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The application of this screen led to the identification of multiple mutants, which exhibit some distinct phenotypes relative to one another and to previously identified maize mutants that can reactivate the silent transgene.
Multiple mutants which we constructed were confirmed by genotyping, using wild-type and T-DNA allele-specific PCR for insertional alleles, and CAPS [ 124] or dCAPS [ 125] markers for point mutation alleles; except for max4-1, whomozygosityosity was confirmed by testing progeny for uniform BASTA-resistance.
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To dissect the role of each putative member of MFS superfamily, an essential part of the process will now involve construction of multiple knockout mutants, which will enable to unravel their role in drug or nutrient transport.
However, N. crassa can be easily sexually crossed to generate double-, triple-, and multiple-gene mutants, which would help to further investigations of the mechanism of hypercellulase production in RUT C30 in future.
Hence, we conducted a genetic screen and isolated multiple mutants in which GFP RAB-3 lost its punctate localization pattern.
Of note is that in several cases our analysis identified multiple mutant loci, which then had to be individually assessed as candidate loci.
Each of these expression responses was previously identified in association with multiple long-lived dwarf mutants, which have reduced levels of GH and/or IGF-1 in circulation [ 24].
Other multiple mutants, all of which contained the K227Q mutation, formed dramatically reduced levels of undesired ligation products.
It also allows for higher-level replication of intermediate mutants, which contributes to the faster accumulation of multiple mutations by individuals.
The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized.
Furthermore, the ttx-3 mutants, which are incapable of differentiating AIY interneurons critical for multiple sensory pathways, also accumulate less TAG when feeding on HB101 than on OP50.
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