The effects of nalidixic acid in the yeast Saccharomyces cerevisiae.
thesisposted on 19.11.2015, 08:52 by Agustinus. Yuswanto
Mutants which are simultaneously resistant to nalidixic acid and are temperature-sensitive, have been isolated. Genetic analysis demonstrated the mutation is nuclear. Phenotypic analysis showed that the cell cycle of the mutant [nal(ts)] is arrested at or around the S phase at the non-permissive temperature, although the arrest is not immediate. The mutant is hypersensitive to drugs and chemicals that affect the plasma membrane, suggesting the mutation might alter membrane permeability. The wild-type gene has been cloned by complementation of the Ts phenotype and sequenced. Gene mapping using Southern blots indicates that the NAL gene is on the chromosome XII, although its precise location is still unresolved. The reading frame corresponding to the NAL gene was unambiguously identified by subcloning. The Nal protein, the predicted protein of the NAL gene, has a molecular mass of 63.5 kDa. The Nal protein has a high homology to both the ?-amino-?-ketobutyrate CoA ligase of Escherichia coli and ?-aminolevulinic acid synthetase of various organisms, suggesting that they may share the similar substrates glycine and acyl-CoA. Polyclonal antibodies against the Nal protein have been raised in a chicken and rabbit. These antibodies recognise in yeast extract protein with a molecular mass of approximately 70 kDa, a protein in line with the predicted size of the protein. Indirect immunoflourescence microscopy demonstrated that the Nal protein is mainly localised in the mitochondria.