Functional Complementation


Many studies on gene cloning, particularly on genes with a very low level of expression, have benefited from the so‐called functional cloning of genes, mostly complementary DNAs (deoxyribonucleic acids) in higher eukaryotes including mammals, by phenotype‐complementation assay, using cell mutants deficient in biological pathways. Successful gene‐cloning studies using 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 14 complementation groups (CGs). Such a forward genetic approach using a dozen CGs of peroxisome‐deficient Chinese hamster ovary (CHO) cell mutants led to isolation of human peroxin (PEX) genes. Search for pathogenic genes responsible for PBDs of all 14 CGs is now accomplished with another approach, the homology search by screening the human expressed sequence tag (EST) database using yeast PEX genes.

Key concepts

  • 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 and 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. Because 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 mutations are genetic alterations 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 (e.g. 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.

Keywords: cell mutants; phenotype; complementation; peroxisome biogenesis disorders; PEX genes

Figure 1. Schematic view of peroxisome biogenesis in mammals. The intracellular locations and molecular properties of peroxins are shown. Peroxins are divided into three groups: (1) peroxins that are required for matrix protein import; (2) those including Pex3p, Pex16p and Pex19p, responsible for peroxisome membrane assembly; (3) those such as three forms of Pex11p, Pex11pα, Pex11pβ and Pex11pγ, apparently involved in peroxisome proliferation where DLP1, Mff and Fis1 coordinately function. PTS1 and PTS2 proteins are recognised by Pex5p and Pex7p, respectively, in the cytoplasm. Two isoforms, Pex5pS and Pex5pL, of Pex5p are identified in mammals. PTS1 proteins are transported by homo‐ and hetero‐oligomers of Pex5pS and Pex5pL to peroxisomes, where Pex14p functions as a convergent, initial docking site of the ‘protein import machinery’ translocon. Pex5pL directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol and translocates the Pex7p–PTS2 protein complex to Pex14p. PTS1 and PTS2 proteins are then released at the inner surface and/or inside of peroxisomes, downstream Pex14p and upstream Pex13p. Pex5p and Pex7p subsequently translocate to other translocon components such as the RING peroxins, Pex2p, Pex10p and Pex12p. Both Pex5p and Pex7p finally shuttle back to the cytosol. In regard to peroxisome‐cytoplasmic shuttling of Pex5p, Pex5p initially targets to an 800‐kDa complex containing Pex14p and then translocates to a 500‐kDa complex comprising RING peroxins. At the terminal step of the protein import reaction, Pex1p and Pex6p of the AAA family catalyse the export of Pex5p, where ubiquitination of Pex5p is a prerequisite for the Pex5p exit. A cytosolic factor, AWP1/ZFAND6 (p40), is required for the export and recycling of Ub‐Pex5p in mammals.


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Further Reading

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Faust PL and Hatten ME (1997) Targeted deletion of the PEX2 peroxisome assembly gene in mice provides a model for Zellweger syndrome, a human neuronal migration disorder. Journal of Cell Biology 139: 1293–1305.

Maxwell M, Bjorkman J, Nguyen T, et al. (2003) Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype. Molecular and Cellular Biology 23: 5947–5957.

Web Links

Peroxisome biogenesis disorders; PeroxisomeDB:

OMIM database in NCBI; OMIM Entry # 214100 (Zellweger syndrome)

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Fujiki, Yukio(Sep 2017) Functional Complementation. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0005676.pub3]