Ga. De Souza et al., Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein, PLANT MOL B, 39(1), 1999, pp. 95-104
Transient expression and electrophoretic mobility shift assay were used to
investigate the cis elements and the DNA-binding proteins involved in the r
egulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of u
nidirectional deletions was generated in a 962 bp fragment of the alpha-coi
xin promoter that had been previously fused to the reporter gene GUS. The c
onstructs were assayed by transient expression in immature maize endosperm.
There was no significant decrease in GUS activity as deletions progressed
from -1084 to -238. However, deletion from -238 to -158, which partially de
leted the O2c box, resulted in a dramatic decrease in GUS activity emphasiz
ing the importance of the O2 box in the quantitative expression of the gene
. The -238 promoter fragment interacted with Coix endosperm nuclear protein
s to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same ret
arded complexes were observed when the -158 promoter fragment was used in t
he binding reactions. Reactions with nuclear extracts isolated from Coix en
dosperms harvested from 6 to 35 days after pollination revealed that the 5
DNA-protein complexes that interact with the alpha-coixin promoter are diff
erentially assembled during seed development. Deletion analysis carried out
on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -
51 is essential for the formation of the complexes observed. When nuclear e
xtracts were incubated with an antiserum raised against the maize Opaque-2
protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented ind
icating that an Opaque-2 like protein participates in the formation of thos
e complexes. Complex C2 was not affected by the addition of the O2 antibody
, suggesting the existence of a novel nuclear factor, CBF1, that binds to t
he promoter and makes protein-protein associations with other proteins pres
ent in Coix endosperm nuclei.