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High-frequency deletion event at aprt locus of CHO cells: detection and characterization of endpoints
1Institut du Cancer de Montréal, Québec, Canada.
Abstract:
Two mechanisms are implicated in generating recessive drug resistance mutants at the adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary (CHO) cells, one of which is a spontaneous high-frequency deletion of the entire gene. We have isolated and mapped a 19-kb fragment carrying aprt and its flanking sequences. A Southern blot study of 198 independent deletion mutants revealed that two different mutants have one of their breakpoints within the 19-kb region analyzed. One of these has an upstream breakpoint which could be narrowed down to a 4-kb fragment containing repetitive sequences. The other mutant has a breakpoint within a 410-bp sequence located 8.5 kb downstream of the aprt gene and which carries several elements similar to those signaling V-(D)-J joining in immunoglobulin and T-cell receptor gene rearrangements. In each case the other breakpoint lay outside of the analyzed region. These results support the previous indications that the deletions created by this spontaneous event are large.
Insights
Spontaneous deletions generate drug resistance in Chinese hamster ovary (CHO) cells. Researchers mapped breakpoints within a 19-kb fragment containing the adenine phosphoribosyltransferase (aprt) gene, revealing large deletions responsible for resistance.
Area of Science:
- Molecular Genetics
- Cell Biology
- Genomics
Background:
- Recessive drug resistance in Chinese hamster ovary (CHO) cells arises from multiple mechanisms.
- A high-frequency spontaneous deletion of the adenine phosphoribosyltransferase (aprt) gene is a key mechanism.
- Understanding the precise nature and breakpoints of these deletions is crucial for elucidating resistance pathways.
Purpose of the Study:
- To isolate and map a DNA fragment containing the aprt gene and its flanking regions.
- To identify the specific breakpoints of spontaneous deletions in aprt mutants.
- To characterize the sequences at these breakpoints and their potential role in deletion formation.
Main Methods:
- Isolation and mapping of a 19-kb DNA fragment encompassing the aprt gene.
- Southern blot analysis of 198 independent recessive drug-resistant mutants.
- Fine mapping of deletion breakpoints within the analyzed 19-kb region.
Main Results:
- Two distinct deletion mutants exhibited breakpoints within the 19-kb aprt-containing fragment.
- One mutant displayed an upstream breakpoint within a 4-kb region containing repetitive sequences.
- The second mutant showed a downstream breakpoint within a 410-bp sequence with V-(D)-J-like elements, located 8.5 kb from aprt.
Conclusions:
- The spontaneous deletion event creating aprt mutants involves large-scale genomic rearrangements.
- Repetitive sequences and V-(D)-J-like elements may contribute to the initiation or processing of these deletions.
- These findings provide detailed insights into the molecular mechanisms underlying spontaneous gene deletions and drug resistance.