Characterization of gata1 mutations: T301K, K333R, and vlt. (A) Schematic representation of Gata1 protein with N- and C-zinc fingers marked as NF and CF, respectively. Multispecies alignment of the region containing the mutations: T301K (blue box), K333R (green box), and vlt (R339X, red box). Dr indicates zebrafish; Hs, human; Mm, mouse. (B) Shown is a 3-dimensional protein model of the zebrafish Gata1 C-terminal zinc finger domain. The ribbon structure depicts a 60 amino acid fragment of the zebrafish Gata1 protein (purple) bound to DNA (gray). The location of substituted residues (T301 and K333; yellow ball-and-stick) and the region truncated in vlad tepes (vlt; red) are indicated. Residue T301 is in close proximity to the DNA backbone of the major groove, suggesting its missense mutation may alter DNA binding. In contrast, the side chain of residue K333 points outward from an extended loop, an orientation that may be tolerant of conservative substitution. (C) DNA-binding activity of wild-type (WT) and mutant forms of Gata1 as determined by electrophoretic mobility shift assay with a ³²P-labeled probe for a β/ϵ globin promoter sequence and in vitro–translated proteins. The DNA–protein complexes are seen as slower migrating bands marked by black and white arrowheads. The first lane on the left contains only the probe, and the next lane also contains lysate but no Gata1 protein. Similar amounts of the Gata1 proteins were used in the experiments, as demonstrated by the Western blot (bottom). (D) Transcriptional activity of mutant forms of Gata1 relative to the wild-type (WT) Gata1 protein as measured by a Dual Luciferase Assay. T301K substitution reduced the ability of Gata1 to activate transcription in vitro, whereas K333R substitution did not affect Gata1 activity in vitro.