Functional Complementation

Abstract Many studies on gene cloning, particularly on genes with a very low level of expression, have benefitted from the so‐called functional cloning of genes, mostly complementary deoxyribonucleic acids (cDNAs) in higher eukaryotes including mammals, by phenotype‐complementation assay, using cell mutants deficient in biological pathways. Successful gene‐cloning studies utilised a rapid functional complementation assay of mammalian somatic cell mutants include the search for pathogenic genes responsible for peroxisome biogenesis disorders (PBDs), autosomal recessive, progressive disorders characterised by loss of multiple peroxisomal metabolic functions and defects in peroxisome assembly, consisting of 13 complementation groups (CGs). Such a forward genetic approach using a dozen CGs of peroxisome‐deficient Chinese hamster ovary cell mutants led to isolation of human peroxin ( PEX ) genes. Search for pathogenic genes responsible for PBDs of all 13 CGs is now accomplished with another approach, the homology search by screening the human expressed‐sequence tag database using yeast PEX genes. Key Concepts: A mutation is a permanent change in the DNA sequence of a gene. Mutations in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene. Mutations may lead to changes in phenotype. Cell mutants are a highly useful tool in genetic, biochemical as well as cell biological research. Eukaryotic organisms have two primary cell types – germ and somatic. Mutations can occur in either cell type. If a gene is altered in a germ cell, the mutation is termed a germinal mutation. As germ cells give rise to gametes, some gametes will carry the mutation and it will be passed on to the next generation when the individual successfully mates. Somatic mutation refers to genetic alteration acquired by a cell that can be passed to the progeny of the mutated cell in the course of cell division. Somatic mutations differ from germ‐line mutations, which are inherited genetic alterations that occur in the germ cells (i.e. sperm and eggs). Somatic mutations are frequently caused by environmental factors, such as exposure to ultraviolet radiation or to certain chemicals. In genetics, complementation occurs when two strains of an organism with different homozygous recessive mutations that produce the same phenotype (for example, a change in wing structure in flies) produce offspring with the wild‐type phenotype when mated or crossed. Complementation will occur only if the mutations are in different genes. A genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions. Several methods including the most recent lipofection have been developed for transfecting DNA into animal cells. Genetic phenotype‐complementation of peroxisome assembly‐defective mutants of mammalian somatic cells such as Chinese hamster ovary (CHO) cells and of several yeast species including Saccharomyces cerevisiae and Pichia pastoris would lead to identification and characterisation of numerous genes that are essential for peroxisome biogenesis.