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 amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate 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 remained inconclusive, dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, we report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a "transcript selection" strategy, we cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, we constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp.(ABSTRACT TRUNCATED AT 250 WORDS)
Sen
S
S
Division of Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Sen
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P
Mulac-Jericevic
B
B
Zhou
H
H
Pirrotta
V
V
Stass
S A
SA
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics*; D005111:Extrachromosomal Inheritance; D005784:Gene Amplification; D005786:Gene Expression Regulation; D015973:Gene Expression Regulation, Leukemic; D016259:Genes, myc; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000473:pathology; D008214:Lymphocytes / Q000378:metabolism; D008846:Micromanipulation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm