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Fig. 2. Structure and function of the dimA gene. (A) Site of insertion of
the disruption plasmid and structure of the dimA gene. The pBSR1
disruption vector was recovered from the dimA–
mutant by plasmid rescue. The insertion lies in the second exon of a 3846 bp
gene, encoding a putative 1242 amino acid protein. The protein contains long
stretches of asparagine (N) and glutamine (Q) residues as indicated. The
region between amino acids 545-676 shows extensive homology to the DNA-binding
and dimerization domains of bZIP and bRLZ transcription factors. (B) Sequence
alignment of the putative dimA DNA-binding and dimerization domain
with examples of bZIP and bRLZ proteins from human, mouse, Drosophila
and yeast (gi:19745184, gi:10835484, gi:135304, gi:17647933 and gi:135867).
(C) DimA binds DNA. Binding of total soluble protein extracts prepared from
bacteria expressing the putative DimA DNA-binding/dimerization domain
(dimA) fused to GST was compared with extract from cells expressing
GST alone (pGEX). Equal amounts of total protein were assayed and loaded. A 48
bp fragment from the 3' half of the minimal ecmO/lacZ promoter
was used as a probe and poly dAdT was included as a non-specific competitor.
The probe is only retarded when mixed with DimA-expressing extract. The amount
of binding is reduced by the addition of a 10-fold excess of unlabeled
oligonucleotide (CC). (D) The effects of varying non-specific competitor
species on DNA binding. Strongest binding is evident in the absence of
non-specific competitor. The addition of poly dIdC strongly reduces binding,
whereas poly dAdT addition results in a small reduction in binding. Fewer
retarded bands are visible (compared with C), as electrophoresis was performed
at 4°C to stabilize protein DNA interactions.
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