First, we analyzed the expression of SlCaMs at 45 and 50 DPA in these mutants which corresponded to the mature green and breaker stages in wild-type fruit.
In particular, line 2# showed almost identical resistance as the cyn-1 mutant, which corresponded with similar AtCYN transcript levels between the two lines (Figure 4B, C).
The distribution of this truncated mutant, which corresponds to the full-length protein lacking the long central coiled-coil, was almost identical to that of the endogenous GMAP210 in both control cells and isolated Golgi ministacks induced by nocodazole treatment.
Solving Equations 10 and 11, one could determine that the open-state population increases from ∼30% for the wild type to ∼65% for the mutant, which corresponds to a conformational energy change of −0.88 kCal/mol.
In these experiments, bubR1rev1 and bubR1D1326N mutants were used, which correspond to a deletion and a point-mutation at the catalytic kinase-domain, respectively [20], [21].
Figure 2 shows the difference plots (A) and melting peaks (B) for different dilutions of mutant DNA, and demonstrates that this assay can detect 2.5% mutant alleles, which corresponds to 5% tumour cells carrying a heterozygous mutation.
Intriguingly, the Dvl2 mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh ) that results in defective PCP pathway, fails to antagonize SG assembly.
Intriguingly, the mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh ) that results in defective PCP pathway, failed to antagonize SG assembly (Fig. 1D,E).
Interestingly in wild species tomato (Solanum penelli), the aconitase mutant allele (which corresponds to SlAco3b) is deficient in both cytosolic and mitochondrial aconitase proteins (Carrari et al. 2003), suggesting that at least in tomato this gene product is dual targeted.
This approach yielded the mutants V19I, G138A, E139N, and N148E, which correspond to the AsLOV2 mutants V416I, G528A, V529N, and N538E [ 19, 28, 29].
The mutant clones will be referenced according to the abbreviations listed in Table 1; mutant clone 1, which corresponds to the triple mutant W559A/K560A/G561A, will be referred to as TAp63 αWKG>AAA.
