Deletion of the nuclear gene encoding the mitochondrial citrate transport protein from Saccharomyces cerevisiae

The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) teas been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 1996-09, Vol.226 (3), p.657
Main Authors: Kaplan, R.S. (University of South Alabama, Mobile, AL.), Mayor, J.A, Kakhniashvili, D, Gremse, D.A, Wood, D.O, Nelson, D.R
Format: Article
Language:English
Subjects:
Carrier Proteins - genetics
Cell Nucleus - metabolism
EXPRESION GENICA
EXPRESSION DES GENES
GENE
Gene Deletion
GENES
Genes, Fungal
Genetic Complementation Test
GENETICA
GENETIQUE
Haploidy
Mitochondria - metabolism
MITOCHONDRIE
MITOCONDRIA
Molecular Sequence Data
MUTACION
MUTATION
Open Reading Frames
Phenotype
Polymerase Chain Reaction
PROTEINAS
PROTEINE
RECOMBINACION
RECOMBINAISON
Recombination, Genetic
Reproducibility of Results
SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
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Summary:The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) teas been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot analysis provided the first quantitative estimate of the level of the CTP in wild-type yeast mitochondria and indicated the absence of expressed CTP in mitochondria isolated from the deletion strain. Deletion of CTP1 did not lead to a phenotype on any carbon source tested, indicating that CTP1 is wt an essential gene. This suggests that either known alternative pathways are able to produce sufficient acetyl-CoA to support biosynthetic reactions, or there exists a second CTP gene. The ability of the deletion strain to serve as a host for the correct targeting and overexpression of a mutated CTP was then demonstrated. These studies provide a system which permits the use of site directed mutagenesis to examine both CTP targeting to mitochondria, as well as the molecular basis underlying CTP function. Moreover, this system will not only facilitate the study of the yeast CTP, but also CTPs expressed from the cDNAs of higher eukaryotes
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1996.1411