Cell mutant isolation

Escherichia coli can he adapted to grow on PPi as the sole source of phosphorus in this situation the cells are dependent upon intracellular inorganic pyrophosphatase activity to make Pi available. To avoid possible confusion from the inducible alkaline phosphatase of E. coli, which also has pyrophosphatase activity (see Chapter 17, by Reid and Wilson, this volume), all mutant isolation studies began with an E coli strain unable to synthesize this inducible protein. 

A fourth type of WgaR CHO cell mutant has now been isolated (Stanley, manuscript in preparation). This mutant is more highly resistant to WGA than the previously described mutants and it has been shown to belong to a new complementation group (group VIII). 

Urlaub G, Chasin LA (1980), Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity, Proc. Natl Acad. Sci. USA 77 4216-4220. 

Hasegawa, K., Kuge, O., Nishijima, M., and Akamatsu, Y., 1989, Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis. J. Biol. Chem. 264 19887-19892. 

Rusinol, A.E., Yang, L, Thewke, D., Panini, S.R., Kramer, M.F., and Sinensky, M.S., 2000, Isolation of a somatic cell mutant resistant to the induction of apoptosis by oxidized low density lipoprotein, J. Biol. Chem. 275 7296-7303. 

P-gp mutants isolated from drug-resistant cell lines. Photolabeling with [207, 208]... 

PG is made in mitochondria and microsomes of animal cells and appears to be primarily converted to DPG. DPG is biosynthesized exclusively on the matrix side of the mitochondrial inner membrane and is found only in this organelle. There is evidence that the rate-limiting step in DPG biosynthesis is the conversion of PA into CDP-DG (G.M. Hatch, 1994). Consistent with this idea, the levels of CTP regulate DPG biosynthesis in cardiac myoblasts (G.M. Hatch, 1996). Using techniques developed by Raetz and co-workers [14], a temperature-sensitive mutant of PG-P synthase in CHO cells was isolated (M. Nishijima 1993). The mutant had only 1% of wild-type PG-P synthase activity at 40°C and exhibited a temperature-sensitive defect in PG and DPG biosynthesis. This mutant was used to show that DPG is required for the NADH-ubiquinone reductase (complex I) activity of the respiratory chain. 

Zoeller, R.A., Raetz, C.R. 1992. Strategies for isolating somatic cell mutants defective in lipid biosynthesis. Methods Enzymol. 209 34-51. 

Figure 26 Compartmentalized self-replication. The polymerase gene library is cloned into an expression vector and subsequently transformed into an E. coli cell. After the polymerase library is expressed, the cells are isolated and encapsulated with PCR reagents using a water and oil emulsion. The emulsion is then thermocycled, destroying the E. co//cell wall and allowing amplification to occur. Post-CSR, the amplicons enriched in genes encoding active mutants can be collected and purified for the next round of selection.

Table 1. Comparison between the photosynthetic activity of wild-type and several mutants at amino acid position 250 in the M-subunit of Rb. capsulatus in both whole cells and isolated reaction centers. [MKq] 1 mM.

Five substitutions for Y-L162 were made and the kinetics of the re-reduction of the special pair were measured in intact cells and isolated mutant RCs using cytochrome C2 from R. sphaeroldes. 

Liu Y, Childs RA, Matrosovich T, Wharton S, Pahtia AS, Chai W, Daniels R, Gregory V, Uhlendorff J, Kiso M et al. (2010) Altered receptor specificity and cell tropism of D222G hcanagglutinin mutants isolated Irom fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol 84 12069-12074... 

Maeda, S., Hashimoto, K., and Simizu, B., 1979, Complementation between temperature-sensitive mutants isolated from Aedes albopictus cells persistently infected with western equine encephalitis virus. Virology 92 532. 

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