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Ultrastructural features of minute chromosomes in a methotrexate-resistant mouse 3T3 cell line
Abstract:
The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 X 10(3) kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, suggesting that they are topologically interlocked. They are often closely apposed to chromosomal telomeres or arms, a configuration that may be involved in their distribution at mitosis. In addition to typical minutes, which do not possess kinetochores, a small marker chromosome possessing all of the features of a centromere region is present in parental and resistant cells. An unusual feature of this cell line is the retention of resistance, minute chromosomes, and amplified dihydrofolate reductase genes; most methotrexate-resistant mouse cell lines with minute chromosomes lose these properties when grown in the absence of methotrexate.
Insights
Minute chromosomes in methotrexate-resistant 3T3 cells, though small, possess typical chromatin organization. These minutes, along with amplified dihydrofolate reductase genes, are stably retained, unlike in other cell lines.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Methotrexate-resistant 3T3 cells harbor amplified dihydrofolate reductase (DHFR) genes and minute chromosomes.
- Minute chromosomes are often lost in resistant cell lines when selection pressure is removed.
- Standard light microscopy may not detect the smallest minute chromosomes.
Purpose of the Study:
- To characterize the structure and organization of minute chromosomes in methotrexate-resistant 3T3 cells.
- To investigate the stability of minute chromosomes and amplified DHFR genes in these cells.
- To explore the potential role of minute chromosome configuration in mitotic distribution.
Main Methods:
- Miller spreading procedure applied to mouse metaphase spreads.
- Analysis of minute chromosome size, number, and chromatin organization using microscopy.
- Comparison of minute chromosome characteristics in parental and resistant cell lines.
Main Results:
- Minute chromosomes exhibit typical nucleosomal chromatin organization and higher-order fiber structures, similar to normal chromosomes.
- No evidence of linear DNA was found within the minute chromosomes.
- Minutes are often paired and connected by chromatin fibers, suggesting topological interlocking.
- A small marker chromosome with centromeric features is present.
- The cell line uniquely retains resistance, minute chromosomes, and amplified DHFR genes without methotrexate selection.
Conclusions:
- Minute chromosomes in this cell line possess a complex, non-linear chromatin structure.
- The stable co-retention of minute chromosomes, amplified DHFR genes, and drug resistance is an unusual characteristic.
- The observed configuration of minute chromosomes may influence their segregation during mitosis.