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DNA content and structure of (double) minutes of a methotrexate-resistant cell line
A P Jongsma1, W A Duijndam, P Borst
1Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Abstract:
We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.
Insights
Single minutes (SMs) contain one 2.5 Mb DNA circle, while double minutes (DMs) contain two. This study quantifies DNA content in these amplified DNA structures in methotrexate-resistant cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Double minutes (DMs) and single minutes (SMs) are extrachromosomal DNA elements associated with gene amplification.
- Understanding the DNA content of DMs and SMs is crucial for studying drug resistance mechanisms.
Purpose of the Study:
- To accurately determine the DNA content of individual intact double minutes (DMs) and single minutes (SMs).
- To elucidate the molecular composition of DMs and SMs in the methotrexate-resistant mouse cell line 3T6R50.
Main Methods:
- Fluorometric quantification of DNA in individual chromatin bodies within metaphase spreads.
- Feulgen-Schiff pararosaniline staining for DNA visualization and measurement.
- Utilizing *E. coli* DNA content as a reference standard (4.7 Mb).
Main Results:
- Intact DMs in 3T6R50 cells contain approximately 4.4 megabase pairs (Mb) of DNA.
- Intact SMs in 3T6R50 cells contain approximately 2.6 Mb of DNA.
- Findings suggest SMs consist of one circular DNA molecule and DMs consist of two.
Conclusions:
- A single minute (SM) comprises a single circular double-stranded DNA molecule of about 2.5 Mb.
- Double minutes (DMs) are composed of two such circular DNA molecules.
- These findings provide quantitative insights into the structure of amplified DNA elements in drug-resistant cells.