Complementation analysis

Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12. In the absence of normal B12 activation, homocystinuria results from a failure of normal vitamin B12 metabolism. Complementation analysis classifies defects in vitamin B12 metabolism into three groups cblC (most common), cblD and cblF. Most individuals become ill in the first few months or weeks of life with hypotonia, lethargy and growth failure. Optic atrophy and retinal changes can occur. Methylmalonate excretion is excessive, but less than in methylmalonyl-CoA mutase deficiency, and without ketoaciduria or metabolic acidosis. 

Hoffmann GF, Brendel SU, et al. (1992) Mevalonate kinase assay using DEAE-cellulose column chromatography for first-trimester prenatal diagnosis and complementation analysis in mevalonic aciduria. J Inherit Metab Dis 15 738-746... 

The next phase in pathway analysis involves biochemical procedures. As many metabolic intermediates as possible are isolated, their structures are determined, the order of reactions is determined, and the enzymes that catalyze the different reaction steps are isolated and characterized. The mutants isolated for the complementation analysis are also... 

Different procedures are used for analysis of simple and complex pathways. Analysis of single-step pathways often begins with the isolation and characterization of the enzyme involved. During enzyme isolation each purification step is monitored by a specific assay that measures the conversion of substrate to product. Multistep pathway analysis ideally begins with complementation analysis, a genetic technique that entails the isolation of mutants with genetic blocks in each step of the pathways. Once the numbers of enzymes and intermediates are established, each enzyme can be isolated with the help of a specific assay. 

The Number of Proteins Participating in a Pathway Is Known through Genetic Complementation Analysis... 

The investigation of an amino acid biosynthetic pathway in E. coli begins with the accumulation of mutants that are deficient in the capacity to synthesize that amino acid. Mutants of this sort are known as auxotrophs. Each auxotroph bears a mutation in one of the genes that encodes an enzyme required for a step in the biosynthetic pathway of the amino acid. It is possible to determine how many steps are in a particular pathway by the process of complementation analysis (see fig. 11.14). 

Using Tn5 mutagenesis, complementation analysis, and DNA-sequencing experiments, a gene designated lag-1 (lipopolysaccharide-associated gene)... 

Ben-Yoseph Y, Pack BA, Mitchell DA, et al. Characterization of the mutant A -acetylglucosaminyl-phosphotransferase in I-cell disease and pseudo-Hurler polydystrophy complementation analysis and kinetic studies. Enzyme 35 106-116,1986. 

Brul S, Westerveld A, Stri jland A. Wanders RJ, Schram AW, Heymans HS, et al. Genetic heterogeneity in the cerebrohepatorenal (Zel I weger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest 1988 81(6) 1710—1715. 

Complementation analysis determines whether recessive mutations are in the same or different genes. Complementation tests in yeast are performed by mating haploid a and a cells carrying different recessive mutations to produce diploid cells. In the analysis of cdc mutations, pairs of different haploid temperature-sensitive cdc strains were systematically mated and the resulting diploids tested for growth at the permissive and nonpermissive temperatures. In this hypothetical example, the cdcX and cdcY mutants complement each other and thus have mutations in different genes, whereas the cdcX and cdcZ mutants have mutations in the same gene. 

The number of functionally related genes involved in a process can be defined by complementation analysis (see Figure 9-7). 

A number of experimental approaches can be used to analyze mutations. Describe how complementation analysis can be used to reveal whether two mutations are in the same or in different genes. What are suppressor mutations and synthetic lethal mutations ... 

McGuinness, M.C., Moser, A.B., Poll-The, B.T. Watkins, P.A. (W) )Biochem. Med Metab. Biol , 228-242. Complementation analysis of patients with intact peroxisomes and impaired peroxisomal beta-oxidation. 

Wanders, R.J., van Roermund, C.W., Brul, S., Schutgens, R.B. Tager, J.M. (1992) J. Inherit Metab. Dis. 15, 385-388. Bifunctional enzyme deficiency identification of a new type of peroxisomal disorder in a patient with an impairment in peroxisomal beta-oxidation of unknown aetiology by means of complementation analysis. 

Complementation analysis, in which cells showing similar alterations in their surface saccharides are tested, either by fusion or genetic transfer, to see if their genetic defects will correct each other or not, affords a useful means of getting closer to the basic biochemical lesions. For example, deficits of sugar nucleotide synthetase and glycosyltransferase should complement each other, while two different mutations in the transferase gene should not and it now seems clear that both types of mutant do arise. For a detailed recent review see Stanley (1980). 

Szczepankowska AK, Prestel E, Mariadassou M, Bardowski JK, Bidnenko E. Phylogenetic and complementation analysis of a single-stranded DNA binding protein family from lactococcal phages indicates a non-bacterial origin. PLoS One. 2011 6(ll) e26942. 

Figure 7 Schematic diagram of the external bombardment mode of electron gas SNMS, including an Auger electron spectrometer for complemental analysis.

Treatment of CBS deficiency includes a low-methionine diet, vitamin Bs, folic acid, and betaine (N-trimethylglycine) [1]. Betaine works by (re)methy-lating homocysteine to methionine, and it is used in conjunction with folic acid in the treatment of MTHFR deficiency [3]. Hydroxycobalamin should be given to patients with methylmalonic aciduria protein restriction, folic acid, vitamin Bg, betaine and other measures may also be appropriate, depending on which mutant class (cblA through cblG) a patient is assigned to by complementation analysis [3, 6]. 

---