Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
Molecular, Cellular, and Developmental Biology
Kristen M. Johansen
The mAb 2A labels Drosophila embryos in a novel, dynamic staining pattern that identifies a nuclear meshwork during interphase, which is remodeled to form a spindle structure at metaphase. This intriguing pattern made the antigens recognized by mAb 2A interesting targets for cloning and analysis, Skeletor was found to be encoded by a complex locus. Two alternatively spliced transcripts from the Skeletor locus were identified, Orf1b/Orf2 and Orf1a. Orf1b/Orf2 has an unusual structure, containing two overlapping open reading frames with coding potentials of 87 kD and 81 kD respectively. Analysis of Orf2 amino acid content reveals overall high proline content, several low complexity regions, and a bipartite nuclear localization signal. Nuclear localization was confirmed by generating Skeletor-specific antibodies, resulting in a staining pattern that closely recapitulates the original mAb 2A staining pattern. As well, staining of whole mount salivary nuclei and immunolabeling of polytene chromosome squashes suggest that Skeletor is chromatin-associated in interphase nuclei. Triple-labeling studies comparing the localization of Skeletor, chromatin, and microtubules indicate that Skeletor spindle formation may precede that of the microtubule spindle apparatus. Perturbation of Skeletor structures using the anti-Skeletor mAb 1A1 showed an effect on nuclear morphology and division. These observations led to the hypothesis that Skeletor protein forms a nuclear structure that dynamically reorganizes during the cell cycle, and may act both as an organizing guide or scaffold for the microtubules and as a determinant of nuclear morphology. Orf1a is a truncated form of Orf1b/Orf2, encoding a protein of 31 kD, which contains a transmembrane spanning domain and an N-terminal repeat of 102 amino acids. Antibodies show the Orf1a protein localizes to a reticulated meshwork in the cytoplasm. Interestingly, Orf1 appears to have homologues in at least two species, and based on homology within the Drosophila genome and with other species, the repeat motif may define an as yet uncharacterized family of proteins or a conserved functional domain.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Diana Lee Walker
Walker, Diana Lee, "Characterization of Skeletor: a novel nuclear protein implicated in mitotic spindle assembly " (2000). Retrospective Theses and Dissertations. 12293.