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Fig. 1. Identification of a DimA-interacting protein. The
Dictyostelium genome contains 19 potential bZIP genes. Alignments and
phylogenetic trees of the DimA basic DNA-binding domain (A) and leucine
zipper dimerisation domain (B) are shown. Identifiers from the
Dictyostelium database
(www.dictybase.org)
are shown with DimB highlighted in blue. Basic residues are shown in red,
hydrophobic in blue, small and polar in white, and secondary structure
breakers in orange. The basic regions were defined as the first 26 amino acids
from the basic DNA-binding domain, and all 19 sequences were aligned using
CLUSTALX (default setting) and a phylogenetic neighbour-joining tree was
plotted using NJ-Plot. Scale bars above the alignment indicate 0.5 estimated
amino acid substitutions per site. Multiple sequence alignment and
phylogenetic analysis were performed based on the dimerization domain,
represented by the first four hepatads from the leucine zipper region of each
protein. The results indicated a greater sequence identity between DimA and
DimB bZIP domain. (C) DimA and DimB form homo- and heterodimers in
vitro. Fusion proteins were expressed in E. coli and purified by
affinity chromatography. DimA or DimB GST fusion proteins were mixed with His
tagged versions before incubating in the presence of glutathione sepharose
beads. Presence of His fusion proteins was monitored in the supernatant (S,
unbound) and pellet (P, bound).
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