We have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosomepositive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination.Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy afflicting 1 in 30,000 children annually (1). Though therapeutic advances have resulted in long-term disease-free survival rates of 60% (2), those tumors that contain a karyotypic abnormality have a significantly poorer prognosis (3). This would suggest that neoplastic mechanisms may be at work as a consequence of chromosomal rearrangement that make these tumors more resistant to treatment. The most common karyotypic abnormality seen in ALL is the Philadelphia chromosome (Ph). It is this same chromosomal translocation that is characteristically linked to >90% of patients with chronic myelogenous leukemia (CML) (4). In ALL, however, the Ph is less frequently observed and is present in =10% of patients (5). Karyotypic analysis has revealed that this marker chromosome is a result of a reciprocal translocation between chromosome 9 band q34 and chromosome 22 band qll.2: t(9;22)(q34;qll.2) (6).Though these rearrangements in ALL and CML are karyotypically indistinguishable, their molecular structures differ. In both instances this translocation results in the juxtaposition of upstream regions of the BCR gene located on chromosome 22 to a distal portion of the ABL protooncogene on chromosome 9 (7-10). This results in the formation of both hybrid BCR/ABL transcripts and proteins (11,12). In CML, this rearrangement has been mapped to within a 5-to 6-kilobase pair (kb) BCR (23,24). Breakpoint locations of the majority of Ph' ALL have eluded molecular definition. Since only portions of the large first intron have been isolated, it has been postulated that these mol...
The study of tumor-specific chromosomal abnormalities has been severely impeded by an inability to link cytogenetic to molecular data. Restriction fragment length polymorphism mapping of any particular chromosomal rearrangement to the resolution limit of genetic methodology generates sets of probes that frequently are still too widely spaced to render the rearrangement breakpoints accessible to molecular isolation. (7). The localization of genomic sequences on such large contiguous fragments of the human genome has made it possible to construct long-range physical maps over large chromosomal domains (8, 9). Despite the broad utility of such a reagent, the construction of only three complete human genomic YAC libraries has been described (4-6). All these libraries used karyotypically normal human cell lines making them unsuitable for the direct isolation of specific chromosomal rearrangements. We present a feasible streamlined approach for generating individual YAC libraries that contain tumor-specific chromosomal abnormalities. Recent advances in YAC vector construction as well as genomic insert DNA preparation have been applied to create a three-haploid-genomic-equivalent human YAC library from a neuroepithelioma cell line containing a characteristic t(11;22) chromosomal translocation. In contrast to previous YAC library constructions all manipulations of very high molecular weight DNAs were performed in agarose. Modification of the conditions for yeast spheroplast preparation has enhanced transformation efficiency. Clones were grown in 96-well microtiter dishes and screened with two single-copy genomic probes that bracket the t(11;22) translocation breakpoint. Each genomic probe yielded at least one positive YAC clone ranging in size from 370 kb to 550 kb.Definitive analysis of tumor-specific chromosomal abnormalities has resulted in the discovery of important genes and events involved in malignant transformation (for reviews, see refs. 1 and 2). The molecular isolation of the genetic loci involved in such rearrangements has been hampered by the size and complexity of the human genome. Conventional cloning techniques using phage or cosmid vectors have been limited to fragments less than 50 kilobases (kb). On the other hand chromosome mapping by either karyotypic banding or restriction fragment length polymorphism recombination analysis has a resolution limit on the order of millions of base pairs. This physical disparity poses an obstacle for linking mammalian genetic and molecular data. As a result the complete characterization of tumor-specific chromosomal abnormalities has been limited to those rearrangements that involve a genomic locus that has fortuitously been previously isolated.The strategy of cloning very large genomic fragments in yeast holds considerable promise of closing the gap between genetic and molecular analyses (3). A few laboratories have reported the construction of recombinant human genomic libraries as yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae (4-6). In th...
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