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Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: Facilitation by fluorescence-activated flow sorting of metaphase chromosomes

Kanda, N. and Schreck, R. and Alt, F. and Bruns, G. and Baltimore, D. and Latt, S. (1983) Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: Facilitation by fluorescence-activated flow sorting of metaphase chromosomes. Proceedings of the National Academy of Sciences of the United States of America, 80 (13). pp. 4069-4073. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:KANpnas83

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Abstract

Human neuroblastoma IMR-32 cells have large homogeneously staining regions (HSRs), primarily in the short arms of chromosome 1. We have constructed a recombinant phage library that is enriched for DNA present in the HSR of this chromosome by using fluorescence-activated flow sorting for initial chromosome purification. Eleven distinct cloned DNA segments were identified that showed significantly greater hybridization to IMR-32 genomic DNA, detected by Southern blotting, than to normal human genomic DNA. These sequences have also been localized to the HSR of chromosome 1 by in situ hybridization. Based on an approximate 50-fold sequence amplification for each cloned segment and a total HSR size of 150,000 kilobases, the amplified unit in the HSR is estimated to be 3,000 kilobases. Sequences homologous to all cloned HSR DNA segments were mapped to human chromosome 2 by using human-mouse hybrid cells. Further work using in situ hybridization demonstrated that cloned HSR segments were localized in the short arm of chromosome 2 in both normal and IMR-32 cells. Thus, the amplification of these sequences in IMR-32 cells may have involved transposition from chromosome 2 to chromosome 1.


Item Type:Article
Additional Information:© 1983 by the National Academy of Sciences. Contributed by David Baltimore, April 1, 1983. We thank Ms. Nancy Kohl and associates for providing unpublished data about DNA sequence amplification in neuroblastoma cells lines other than IMR-32. We also thank Dr. L. Kunkel for technical advice for analysis of cloned DNA segments, Drs. U. Tantravahi, C. Morton, and G. Wahl for advice about in situ hybridization, and Ms. Elise Thomas for technical assistance. This research was supported with funds from the American Cancer Society (CD-36) and the National Institutes of Health [HD-04807, CA-26717, and CA-14501 (core grant to S.E. Luria)]. N.K. is a Fogarty International Fellow, F.A. was a Special Fellow of the Leukemia Society, and D.B. is an American Cancer Society Research Professor. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:recombinant phage library; gene mapping; amplification and rearrangement
Record Number:CaltechAUTHORS:KANpnas83
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:KANpnas83
Alternative URL:http://dx.doi.org/10.1073/pnas.80.13.4069
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7631
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Mar 2007
Last Modified:26 Dec 2012 09:33

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