vaginalis and T. tenax. Conclusion Using two approaches did not yield any T. vaginalis unique genes, suggesting strongly there is a high genetic identity between T. vaginalis and T. tenax. For all of the genes originally identified and examined as unique to T. LDK378 supplier vaginalis, the genes were found to be identical in T. tenax. We found higher rates of
transcription in T. vaginalis compared with T. tenax. Our data may help explain recent reports on the respiratory infections by both of these trichomonal species. Finally, attention needs to be given to the possibility that T. tenax is a genetic variant of T. vaginalis. Methods Parasites The fresh clinical isolates of T. vaginalis UT00-40 and T016 were grown in batch culture at
37°C no more than three weeks in trypticase-yeast extract-maltose (TYM) medium supplemented with 10% heat-inactivated horse serum . The isolate T016 was used for construction of the expression cDNA library that was used for screening with T. tenax-adsorbed pooled patient sera, as described below. The T. tenax Hs-4:NIH was grown in LYI Entamoeba medium supplemented with 10% heat-inactivated fetal bovine serum as recommended by ATCC. The T. tenax BX-795 solubility dmso isolate was confirmed using the PT3 sense primer (5′-AGTTCCATCGATGCCATTC-3′) and the PT7 antisense primer (5′-GCATCTAAGGACTTAGACG-3′) . PCR-based cDNA subtractive hybridization Total RNA was extracted from T. vaginalis UT00-40 and T. tenax organisms using Trizol (Invitrogen, Carlsbad, CA). The double-stranded cDNAs were synthesized from 1 μg total RNA of each group using a Smart PCR cDNA synthesis kit (BD Clontech, Mountain View, CA) and were used for suppression PCR-based cDNA subtractive hybridization using a PCR-select cDNA subtraction
kit (BD Clontech). The cDNAs prepared from T. tenax and T. vaginalis were regarded as driver and tester, respectively, and the driver cDNA population was subtracted from the tester cDNA population. Suppression PCR was performed to prepare the cDNA pool, enriched for genes accumulated in T. vaginalis (forward-subtracted). 5-Fluoracil ic50 The resultant tester-specific cDNAs were amplified by PCR, and cloned into pGEM-T-easy vector (Promega Corp., Madison, WI). The detailed Cilengitide procedures were described in the protocol of the PCR-select cDNA subtraction kit (BD Clontech). The subtracted cDNA fraction was cloned into a TA vector and transformed into Escherichia coli to create an enriched T. vaginalis cDNA library. Sequencing and analysis Colonies were randomly selected, and plasmids were prepared using a Miniprep kit (QIAGEN, Valencia, CA). The cDNA inserts were verified by restriction digestion, and the clones were sequenced in the Washington State University institutional DNA-sequencing facility. Sequence data was compared with the GenBank database using a BLAST program. RT-PCR analysis of selected genes Differential expression of a subset of cloned genes was confirmed by semi-quantitative RT-PCR.