Zweifel, Adrienne Elizabeth (2010-05). Phenotypic Characterization of Self- Assembling Protein Fragments Using Negative Dominance. Doctoral Dissertation. | Thesis individual record
abstract

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.

etd chair
publication date
2010