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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mutant cell lines resistant to N-(phosphonacetyl)-L-aspartate exhibit amplified genes for aspartate transcarbamylase (CAD).
• The amplified DNA region is significantly larger than the CAD gene itself, suggesting complex amplification events.

Purpose of the Study:

• To develop a method for isolating and cloning DNA fragments from amplified regions in mutant cell lines.
• To investigate the structure and gene content of amplified DNA associated with drug resistance.

Main Methods:

• Differential screening using genomic DNA probes enriched for amplified sequences.
• Hydroxyapatite chromatography to isolate double-stranded DNA fragments.
• Nick translation and hybridization to remove repetitive sequences.
• Screening of a recombinant phage library with specific probes.

Main Results:

• Successfully isolated nine independent DNA fragments totaling approximately 165 kilobases of amplified DNA, including the CAD gene.
• Identified an overexpressed gene for a 3.8-kilobase RNA within the amplified region.
• Demonstrated the method's general applicability for cloning DNA amplified 10-fold or more.

Conclusions:

• The developed method enables the cloning of amplified DNA, facilitating the study of gene amplification.
• This technique can be broadly applied to investigate gene amplification in drug resistance, transformation, and differentiation.
• The cloned fragments provide tools for studying the structure, amplification mechanism, and gene expression of amplified DNA regions.