Vectors adducted with the corresponding nonreduced cross-links did yield transformants under these conditions, suggesting that the native cross-links reverted at least partially to noncross-linked N-dG lesions in the NER-deficient host.
The transformation of high molecular weight DNA extracted from 1974M1 into S. pneumoniae sensitive strains followed by the selection of the transformants under LNZ pressure should allow discriminating the mutations actually involved in resistance from bystander mutations that may have been randomly selected during the selection process.
The recipient genomic DNA was isolated from transformants obtained under the following co-cultivation conditions: the ratio of bacterial cells to target conidia was 5 × 103 or 1 × 104 on solid IM (Table 3, 48 h) and 50 in liquid IM (Table 5, 48 h).
For each experiment three transformants cultivated under the same conditions were assayed.
To assess whether the increase in the crtS gene dose effectively leads to an increase in the crtS mRNA levels, RT-qPCR analysis was performed for the wild-type, heterozygous and homozygous transformants grown under the same conditions.
Leaky expression from the cbhI promoter in glucose medium could explain the increased number of targeted integration transformants under repressed conditions, as compared to the control transformants.
The β-galactosidase activity of transformants under HIS3 selection is generally enhanced, probably because the transformants are forced to produce the hybrid proteins in quantities that enable the HIS3 gene to be expressed at levels that permit growth.
The number of transformants under such a test ranged from 0 60, whereas 100 ng of ccDNA typically gives 104 colonies, or greater, under similar transformation conditions.
At the end of the day, a first plate containing 200 μl of liquid medium was inoculated with cells taken from the different yeast transformants under study, and incubated at 30 °C overnight with orbital agitation at 250 rpm.
TFs with bHLH motifs are well known as stress regulator in plants, so we investigated the phenotypes of NsbHLH2 transformants under stressful conditions (N limitation and osmotic stress).
