Masamitsu Yamaguchi

THE DNA REPLICATION-RELATED ELEMENT (DRE)/DRE-BINDING FACTOR SYSTEM IS A TRANSCRIPTIONAL REGULATOR OF THE DROSOPHILA E2F GENE

Two mRNA species were observed for theDrosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. We found three tandemly aligned sequences, similar to the DNA replication-related element (DRE; 5′-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathioneS-transferase fusion protein with the N-terminal fragment (1–125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. We have established transgenic lines of Drosophila in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity ofdE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene.

DNA Chain Elongation Mechanism of DNA Polymerases α, β and γ

There are many lines of evidence that indicate the existence of at least two kinds of DNA replication mechanisms in eukaryotic cells. (1) One is that observed in nuclear DNA replication, where DNA chains (at least the lagging strand) are synthesized in relatively short pieces (3–5s) that are later elongated and joined together1–6. These short DNA intermediates are also observed in the replication of viral DNA such as polyoma virus7,8 and simian virus (SV)4O9,10. (2) The other is that for adenovirus DNA11-12 and mitochondrial DNA13. These DNA’s are not replicated via Okazaki pieces as the intermediates, but replicated in a continuous mode.