ABSTRACT
Sporadic childhood tumors associated with Beckwith-Wiedemann syndrome (BWS) all show abnormalities of the same region on chromosome 11. In addition to chromosome 11, other chromosome regions are affected in some of these tumor types. In this study we analyzed the region on chromosome 1p involved in the etiology of BWS-associated tumors, Wilms tumor, rhabdomyosarcoma, and hepatoblastoma. For this purpose we determined the location of two novel translocation breakpoints in this chromosome region in cells from a Wilms tumor and cells from a rhabdomyosarcoma. We constructed a map of the region and found that both breakpoints are separated by at least 875 kb. We identified a PAC clone which crosses the rhabdomyosarcoma breakpoint and found several exons within this clone. We established that this breakpoint is located proximal to the PAX7 gene and, therefore, identified a new region involved in the etiology of rhabdomyosarcomas.
Subject(s)
Chromosome Breakage/genetics , Chromosomes, Human, Pair 1/genetics , Hepatoblastoma/genetics , Homeodomain Proteins , Rhabdomyosarcoma/genetics , Translocation, Genetic/genetics , Wilms Tumor/genetics , Beckwith-Wiedemann Syndrome/complications , Child , Chromosome Walking , Chromosomes, Artificial, Yeast/genetics , Cloning, Molecular , Contig Mapping , Electrophoresis, Gel, Pulsed-Field , Exons/genetics , Genetic Linkage/genetics , Hepatoblastoma/complications , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Muscle Proteins/genetics , Nerve Tissue Proteins/genetics , PAX7 Transcription Factor , Receptors, Cannabinoid , Receptors, Drug/genetics , Rhabdomyosarcoma/complications , Tumor Cells, Cultured , Wilms Tumor/complicationsABSTRACT
We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin.
