General Method for Cloning Amplified DNA by Differential Screening with Genomic Probes

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1982 May 1

PMID 6896736

Citations 18

Authors

O Brison

F Ardeshir

G R Stark

Affiliations

Soon will be listed here.

Abstract

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite. This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.

Citing Articles

Localization of amplified CAD genes on rearranged chromosomes of Chinese hamster cells.

Ottaggio L, Agnese C, Bonatti S, Cavolina P, Di Leonardo A, Miele M Cytotechnology. 2012; 1(1):25-31.

PMID: 22358436 DOI: 10.1007/BF00351118.

Identification and characterization of transcribed sequences on human chromosome 9q32-34.

Falk J, Usui H, Sutcliffe J J Mol Neurosci. 1994; 5(3):165-79.

PMID: 7654519 DOI: 10.1007/BF02736731.

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Zieg J, Clayton C, Ardeshir F, Giulotto E, SWYRYD E, Stark G Mol Cell Biol. 1983; 3(11):2089-98.

PMID: 6656764 PMC: 370075. DOI: 10.1128/mcb.3.11.2089-2098.1983.

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Ardeshir F, Giulotto E, Zieg J, Brison O, Liao W, Stark G Mol Cell Biol. 1983; 3(11):2076-88.

PMID: 6656763 PMC: 370074. DOI: 10.1128/mcb.3.11.2076-2088.1983.

Specific DNA sequence amplification in human neuroblastoma cells.

Montgomery K, Biedler J, Spengler B, Melera P Proc Natl Acad Sci U S A. 1983; 80(18):5724-8.

PMID: 6577451 PMC: 384331. DOI: 10.1073/pnas.80.18.5724.

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