General method for cloning amplified DNA by differential screening with genomic probes

Journal Article · Sat May 01 04:00:00 EDT 1982 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.2.5.578· OSTI ID:5877678

Brison, O; Ardeshir, F; Stark, G R

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. 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.

• Research Organization: Dept. of Biochemistry, Stanford Univ. School of Medicine, Stanford, CA 94305
• OSTI ID: 5877678
• Journal Information: Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 2:5; ISSN MCEBD
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
ADENYLIC ACID
AMINO ACIDS
ANIMALS
ANNEALING
ASPARTIC ACID
BIOLOGICAL FUNCTIONS
BIOSYNTHESIS
BIOTECHNOLOGY
CARBAMATES
CARBONIC ACID DERIVATIVES
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CELL CULTURES
CELL TRANSFORMATIONS
CHROMOSOMAL ABERRATIONS
CLONING
DNA
DNA SEQUENCING
DNA-CLONING
DRUGS
ENZYME INHIBITORS
ENZYMES
FUNCTIONS
GENE AMPLIFICATION
HAMSTERS
HEAT TREATMENTS
HYBRIDIZATION
IMMOBILIZED CELLS
MAMMALS
MUTANTS
MUTATIONS
NUCLEIC ACIDS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
RECOMBINANT DNA
RIBOSOMAL RNA
RNA
RODENTS
SCREENING
SENSITIVITY
STRUCTURAL CHEMICAL ANALYSIS
SYNTHESIS
TRANSFERASES
VERTEBRATES