Identification of the minimum regulatory region of a XXXX A-signal-dependent developmental gene.

@entity4986 @entity1021
Developmental expression of the @entity4986 gene 4521 requires extracellular A-signal. This signal is generated in response to nutrient limitation and functions in cell density sensing. To identify the upstream limit of the minimum region required in vivo for A-signal-dependent 4521 expression, a 5' deletion analysis of the 4521 regulatory region was performed. A new vector, pHBK280, was designed to facilitate this analysis. This vector creates tandem copies of the 4521 gene in the @entity4986 chromosome, such that the regulatory region to be tested is upstream of a single copy of the lacZ reporter gene. The 5' deletion analysis revealed that at most, 146 bp of DNA upstream of the @entity1021 ( @entity1021 ) was required for full developmental expression of 4521. Basal expression levels were observed with constructions containing 90 bp of DNA upstream of the @entity1021 . In vitro gel retardation assays revealed that DNA fragments with 5' ends of 146 and 125 bp upstream of the @entity1021 and a common 3' end of +24 bp were retarded in their mobility after incubation with all of the @entity4986 developmental crude cell extracts tested. In contrast, a fragment starting at 90 bp upstream of the @entity1021 and ending at +24 bp was not retarded in its mobility after incubation with the same cell extracts. These in vivo and in vitro data suggest that cis-acting elements located between 146 and 90 bp upstream of the @entity1021 serve as binding sites for one or more trans-acting regulatory factors required for 4521 developmental expression.
