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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.

In a wide variety of organisms, gametes develop within clusters of interconnected germline cells called cysts. Four major principles guide the construction of most cysts: synchronous division, a maximally branched pattern of interconnection between cells, specific changes in cyst geometry, and cyst polarization. The fusome is a germline-specific organelle that is associated with cyst formation in many insects and is likely to play an essential role in these processes. This review examines the cellular and molecular processes that underlie fusome formation and cyst initiation, construction, and polarization in Drosophila melanogaster. The studies described here highlight the importance of cyst formation to the subsequent development of functional gametes.

The synaptonemal complex (SC) is intimately involved in the process of meiotic recombination in most organisms, but its exact role remains enigmatic. One reason for this uncertainty is that the overall structure of the SC is evolutionarily conserved, but many SC proteins are not. Two putative SC proteins have been identified in Drosophila: C(3)G and C(2)M. Mutations in either gene cause defects in SC structure and meiotic recombination. Although neither gene is well conserved at the amino acid level, the predicted secondary structure of C(3)G is similar to that of transversefilament proteins, and C(2)M is a distantly related member of the ␣-kleisin family that includes Rec8, a meiosis-specific cohesin protein. Here, we use immunogold labeling of SCs in Drosophila ovaries to localize C(3)G and C(2)M at the EM level. We show that both C(3)G and C(2)M are components of the SC, that the orientation of C(3)G within the SC is similar to other transverse-filament proteins, and that the N terminus of C(2)M is located in the central region adjacent to the lateral elements (LEs). Based on our data and the known phenotypes of C(2)M and C(3)G mutants, we propose a model of SC structure in which C(2)M links C(3)G to the LEs. meiosis ͉ recombination ͉ chromosome ͉ immunogold ͉ electron microscopy I n general terms, the structure of the synaptonemal complex (SC) is conserved among diverse organisms with two lateral elements (LEs) that run along the length of each pair of homologous chromosomes, a central element (CE) that is located midway between the two LEs, and transverse filaments (TF) that connect the LEs to the CE (reviewed in ref. 1). However, distinct differences exist among organisms, particularly in the degree of organization of the CE (2). The conditions required for SC assembly also differ, with DNA double-strand breaks being required for SC formation in some species (e.g., budding yeast, mammals, and plants) but not in others (Drosophila and Caenorhabditis elegans) (3-5). These differences may be useful in defining nonconserved features of SC as well as in highlighting conserved functions.The morphological structures of CEs from a mammal (rat) and two insects (Drosophila and a beetle, Blaps cribrosa) were analyzed at high resolution by using EM tomography (2). In these organisms, the CE structure is essentially the same, but the degree of organization varies considerably. The CE in insects is highly organized, with two (and sometimes more) distinct longitudinal components. These dense longitudinal components appear to be composed of vertical ''pillars'' that link multiple layers of CE together. In comparison, the CE of mammals is less well organized; multiple layers of CE are not obvious, and the longitudinal components are so discontinuous that they typically appear as a single, rather broad, dark structure midway between LEs (2, 6). Some investigators have suggested that the longitudinal components are formed, at least partially, by the Nterminal domains of TFs (7, 8). Whether this difference among species in th...

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Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. The maintenance of the prophase I arrest requires the silencing of Cdk1 activity. Drosophila oocytes inhibit the accumulation of the mitotic cyclins, the activating subunits of Cdk1, via a poorly defined posttranscriptional mechanism. Here, we demonstrate that the translational repressor Bruno binds the 3' UTR and inhibits the translation of the mitotic cyclin Cyclin A during prophase of meiosis I. In the absence of Bruno, ovarian cysts enter meiosis but rapidly accumulate high levels of mitotic cyclins and return to the mitotic cycle. Based on our results, we propose a model in which Bruno and the anaphase-promoting complex/cyclosome act together to restrict the accumulation of the mitotic cyclins, and thus Cdk1 activity, during the prophase I arrest of the Drosophila oocyte.