Figure 5
Figure 5. The booreana ENU strain harbors a point mutation in the transactivation domain of c-Myb. (A) Homozygous booreana mutants (boo/boo) appear normal at E14.5. (B) Resequencing of c-Myb (ENSMUSG00000019982) from 2 phenotype-positive embryos identified an A-to-G transition in both cases in exon 8 at base pair 923, changing amino acid 308 from glutamic acid to glycine. (C) Domain structure of c-Myb showing the E308G point mutation in the transactivation domain. DNB indicates DNA binding; TA, transactivation/acidic; and NRD, negative regulatory. (D) Complete blood counts of 8-week-old booreana homozygous and heterozygous mutants and wild-type littermate controls as measured by ADVIA 2120 machine. *Statistically significant differences (P < .05) between boo/boo and +/+ groups. **Significance between boo/boo and both +/+ and boo/+ groups. (E-F) Blood films of 12-week-old boo/boo mice (E) and boo/+ littermates (F). The mutant animals have increased platelets, polychromasia (p), and Howell-Jolly bodies (arrowheads) and basophilic stippling (s). For panels E and F, images were generated using an Olympus BX51 microscope with a U PlansApo 60× lens, 1.35 NA, under Olympus imersion oil. Images were collected using an Olympus DP70 camera and DP controller software. Digital images were adjusted and labeled using Photoshop CS4. (G) Reporter assays in 293T cells cotransfected with the myeloperoxidase gene promoter linked to lacZ and various HA-tagged c-Myb constructs, including wild-type (WT), L302A, M303V, and E308G (Booreana) mutations within the TA domain. Bars represent mean normalized relative light units (RLU), and error bars represent SD of the mean of triplicate biologic assays. (H) Western blot of transfected 293T cells for the HA tag and for endogenous β-actin showing equivalent transfection efficiency. (I) GST pull-down assays showing interactions between GST-CBP-KIX (KIX) and in vitro translated c-Myb mutants. Input (ipt) and bound wild-type (WT), E308G, or L302A radiolabeled c-Myb proteins eluted from the indicated GST fusions are shown in top panel; the bottom panel shows the presence of the relevant GST proteins, visualized by Coomassie blue staining, in each binding reaction.

The booreana ENU strain harbors a point mutation in the transactivation domain of c-Myb. (A) Homozygous booreana mutants (boo/boo) appear normal at E14.5. (B) Resequencing of c-Myb (ENSMUSG00000019982) from 2 phenotype-positive embryos identified an A-to-G transition in both cases in exon 8 at base pair 923, changing amino acid 308 from glutamic acid to glycine. (C) Domain structure of c-Myb showing the E308G point mutation in the transactivation domain. DNB indicates DNA binding; TA, transactivation/acidic; and NRD, negative regulatory. (D) Complete blood counts of 8-week-old booreana homozygous and heterozygous mutants and wild-type littermate controls as measured by ADVIA 2120 machine. *Statistically significant differences (P < .05) between boo/boo and +/+ groups. **Significance between boo/boo and both +/+ and boo/+ groups. (E-F) Blood films of 12-week-old boo/boo mice (E) and boo/+ littermates (F). The mutant animals have increased platelets, polychromasia (p), and Howell-Jolly bodies (arrowheads) and basophilic stippling (s). For panels E and F, images were generated using an Olympus BX51 microscope with a U PlansApo 60× lens, 1.35 NA, under Olympus imersion oil. Images were collected using an Olympus DP70 camera and DP controller software. Digital images were adjusted and labeled using Photoshop CS4. (G) Reporter assays in 293T cells cotransfected with the myeloperoxidase gene promoter linked to lacZ and various HA-tagged c-Myb constructs, including wild-type (WT), L302A, M303V, and E308G (Booreana) mutations within the TA domain. Bars represent mean normalized relative light units (RLU), and error bars represent SD of the mean of triplicate biologic assays. (H) Western blot of transfected 293T cells for the HA tag and for endogenous β-actin showing equivalent transfection efficiency. (I) GST pull-down assays showing interactions between GST-CBP-KIX (KIX) and in vitro translated c-Myb mutants. Input (ipt) and bound wild-type (WT), E308G, or L302A radiolabeled c-Myb proteins eluted from the indicated GST fusions are shown in top panel; the bottom panel shows the presence of the relevant GST proteins, visualized by Coomassie blue staining, in each binding reaction.

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