ABSTRACT
Mutation of the Drosophila maternal cell cycle regulator, Gnu, results in loss of embryonic mitosis and the onset of excessive nuclear DNA replication. The Gnu phosphoprotein is normally synthesized in nurse cells and transported to the developing oocyte. We created a gnuGFP-bcd3'UTR transgene using the gnu promoter and bicoid 3'UTR, that translates GnuGFP only on egg activation from a localized anterior source. This transgene was able to rescue the sterility of gnu mutant females. Gnu is therefore first required after egg activation for polar body condensation and zygotic mitoses. Embryos containing pronounced anterior-posterior gradients of Gnu activity demonstrate that Gnu regulates mitotic activity by promoting cyclin B stability. Our gnuGFP-bcd3'UTR vector provides a novel experimental strategy to analyse the temporal requirement and role of cell cycle regulators including potential sperm-supplied factors in eggs and embryos.
Subject(s)
Cell Cycle Proteins/physiology , Cyclins/metabolism , Drosophila/embryology , Mitosis/physiology , Animals , Base Sequence , DNA Primers , Drosophila/cytology , FemaleABSTRACT
At the transition from meiosis to cleavage mitoses, Drosophila requires the cell cycle regulators encoded by the genes, giant nuclei (gnu), plutonium (plu) and pan gu (png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Meiosis/physiology , Mitosis/physiology , Actins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , DNA Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/physiology , Epistasis, Genetic , Female , Gene Expression Regulation, Developmental , Infertility , Male , Molecular Sequence Data , Oocytes/cytology , Oocytes/physiology , Ovary/cytology , Ovary/physiology , Phenotype , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , TransgenesABSTRACT
In Drosophila melanogaster, the cell cycle control gene, plutonium (plu), is located between the PCNA and RpS18 genes at position 56F on chromosome arm 2R. We have used a comparative genomic approach to investigate the evolution of the plu gene and to locate conserved cis-acting elements for plu, RpS18 and PCNA. Using primers within coding regions of PCNA and RpS18, we amplified and sequenced the intervening region from twelve Drosophila species. In each species, this region contains a plu gene resembling the D. melanogaster gene in size and in the number and position of introns. The predicted Plu sequence from the different species demonstrates that the first two ankyrin repeats are conserved. Of the transcriptional control elements of D. melanogaster PCNA, we found that three motifs 5' to the PCNA transcription unit are conserved in Drosophila species.
