trachomatis strains. Statistical significance is indicated with the asterisk above the individual
treatment groups, as compared to pcDNA-transfected cells (Student’s t-test, p < 0.01). The multinuclear phenotype was manifested by the carboxy-terminal 179 amino acids of CT223p (Fig. 4). A reduced but still significant level of multinuclear cells were identified in cells transfected with a plasmid encoding only the carboxy-terminal 56 amino acids of CT223p, but, transfection of a plasmid encoding an internal fragment of CT223p (CT223/CT91p) did not lead to a significant level of multinuclear cells. These data suggest that the FK228 domain of the protein responsible for blocking cytokinesis was present in the carboxy-terminal 56 amino acids. Cytosolic expression of other incs The orf encoding CT223p is within a likely operon encoding known and candidate inc genes CT223-CT227, and is adjacent to a very early operon containing two inc genes (CT228 and CT229). We tested each orf in these operons for an association with a polynuclear phenotype. Each orf was expressed in transfected cells and there was no
apparent difference in expression level, based on fluorescence microscopy of transfected cells (not shown). Orfs CT224 and CT225 also were associated with a reduced but still statistically significant percentage of polynuclear cells Tacrolimus (FK506) in a transfected population (Fig. 4). None of the other tested orfs were associated with an increased number of polynuclear cells. The same approach was used for testing the effects SB202190 chemical structure on cell cytokinesis of other Inc proteins. HeLa or McCoy cells transfected with plasmids encoding each protein from C. trachomatis incA and incC, and C. caviae incA, incB and incC were compared with cells expressing full length and truncated CT223. None of these plasmids led to an increase in polynuclear cells relative to AZD3965 ic50 controls (Fig. 4). The CT223 coding sequence
from different C. trachomatis strains encode proteins with up to 5% difference in amino acid sequence (22). We therefore tested plasmids encoding CT223p from strains with known amino acid sequence differences for their ability to block cytokinesis. Transfection of plasmids encoding each CT223p sequence was associated with an increase in multinucleate cells (Fig. 5). In contrast, transfection of a plasmid expressing C. muridarum TC0495, which is a syntenous, apparent CT223 homolog (less than 30% predicted amino acid sequence identity), did not lead to an increase in the number of multinucleate cells relative to controls (Fig. 5). Cells producing CT223p and CT223/179p have increased numbers of centrosomes To further explore the multinuclear phenotype, cells expressing CT223 were labeled with antibodies specific against γ-tubulin.