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In particular, the M2 clones from hMLH1−/− TGFBR2 OF cells revealed that all clones underwent −1 bp frameshift mutation, indicating that the M2 clones are fully mutant cells containing a frameshifted A9/T9 microsatellite.
Clones from M1 populations in hMLH1−/− and hMSH6−/− backgrounds revealed the coexistence of mutated (65±14%, A9 for TGFBR2 and A7 for ACVR2) and WT (35±14%, A10 and A8) microsatellite sequences, indicating the existence of intermediate mutant cells containing A8/T7 or A10/T9 heteroduplexes within the M1 population transferring into full mutant cells as previously observed [25].
The cta1Δ mutant cells containing the empty plasmid failed to grow in medium containing 5 mM H2O2.
Several studies reported that genes crucial to the viability of Synechocystis cannot be successfully and completely inactivated, resulting in heteroploid mutant cells, containing both chromosomes harbouring the wild-type gene and the knockout locus.
In vivo, Ndc80/ Hec1 mutant cells containing K166D and K166E substitutions fail to accumulate in metaphase and have weak kinetochore-microtubule attachments (Sundin et al. 2011; Tooley et al. 2011).
Among these ts mutants, pcp1 14 mutant cells (containing two point mutations, K848R and L1187P, Fig. 4A and Supplementary Fig. S1) showed reduced protein levels even under the permissive condition (27 °C) (reduced by >60%, Fig. 4B) [35,40].
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Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p.
Thus, vps22, vps25, and vps36 mutant cells contain abnormally large early endosomes that accumulate ubiquitin-conjugated proteins.
The Northern blot revealed that both clpX and clpP mutant cells contained rot-transcript levels that were slightly reduced compared to wild-type levels.
As shown in Fig. 4A, almost all single mutant cells contained the typical dis-like pre-anaphase nuclei (see Fig. 1A).
In contrast, heparan sulfate produced by the mutant cells contained fewer of these smaller oligosaccharides and more extended structures rich in N-acetylated disaccharides (dp>18) were present.
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Justyna Jupowicz-Kozak
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