Bromodeoxyuridine 5'-monophosphate incorporation into yeast nuclear and mitochondrial deoxyribonucleic acid
Abstract:
Standard laboratory yeast strains can be enriched for thymidine 5'-monophosphate (TMP) uptake derivatives that generate only a low percentage of respiratory-deficient colonies (petites) under inhibition of TMP biosynthesis. Such mutants incorporated bromodeoxyuridine 5'-monophosphate (BrdUMP) into both nuclear and mitochondrial deoxyribonucleic acid (mtDNA); however, they showed a selectivity for TMP over BrdUMP incorporation. The preferential incorporation of [3H]TMP or BrdUMP into mtDNA was strain dependent. The density increments after growth in the presence of BrdUMP reached 50 mg/ml for nuclear DNA and 22 mg/ml for mtDNA in CsCl gradients. Density shifts corresponding to 4% bromouracil substitution were easily detected. Preliminary density transfer experiments confirm that mtDNA does not replicate in synchrony with nuclear DNA.
Insights
Yeast mutants efficiently incorporate bromodeoxyuridine 5'-monophosphate (BrdUMP) into nuclear and mitochondrial DNA, showing selectivity for thymidine 5'-monophosphate (TMP). Mitochondrial DNA replication is asynchronous with nuclear DNA replication.
Area of Science:
- Molecular biology
- Yeast genetics
- Mitochondrial DNA research
Background:
- Standard yeast strains can be modified to uptake thymidine 5 '-monophosphate (TMP).
- These modified strains exhibit low percentages of respiratory-deficient colonies (petites) when TMP biosynthesis is inhibited.
- This study investigates the incorporation of BrdUMP in these yeast mutants.
Purpose of the Study:
- To analyze the incorporation of bromodeoxyuridine 5 '-monophosphate (BrdUMP) into nuclear and mitochondrial DNA (mtDNA) in yeast mutants.
- To determine the selectivity of these mutants for TMP versus BrdUMP incorporation.
- To investigate the replication patterns of mtDNA relative to nuclear DNA.
Main Methods:
- Enrichment of yeast strains for TMP uptake derivatives.
- Incorporation of BrdUMP and [3H]TMP into DNA.
- Cesium chloride (CsCl) density gradient centrifugation.
- Density transfer experiments.
Main Results:
- Mutants incorporated BrdUMP into both nuclear and mtDNA.
- A selectivity for TMP over BrdUMP incorporation was observed.
- Preferential incorporation of TMP or BrdUMP into mtDNA was strain-dependent.
- Significant density shifts indicating bromouracil substitution were detected in both nuclear and mtDNA.
- mtDNA replication was found to be asynchronous with nuclear DNA replication.
Conclusions:
- Yeast mutants can incorporate BrdUMP into both nuclear and mtDNA, with strain-dependent preferences.
- These findings provide insights into DNA replication and nucleotide metabolism in yeast mitochondria.
- The asynchronous replication of mtDNA relative to nuclear DNA is confirmed.
