MICRODISSECTED DOUBLE-MINUTE DNA DETECTS VARIABLE PATTERNS OF CHROMOSOMAL LOCALIZATIONS AND MULTIPLE ABUNDANTLY EXPRESSED TRANSCRIPTS IN NORMAL AND LEUKEMIC-CELLS

Double-minute (dm) chromosomes are cytogenetically resolvable DNA ampl ification-mediating acentric extrachromosomal structures that are comm only seen in primary tumors, tumor cell lines, and drug-resistant cell s grown in vitro. Selective isolation of dm DNAs with standard molecul ar biological techniques is difficult, and thus, detailed studies to e lucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remai ned inconclusive. dms seen in the acute myeloid leukemia cell line HL- 60 have been shown to harbor the c-nye protooncogene. In this paper, w e report the successful isolation of the dm-specific DNAs from these c ells by the microdissection/polymerase chain reaction technique and de monstrate that the dm DNAs derived from a single discrete normal chrom osome segment 8q24.1-q24.2 reintegrate at various specific locations i n the leukemic cells. The microdissected dm DNA detects multiple abund antly expressed transcripts distinct from c-myc mRNA on Northern blots . By devising a ''transcript selection'' strategy, we cloned the parti al genomic sequence of a gene from the microdissected DNA that encodes two of Obese RNAs. This strategy will be generally applicable for rap id cloning of unknown amplified genes harbored on dms. With DNA from 2 0 microdissected dms, we constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp. The microclones should help in isolating corresponding yeast artificia l chromosome clones for high-resolution physical mapping of dms in HL- 60 cells. Furthermore, application of the microdissection technique ap pears to be an extremely feasible approach to characterization of dms in other cell types. (C) 1994 Academic Press, Inc.