No abstract

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.[Key Words: Drosophila; oogenesis; chorion; amplification; replication; cyclin E] Received November 10, 1997; revised version accepted January 15, 1998.To maintain euploid gene balance, DNA sequences must be replicated every cell cycle but not more than once. Recent evidence indicates that cell cycle control of DNA replication is effected by a two-step mechanism (for review, see Diffley 1996). Origins first become competent to replicate by assembling proteins comprising prereplication complexes onto chromatin in G 1 , and then later, during S, those origins initiate replication (Diffley et al. 1994). Replication from or through an origin dissociates functional prereplication complexes. Once destroyed, these complexes cannot reassemble until the subsequent G 1 , thereby precluding refiring of an origin in a single cycle. Several lines of evidence suggest that cyclin dependent kinases (CDKs), in addition to being required positively for cell cycle progression, act negatively and are responsible for blocking reassembly of replication complexes in S, G 2 , and M (Broek et al. 1991;Hayles et al. 1994;Moreno and Nurse 1994;Dahmann et al. 1995;Sauer 1995;Hua et al. 1997;Jallepalli et al. 1997;Tanaka et al. 1997). It is only after passage through mitosis, during a period in G 1 when kinase levels are low, that complexes can reassemble onto chromatin. This two-step mechanism of assembly and firing linked to kinase levels ensures that each region of the genome is replicated only once per cycle.In Drosophila melanogaster, as in many multicellular eukaryotes including humans, certain tissues become polyploid by entering an endocycle characterized by alternating S and G phases without intervening mitoses (for review, see Carminati and Orr-Weaver 1996). As in other cycles, Cyclin E (CycE), with...

Early during Drosophila oogenesis the 16 interconnected cells of each germ-line cyst choose between two alternative fates. The single future oocyte enters meiosis, arrests, and becomes transcriptionally quiescent. The remaining 15 cells initiate a series of polyploid cell cycles to prepare for their role as nurse cells. Like many other polyploid and polytene cells, during nurse cell growth the major satellite DNAs become highly under-represented by a mechanism that has remained obscure. We implicate the cell-cycle regulator cyclin E in DNA under-representation by identifying a hypomorphic, female sterile cycE mutation, cycE 01672, that increases the amount of satellite DNA propagated in nurse cells. In mutant but not wild-type endomitotic nurse cells, "late S" patterns of bromodeoxyuridine incorporation are observed similar to those in mitotic cells. CycE protein still cycles in cycE °167z germ-line cysts but at reduced levels, and it is found throughout a longer fraction of the cell cycle. Our experiments support the view that oscillating levels of CycE control the polyploid S phase. Moreover, they indicate that a checkpoint linking the presence of unreplicated DNA to the CycE oscillator is lacking, leading to incomplete replication of late-replicating sequences such as satellite DNAs. Unexpectedly, two to three of the 16 cells in cFcE 01672 cysts frequently differentiate as oocytes, implicating cell-cycle programming in oocyte determination.