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Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus
R R Cobb1, J G Burkhart, J S Dubins
1Chemistry and Life Sciences Group, Research Triangle Institute, Research Triangle Park, NC 27709.
Abstract:
We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.
Insights
We analyzed five malic enzyme (Mod-1) mutants, finding that a spontaneous null mutant altered DNA and mRNA, while ENU-induced mutants did not. All mutants were heritable and viable, with null alleles showing reduced enzyme activity but detectable protein.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Malic enzyme (Mod-1) plays a role in cellular metabolism.
- Understanding enzyme mutations is crucial for genetic and biochemical research.
Purpose of the Study:
- To characterize five Mod-1 mutants at the molecular and biochemical levels.
- To investigate the genetic basis and molecular consequences of Mod-1 mutations.
Main Methods:
- Ethylnitrosourea (ENU) mutagenesis and spontaneous mutation induction.
- Restriction endonuclease and Southern blot analysis for DNA alterations.
- Northern blot analysis for mRNA size and levels.
- Enzyme activity assays and Western blot analysis for protein detection.
Main Results:
- Three electrophoretic variants and two null mutants of Mod-1 were identified.
- Spontaneous null mutant showed altered DNA banding patterns and mRNA size.
- ENU-induced mutants had normal DNA and mRNA profiles.
- Null mutants exhibited significantly reduced Mod-1 enzyme activity but detectable protein levels.
Conclusions:
- Spontaneous mutations can lead to significant molecular alterations in Mod-1.
- ENU-induced mutations in Mod-1 did not cause detectable DNA or mRNA changes.
- Mod-1 null homozygotes have reduced enzyme activity, but Mod-1 protein is still present.