Write a proposal to address the two specific hypotheses listed below.
For each set of experiments, make sure to include appropriate controls and what results you would expect if the hypothesis is supported. This assignment is based on the information content of two papers: Kearse et al., 2011, and Kearse et al., 2013.
In order to complete the assignment, it 3 other sources will be used to develop the experimental plan requested in the assignment. Include a list of all references used for the paper.
General Hypothesis: eRpL22 family paralogues (eRpL22 and eRpL22-like) retain a conserved set of ribosomal functions, yet specify unique cellular roles in fly development and in spermatogenesis.
Question 1. What experimental approach(es) would you devise to test the specific hypothesis: that specialized ribosomes exist for differential translation of testis-specific mRNAs in Drosophila?
Note that all molecular, biochemical, genetic, and cellular reagents (e.g., DNA clones, antibodies, fly strains) available from Kearse et al. (2011, 2013) are available to you for your experimental approach. If other reagents are needed for the experiments that you devise, you must include a strategy for making those necessary reagents as well as the experimental approach for using the reagents.
Question 2. What experimental approach(es) would you devise to test the specific hypothesis that testis-specific post-translational modifications including SUMOylation and phosphorylation are required to localize eRpL22 to the nucleoplasm of meiotic spermatocytes in Drosophila?
Note that for your proposed experimental plan, you have access to a group of meiotic-arrest mutants where meiosis is incomplete. You are not required to use these mutants in your experimental plan, but this genetic approach may be useful to address the question of interest.
The following background information may be useful.
Meiotic arrest mutant analyses have been instrumental in defining key events in fly spermatogenesis. For example, spermatogenesis is arrested at the G2/M transition of meiosis I in can mutants. Chromosomes are only partially condensed and no post-meiotic cells are present in these mutants (Lin et al., 1996). soti mutants have individualization defects (Kaplan et al., 2010). Western blot analysis of testis extracts from can and soti mutants shows a decrease in accumulation of the testis-specific 55? and 55? species of eRpL22 in can mutants, but not in soti mutants (Kearse, Ph.D. thesis, 2013).
Based on the timing of defects in these mutants, it appears that testis-specific PTMs of eRpL22 occur before the individualization process but after the G2/M transition of meiosis I.
Additional mutants (can, aly5p, chc, mip40, heph2, sotisik, and cbx), with defects in a different stage of spermatogenesis are available. These mutants fall into two classes – meiotic mutants and individualization mutants. The meiotic mutants are can (Lin et al. 1996), aly5p (Lin et al., 1996), and mip40 (Beall et al., 2007). The individualization mutants are chc, (Fabrizio et al., 1998), heph2 (Robida et al., 2010) , sotisik (Kaplan et al., 2010), and cbx (Fabrizio et al., 1998).
These are the two required articles. Please find 3 other sources besides these to relate to the assignment.
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