Title page for ETD etd-01232010-063910

Type of Document Dissertation
Author Rosby, Raphyel Ojomo
Author's Email Address RaphyelRosby@Gmail.com
URN etd-01232010-063910
Title Genetic and Cytological Investigation of Nucleostemin-1 in Drosophila melanogaster
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
DiMario, Patrick Committee Chair
Donze, David Committee Member
Hart, Craig Committee Member
Kato, Naohiro Committee Member
Taylor, Carol Dean's Representative
  • nucleostemin
  • ribosomes
  • large subunit
  • granular component
  • rRNA
  • nucleolus
  • NS-1
  • autophagy
Date of Defense 2010-01-12
Availability unrestricted
Mammalian nucleostemin (NS) is a nucleolar GTP-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). Nucleostemin 1 (NS1) is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity, and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed GFP-NS1 enrich within the nucleolar granular regions as expected, while over-expressed GFP-NS1 localizes throughout the nucleolus and nucleoplasm, and to several transcriptionally active inter-bands of polytene chromosomes. Over-expression caused melanotic tumors and larval and pupal lethality. RNAi depletion of NS1 caused a loss of imaginal (precursor) cells in the larval midgut, and an apparent block in the nucleolar release of large ribosomal subunits in the terminally differentiated larval midgut polyploid cells. Depletion of 60% of NS1 transcripts lead to larval and pupal lethality. Ultra-structural examination of highly differentiated larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes with a concomitant appearance of cytoplasmic pre-autophagosomes. We interpret the appearance of these structures as indicators of cell stress response.
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