Restoration of Mitochondrial Gene Expression Using a Cloned Human Gene in Chinese Hamster Lung Cell Mutant.

BACKGROUND: Gal-32 is a Chinese hamster lung cell nuclear mutant that is unable to grow in galactose due to a defect in mitochondrial protein synthesis. Since the product of the Gal-32 gene was unknown, it was imperative to use phenotypic complementation to clone a human gene that corrected the Gal-32 mutation.
RESULTS: Recessive Gal-32 cells were co-transformed with pSV2-neo plasmid DNA and recombinant DNA from a human genomic library containing the dominant human Gal+ gene and a chloramphenicol-resistance (camr ) gene present in the pSV13 vector. Primary transformants were selected by growth in galactose and the neomycin analog G418. In order to rescue the human Gal+ gene, a genomic library was constructed with primary transformant DNA and the pCV108 cosmid vector. The camr gene was used to identify clones with the nearby human sequences. DNA from two camr , Alu-hybridizing clones was able to transform the recessive Gal-32 cells to the Gal+ phenotype and to restore mitochondrial protein synthesis.
CONCLUSION: These data demonstrate the isolation of two pCV108-transformant recombinant clones containing a human gene that complements the Chinese hamster Gal-32 mutation and restores galactose metabolism.