Rapid detection, cloning and molecular cytogenetic characterisation of sequences from an MRP-encoding amplicon by chromosome microdissection.
Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109.
M E
ME
Ray
X Y
XY
Guan
M L
ML
Slovak
J M
JM
Trent
P S
PS
Meltzer
D001483:Base Sequence; D015139:Blotting, Southern; D018288:Carcinoma, Small Cell / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods; D002879:Chromosomes, Human, Pair 10; D002885:Chromosomes, Human, Pair 16; D002889:Chromosomes, Human, Pair 2; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics*; D005354:Fibrosarcoma / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008175:Lung Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D017422:Sequence Analysis, DNA / Q000379:methods; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D004317:Doxorubicin