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

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.

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 (cam ) 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 cam gene was used to identify clones with the nearby human sequences. DNA from two cam , Alu-hybridizing clones was able to transform the recessive Gal^-32 cells to the Gal⁺ phenotype and to restore mitochondrial protein synthesis.

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.