Phenotypic Characterization of Self- Assembling Protein Fragments Using Negative Dominance
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Protein oligomerization provides a way for cells to modulate function in vivo. In this study, self-assembling protein fragments from ParC, DnaX, and proteins of unknown function were used to generate phenotypes in a dominant negative manner. These fragments were expressed as Thioredoxin (TRX) fusions under the control of the inducible araBAD promoter. Fragments chosen contain only the oligomerization domain of the protein, lacking the regions necessary for catalytic function. Fragments of ParC, a subunit of Topoisomerase (Topo) IV, generated fragment-specific phenotypes. Regions that expressed both the oligomerization domain and CTD of ParC (ParC206-752 and ParC332-752) yielded filamentous cells with several different nucleoid segregation phenotypes. Another ParC fragment containing only the oligomerization domain of ParC (ranging from 333-485) yields a recA-dependent septation defect in a subset of the population. This phenotype suggests that Topo IV may be inhibiting chromosome dimer resolution. The overexpression of DnaX247-455, a fragment containing regions of both the tau and gamma subunits of the DNA Polymerase III holoenzyme, led to a severe plating defect. Upon further investigation, this fragment caused filamentation, a nucleoid defect, and induction of sulA, similar to the effects seen with the dnaX temperature-sensitive alleles. The overexpression of the various y-protein fragments yielded a variety of mediaspecific plating defects on over 50% of the proteins tested. The overexpression of the protein fragments yielded effects that were not seen by other overexpression or deletion experiments, even under similar growth conditions. The results presented here show that the overexpression of self-assembling fragments yield a variety of dominant negative phenotypes. Reducing the activity of protein complexes allows for new aspects of the physiological process to be investigated.
Zweifel, Adrienne Elizabeth (2010). Phenotypic Characterization of Self- Assembling Protein Fragments Using Negative Dominance. Doctoral dissertation, Texas A&M University. Available electronically from