The Drosophila melanogaster gene vasa is known to be necessary for the establishment of a functional pole plasm, as well as for the completion of oogenesis. To further elucidate its role, we have created a null mutation of the vasa gene and examined vasa-null ovaries for defects. Analysis of these ovaries has revealed that vasa is involved in various aspects of oogenesis, including the growth of germ-line cysts, oocyte differentiation, anterior-posterior egg chamber patterning, and dorsal-ventral follicle patterning. In addition, vasa-null oocytes fail to show efficient accumulation of various localized RNAs, such as Bicaudal-C, Bicaudal-D, egl, enc, orb, oskar , and nanos, but still show accumulation of gurken RNA. Interestingly, the accumulation of GURKEN protein in the oocyte is severely reduced, and that of BICAUDAL-C is substantially decreased in null mutants. These results suggest a possible role for vasa in activating translation of targeted RNAs during oogenesis.

Pole plasm in Drosophila melanogaster, through the posteriorly localized determinant nanos, controls the formation of the abdominal segments and, through an unknown mechanism, controls the formation of the germline. oskor, vasa, and tudor are three critical genes in the pole plasm assembly pathway and their gene products, oskar RNA, Vasa protein and Tudor protein are localized in the pole plasm in a precise order. The localization of oskar and nanos mRNAs is closely related to their translational activation. We provide evidence here by in vitro biochemical assays that Vasa protein is an ATP-dependent RNA helicase, an ATPase and an RNA-binding protein, as was predicted from its sequence similarity to mammalian translation initiation factor eIF-4A. The enzymatic activities of Vasa protein are important for its function, but the initial localization of Vasa protein to the pole plasm is independent of its RNA helicase and RNA-binding activities. Further, we cloned Bruno, a Xenopus etr-1 homologue with three ribonucleoprotein-recognition-motifs (RRM), by far-western screening using Vasa protein as bait. Bruno is the product of the gene arrest, which was cloned independently by Webster and Macdonald at Stanford University. The localization of Bruno protein in S1-10 oocytes is similar to that of oskar and gurken RNAs. This is significant as both oskar and gurken RNAs contain Bruno-response-elements at their 3$ sp prime$UTRs. Bruno is mislocalized in vasa mutant ovaries, suggesting that the localization of Bruno protein requires Vasa function. As a translational activator of oskar and nanos RNAs and a regulator of Bruno protein. the participation and the importance of Vasa protein in pole plasm assembly and function is obvious. Whether Vasa acts directly or indirectly to activate translation of specific mRNAs will require further examination.

Loss of germ cells that entered meiosis at different developmental stages was compared. Mice were injected with BrdU at 13.3, 14.3 or 15.3 days post coitum (dpc) and sacrificed either 3 days after BrdU injection or 4 days post partum (dpp). BrdU-labeled germ cells were detected in ovarian sections through double immunofluorescent staining for BrdU and either GCNA-1 or MVH as a germ cell marker. The results show that the loss of germ cells that entered meiosis at 13.3 or 15.3 dpc was excessive compared to the loss of total germ cells. Such preferential elimination was not found for germ cells that entered meiosis at 14.3 dpc. We conclude that oocyte loss during meiotic prophase is influenced by the timing of meiotic onset. / The mechanism of germ cell loss during ovarian development was tested by the presence of markers for apoptosis. Mouse ovaries were isolated at 12.5 dpc, 18.5 dpc and 2 dpp and cultured with doxorubicin (DXR) to induce cell death. Ovarian histological sections were double immunofluorescent stained for GCNA-1 and cleaved caspase-3 or PARP-1. The results suggest that caspase-3 is not activated in germ cells throughout ovarian development whereas PARP-1 is activated in germ cells at 12.5 dpc and 2 dpp but not at 18.5 dpc. Thus, no evidence has yet been provided to support the hypothesis that oocyte death during the meiotic prophase is mediated by apooptosis.

No description available.

Bicaudal-C, a Drosophila gene, is required for centripetal follicle cell migration in the egg chamber and anterior patterning of the embryo; its exact functions are unknown. BICAUDAL-C carries five KH domains required for in vitroRNA-binding and for in vivo activity. Here, I show that Bicaudal-C functions, through RNA-binding, to regulate oskar expression during mid-oogenesis. Females carrying mutations in Bicaudal-C in the regions encoding its KH domains display premature, ectopic translation of OSKAR at the anterior of their stages 8--10 oocytes; this likely accounts for the disruption of anterior patterning in embryos of Bicaudal-C/+ females. Also here, I propose that oskar and gurken share common regulators, including Bicaudal-C. This is based on the discovery of dorsalized progeny of Bicaudal-C/+ females, and on the dominant enhancement of the Bicaudal-C/+ phenotype by specific gurken and cornichon alleles. Finally, I describe a yeast two-hybrid screen used to investigate BICAUDAL-C-protein interaction.

Bicaudal-D (Bic-D) is essential in Drosophila for the establishment of oocyte fate and polarity within the developing oocyte. To study these processes we have engineered a chimeric Bic-D::GFP fusion protein which behaves like the endogenous Bic-D polypeptide. We have identified three genes which are required for the normal subcellular distribution of Bic-D::GFP two genes predicted to encode RNA binding proteins (egalitarian and orb) and Dynein heavy chain. In particular, they affect Bic-D::GFP localization during the early germarial stages of oogenesis during which oocyte fate is established, or later when anterior-posterior polarity is initiated. Our results support the model that Bic-D acts in conjunction with mRNA binding proteins and a negative-end directed microtubule motor in localizing mRNAs. Throughout stages 1--6 of oocyte development, Bic-D::GFP accumulates in the oocyte in a strong posterior cortical focus, resembling a spool, that is aligned with a crater-like indentation in the oocyte nucleus. The aligned focus and crater reveal an early oocyte polarity and a previously undescribed asymmetric subcellular structure that may be involved in tethering the oocyte nucleus. Shape, positioning and orientation of the oocyte nucleus change around stage 6--7, concomitantly with a change in position of the Bic-D::GFP focus to the presumptive dorsoanterior corner. This re-orientation appears to anticipate the establishment of a new dorsoventral polarity in the oocyte and egg chamber. Dhc and Bic-D are both involved in the process of re-orientation of the oocyte nucleus and in polarity formation.

Typescript. / Bibliography: leaves 67-69. / ix, 69 l illus., tables

Thesis (Ph.D.)--Tufts University, 2000. / Adviser: Susan G. Ernst. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 91-118). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Leonard D. Dobens. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed May 23, 2008. Includes bibliographical references (leaves 109-115). Online version of the print edition.

Thesis (M.Sc.)--Victoria University of Wellington, 2007. / Includes bibliographical references.