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Analysis of the DNA binding proteins interacting with specific upstream sequences of the S. purpuratus CyI actin gene
Author(s) -
Ganster Raymond,
Paul Hyacinth,
Katula Karen S.
Publication year - 1992
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080330405
Subject(s) - strongylocentrotus purpuratus , biology , footprinting , oligonucleotide , gene , electrophoretic mobility shift assay , dna , transcription (linguistics) , actin , microbiology and biotechnology , dna binding protein , transcription factor , gene expression , actin binding protein , genetics , actin cytoskeleton , cell , cytoskeleton , linguistics , philosophy
Abstract The Cyl actin gene of the sea urchin, Strongylocentrotus purpuratus , is regulated temporally and spatially within the cells of the early embryo. In an effort to understand the molecular basis for the Cyl actin pattern of expression, we have begun analyzing the protein–DNA interactions within regions previously shown to be of potential functional importance (Katula et al., 1987). Using DNase I footprinting, 10 protected regions were identified containing both conserved and apparently novel protein binding sites. Gel mobility shift competition assays confirmed the presence of multiple protein factors which specifically recognize Cyl actin upstream sequences. Determination of a relative affinity constant value (K r ) indicated that most of the protein factors preferred their respective oligonucleotide sequences vs. a synthetic competitor DNA in a range of 10 4 . The highest affinity binding was observed for proteins binding to the oligonucleotide probe containing theoctamer element (K r ≈ 10 6 ). Heterologous gel shift competition assays were carried out to investigate the interrelatedness of the protein factors. These studies, combined with other data, indicate there are both unique and redundant protein–DNA interactions in the region being examined. Possible alterations in Cyl actin DNA binding proteins were investigated during the period of Cyl transcriptional activation by gel mobility shift analysis. An increase in binding activity was observed for most of the factors, indicating that early transcriptional activity of Cyl actin may involve a general increase in the amount or activity of specific transcription factors. In addition, qualitative changes, as seen by alterations in the shift patterns, were observed for some of the oligonucleotide probes. © 1992 Wiley‐Liss, Inc.