The long-term objectives are to isolate and characterize the genes that code for the components of Complex I of the mammalian electron transport chain and to examine the structure and function of these components.
The specific aims are: 1. To isolate two human X-linked Complex I genes. Our strategy is to isolate these genes by gene transfer and genetic complementation of the defect in Complex I in two different respiration-deficient Chinese hamster cell mutants, followed by molecular cloning of the complementing human genes. Once genomic clones have been isolated they will be used to identify the corresponding cDNA clones. 2. To examine the structure, organization, and expression of these Complex I genes. Experiments will include determination of the nucleotide sequence and composition of both of the Complex I cDNAs and determination of the size, organization (exon-intron structure) and 5' end of each of these X-linked Complex I genes. 3. To characterize the protein encoded by each of these genes. Experiments will include determination of the primary structure of each of these proteins by determination of the nucleotide sequence of each of the cDNA clones. Experiments will also include production of antisera against the cloned gene products. These antisera will be used to determine which subunits of Complex I have been isolated, to examine if these proteins are made as biosynthetic intermediates and to determine if these proteins are missing or altered in charge or molecular weight in the respiration-deficient mutants. These experiments will enable us to isolate and characterize the genes that code for two of the components of Complex I of the mammalian electron transport chain and to examine the structure of the proteins encoded by the genes. A complete understanding of the structure, function, and regulation of each of the components of Complex I is essential since this enzyme complex is fundamental to all aspects of life in eucaryotic organisms.
