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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2010-05-7959 |
| Date | 2010 May 1900 |
| Creators | Zweifel, Adrienne Elizabeth |
| Contributors | Hu, James C. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | application/pdf |
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