A NUCLEOTIDE-SEQUENCE ESSENTIAL FOR THE FUNCTION OF DRE, A COMMON PROMOTER ELEMENT FOR DROSOPHILA DNA REPLICATION-RELATED GENES

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gen e contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE , Drosophila DNA replication-related element), We have generated vario us base substitutions and internal deletions in and around DRE (nucleo tide positions -93 to -100 with respect to the transcription initiatio n site) of the PCNA gene in vitro and subsequently examined their effe cts on the binding to DREF (DRE-binding factor) and PCNA gene promoter activity in cultured Drosophila Kc cells as well as in living flies, Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for comple x formation with DREF, Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the g-bp sequence f rom -93 to -100 is essential for activation of the promoter in Kc cell s, Examination of lacZ expression hom PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformat ion, revealed that the 8-bp sequence is also important for DRE functio n during development, However, we obtained two exceptional mutations i n the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies, Both of these mutations ef fectively reduced the promoter activity in CAT transient expression as say in He cells and the binding to DREF in vitro, Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in Living flies than in the cultured cell or in vitro cases.