chiffon
Genetic analyses suggest that chiffon null mutants are viable. However, there might be a more general requirement for chiffon function that is masked by maternal supply of wild-type chiffon gene product to the mutant embryo. To begin to examine this possibility, experiments were performed to determine if Chiffon mRNA is maternally synthesized and supplied to the embryo. Whole-mount in situ hybridization to RNA in dissected ovaries was used to visualize patterns of RNA expression. Hybridization to the known maternally supplied Bicoid RNA was used as a control. As expected, Bicoid RNA is abundant in nurse cells, and exhibits a characteristic localization in a ring at the anterior of the oocyte. Abundant Chiffon RNA expression is also detected in the germline nurse cells. Faint Chiffon signal could sometimes be detected in the follicle cells of late stage egg chambers, however, the stage at which follicle cell expression is initiated could not be determined. To confirm that the maternally synthesized Chiffon RNA is supplied to the embryo, Northern analysis was performed on RNA isolated from egg chambers and embryos. RNA was isolated from stage 10/11 egg chambers (which comprise nurse cells, follicle cells and the oocyte); stage 13/14 egg chambers (which comprise oocyte and follicle cells), and 0-30 minute-old embryos, which have not yet begun zygotic transcription. The Northern blot was first hybridized with probes specific for several control RNAs. As expected, the maternally supplied Bicoid RNA is present in all three samples. The follicle-cell-specific chorion gene RNAs S36, S15 and S18 are present only in the egg chamber samples, since the follicle cells are degraded prior to egg laying. Abundant S18 and S15 RNA synthesis in the follicle cells does not begin until after stage 11. The 6.5 kb Chiffon transcript was detected in all three samples and therefore is maternally supplied to the embryo (Landis, 1999).
Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-Replication Complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has been characterized as of yet. This paper reports the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact, and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification, but still undergo endocycling. These results show that Cdc7 interacts with Chiffon to form a functional Dbf4-Dependent Kinase (DDK) complex, and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, it was shown that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila (Stephenson, 2014).
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chiffon:
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
date revised: 5 April 2015
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