ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP

Abstract

We isolated mutations in Drosophila E2F and DP that affect chorion gene amplification and ORC2 localization in the follicle cells. In the follicle cells of the ovary, the ORC2 protein is localized throughout the follicle cell nuclei when they are undergoing polyploid genomic replication, and its levels appear constant in both S and G phases. In contrast, when genomic replication ceases and specific regions amplify, ORC2 is present solely at the amplifying loci. Mutations in the DNA-binding domains of dE2F or dDP reduce amplification, and in these mutants specific localization of ORC2 to amplification loci is lost. Interestingly, a dE2F mutant predicted to lack the carboxy-terminal transcriptional activation and RB-binding domain does not abolish ORC2 localization and shows premature chorion amplification. The effect of the mutations in the heterodimer subunits suggests that E2F controls not only the onset of S phase but also origin activity within S phase.

Sequence requirements for function of theDrosophilachorion gene locus ACE3 replicator and ori-β origin elements

“…Endoreplication ceases by stage 10A, and coincidentally the diffuse nuclear ORC2 staining disappears. At this time the ORC staining relocalizes to the chorion gene loci coincident with the initiation of amplification (Royzman et al, 1999) (Fig. 7A).…”

“…Staining early stage egg chambers with antibody specific for ORC2 reveals a diffuse nuclear distribution. At stage 10A, coincident with the initiation of amplification, the diffuse nuclear staining of ORC2 is lost and the ORC localizes into dramatic foci of staining at the chorion gene loci (Royzman et al, 1999) (Fig. 7A).…”

“…Antibody staining procedure was provided by Bosco and Orr-Weaver (Royzman et al, 1999). Dissected ovaries were fixed by 8% formaldehyde/Buffer B solution for 5 minutes, then blocked with 2% normal donkey serum (NDS) in CHIP lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% Na-Deoxycholate).…”

“…Additional conserved DNA replication factor genes required for chorion gene amplification include those encoding Cyclin E (Calvi et al, 1998), E2F (Royzman et al, 1999), DP (Royzman et al, 1999), RBF (Bosco et al, 2001), CDT1 (Whittaker et al, 2000), geminin (Quinn et al, 2001) and MCM6 (Schwed et al, 2002). Antibody to ORC1 or ORC2 reveals that, coincident with the initiation of amplification, ORC moves from a diffuse nuclear distribution into dramatic foci localized at the chorion gene loci (Asano and Wharton, 1999;Calvi et al, 1998;Royzman et al, 1999). The data demonstrate that Drosophila chorion gene amplification uses evolutionarily conserved machinery for initiation; however, some mechanism must exist to uniquely mark the chorion gene loci origins for activation during amplification, when overall genomic replication has ceased.…”

Retinoblastoma family genes

“…This effect is in contrast to that of cyclin E mutant, which showed an all or none effect on BrdU incorporation (Knoblich et al, 1994;Duronio et al, 1998;Follette et al, 1998). Therefore, during normal development dE2F1 is limiting for the expression of replication factors but is not limiting for the expression of cell cycle regulators such as cyclin E. Interestingly, flies with an allele of de2f1 (de2f1 i2 ), which retains the DNA binding and dimerization domain but lacks the transcriptional activation and Rb-binding domain, are viable, but female are sterile due to increased gene amplification in follicle cells (Royzman et al, 1999). As this allele of dE2F1 cannot activate transcription, it was proposed that E2F and Rb proteins may regulate DNA replication directly at the DNA replication origins.…”