Terry L. Orr-Weaver
Massachusetts Institute of Technology
Massachusetts Institute of Technology
Nine Cambridge Center
Cambridge, MA 02142
1988 Searle Scholar
Our laboratory is investigating how cell growth and division are coordinated with key differentiation processes during the development of multicellular organisms. This is achieved by regulating the cell cycle, the cycle of growth (gap phases), DNA replication (S phase), and chromosome segregation (M phase). Specialized cell cycles are required during development. Our goal is to understand the meiotic cell cycle which produces haploid gametes, the rapid early division cycles in embryogenesis and their activation by fertilization, and the endo cell cycle which produces polytene cells. We want to define the shared functions utilized by all types of cell cycles and to identify specific functions that act uniquely in specialized cell cycles. We are using the fruit fly Drosophila melanogaster as a model organism because it permits the combined approaches of genetics, cell biology, and biochemistry.
During meiosis two rounds of chromosome segregation follow a single replication of the DNA. In meiosis I the homologous copies of each chromosome segregate from each other. This requires that the replicated copies of each chromosome, the sister chromatids, defer their segregation until meiosis II and remain attached to each other through meiosis I. We identified two Drosophila proteins, ORD and MEI-S332, that are required to maintain sister-chromatid cohesion during meiosis. We now are unraveling the molecular mechanism through which these proteins control sister-chromatid attachments.
Genetic analysis of mei-S332 suggested that the protein product acts to hold the sister chromatids together at the centromere. We found that MEI-S332 protein localizes to the centromere when the chromosomes condense in prophase I. It persists at the centromere until the sisters separate at anaphase II when it is no longer detectable.>
ORD is required to maintain sister-chromatid cohesion early in meiosis, when the sister chromatids are attached along their lengths. ORD is needed also for proper mitotic divisions in the germline. Although ORD is a novel protein, the positions of mutations within the coding sequence and complex genetic interactions they exhibit define critical functional domains of the protein.
Fertilization and Early Embryonic Divisions
Sperm entry triggers the restart of the cell cycle, but it is unknown how fertilization influences cell cycle regulators. In Drosophila, the early division cycles occur in a nuclear syncytium in the absence of transcription. These rapid cycles lack gap phases during which growth and transcription normally occur. Our goal is to determine how fertilization activates the cell cycle and to elucidate whether novel regulatory mechanisms operate during the rapid S-M cycles.
We identified genes needed to maintain the cell cycle in an arrested state between the completion of meiosis and fertilization. Drosophila females mutant for plutonium (plu), pan gu (png), or a previously identified gene, gnu, produce oocytes that properly complete meiosis but inappropriately undergo DNA replication without being fertilized. The early divisions are also defective in these mutants in that DNA replication is not linked to mitosis. Mutant unfertilized eggs or embryos contain giant, polyploid nuclei. Genetic interactions between the genes show that they regulate the same process. These gene products formally inhibit S phase prior to fertilization and ensure its coupling to mitosis in the early embryo.
PLU protein is similar to a class of inhibitors identified in mammalian cells by their ability to block the onset of S phase. The expression pattern and mutant phenotypes of plu reveal that it acts only during early development. We have a genomic fragment that rescues png and are identifying its protein product. Our immediate aim is to identify the cell cycle targets of these two genes and the regulatory hierarchy between plu, png, and gnu.
The Endo Cell Cycle
Tissues throughout the plant and animal kingdom are polyploid or polytene, one example being the mammalian extraembryonic trophoblast cells. In Drosophila most of the larval tissues become polytene. We found that polytene cells are produced by a modified cell cycle, the endo cell cycle, in which S phase alternates with a gap phase, but mitosis does not occur. The transition from a mitotic cell cycle to the endo cell cycle occurs in late embryogenesis and is under developmental control. There is an invariant pattern of polytene DNA replication dependent on developmental events.
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