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For construction of defined double mutants, V. cholerae strains carrying a deletion mutation was further transformed with genomic DNA fragments of appropriate strains (carrying TnFGL3 inserthe2 in the presence of chitin by using previously described methods26,36.
The presence of the S89G mutation was further verified by sequencing.
The gns4 mutation was further fine-mapped to a 167-kb interval between the markers LYH-91 and LYH-52.
In addition, each mutation was further analyzed by restriction enzyme digestion (see below).
Briefly, CYP21A2 was specifically amplified in two overlapping fragments (from the promoter region to exon 6 and from exon 3 to exon 10, respectively), and each mutation was further analyzed in a second round of PCR.
The uncut band (carrying the mutation) was further amplified and sent to sequence.
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The specificity of defects caused by the R561H mutation is further supported by the analysis of unc-104 /− mutant embryos.
The rate of mutation is further believed to increase in the presence of toxic oxidants that are released during such an inflammatory response (Ness and Cottreau, 1999).
This mutation is further enriched after the fifth sort, and accounts for approximately 80% of AR in that population of cells.
The dominant negative effect of P56S mutation is further supported by the observation that the mutant protein accumulates into intracellular inclusions, which also sequester the wild-type protein (Teuling et al., 2007; Chen et al., 2010).
The dominant pathogenic role of SIMPLE mutation is further supported by our finding of a CMT1C-like peripheral neuropathy phenotype in transgenic mice expressing SIMPLE W116G mutant and the lack of a neuropathy phenotype in SIMPLE knockout mice.
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