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Mutants of yeast specifically resistant to petite induction by fluorinated pyrimidines
Abstract:
Induction of the cytoplasmic petite mutation in yeast by 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) is known to depend on the incorporation of 5FU into some species of RNA; 5FC is active only following deamination to 5FU. Several mutants ahve now been isolated which are resistant to petite mutagenesis by 5FU but remain sensitive to growth inhibition by this analogue. They fall into two classes: those in class I are also resistant to mutagenesis by 5FC, while class II mutants retain partial sensitivity to the latter agent. The growth of both classes is sensitive to 5FC. The behavior of class II mutants requires that exogenous 5FU is specifically excluded from the site of synthesis of the target RNA involved in petite mutagenesis, while 5FC has access to it. The most likely explanation is that the RNA concerned is synthesized in the mitochondria, and that the mitochondrial membranes of class II mutants are impermeable to 5FU but not 5FC. This is supported by the finding that the membrane-active agent dimethylsulfoxide restored 5FU sensitivity to this class of mutants. No such effect was observed with class I mutants, and these are thought to have altered mitochondrial RNA-synthesizing systems which are unable to recognize fluorinated nucleotides.
Insights
Researchers identified yeast mutants resistant to petite mutagenesis by 5-fluorouracil (5FU). Class II mutants show selective resistance, suggesting 5FU is excluded from mitochondrial RNA synthesis sites, unlike 5-fluorocytosine (5FC).
Area of Science:
- Molecular Biology
- Yeast Genetics
- Mitochondrial Biology
Background:
- 5-fluorouracil (5FU) induces cytoplasmic petite mutations in yeast by incorporating into RNA.
- 5-fluorocytosine (5FC) requires deamination to 5FU to be active.
- Mutants resistant to 5FU-induced petite mutagenesis were isolated.
Purpose of the Study:
- To investigate the mechanisms of resistance to petite mutagenesis by 5FU.
- To differentiate between classes of mutants based on their response to 5FU and 5FC.
- To elucidate the role of mitochondrial membranes and RNA synthesis in petite mutagenesis.
Main Methods:
- Isolation and characterization of yeast mutants resistant to petite mutagenesis.
- Testing mutant sensitivity to growth inhibition and mutagenesis by 5FU and 5FC.
- Assessing the effect of dimethylsulfoxide on 5FU sensitivity in class II mutants.
Main Results:
- Two classes of mutants were identified: Class I (resistant to both 5FU and 5FC mutagenesis) and Class II (resistant to 5FU mutagenesis, partially sensitive to 5FC mutagenesis).
- Class II mutants exhibit differential permeability of mitochondrial membranes to 5FU and 5FC.
- Dimethylsulfoxide restored 5FU sensitivity to Class II mutants, supporting the role of membrane permeability.
Conclusions:
- Class II mutants likely possess mitochondrial membranes impermeable to 5FU but permeable to 5FC, directing mutagenesis to mitochondrial RNA synthesis.
- Class I mutants possess altered mitochondrial RNA-synthesizing systems unable to incorporate fluorinated nucleotides.
- These findings highlight the compartmentalization of petite mutagenesis induction in yeast.