However, too much NO signaling is associated with early embryo loss

These animals were maintained in specific pathogen-free conditions in accordance with institutional and national regulations and all protocols were approved by the Floralis Committee on Animal Care. Parasites were inoculated by intraperitoneal injection of 200 mL of a suspension in Hank’s balanced salt solution. To check that an infection had occurred, mice that did not spontaneously die were euthanized in an approved CO2 chamber; whole blood was collected through an intracardiac puncture, and serum was subjected to the Toxoscreen DA assay. To test the ability to induce cysts, brains from these mice were treated as previously described and cysts were observed and numbered microscopically. Bioinformatics Homologs of the Toxoplasma p43 protein were identified by GenTHREADER after splitting the sequence into domains on the basis of the NCBI Conserved Domain Search annotation. These included: Saccharomyces cerevisiae Arc1p, Homo sapiens p18, Homo sapiens p43. In order to generate a multiple-sequence alignment, the corresponding structures were aligned in UCSF Chimera using Match-Maker. The Tg-p43 and Hs-p38 sequences were then manually aligned to the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19630074 resulting structurebased profile, from Match-Align, on the basis of the original GenTHREADER alignment using GeneDoc. The latter program was also used for sequence identity calculations and alignment Lypressin rendering. Domain structure analyses of the GST Cterminal domain containing proteins: Tg-MRS, Tg-QRS, Tg-ERS, Tg-p43, and Tg-YRS were carried out using NCBI Conserved Domain Search with an E-value threshold of 1. In silico predictive disorder analysis was carried out with Genesilico MetaDisorder2. Construction of recombinantly tagged MARS subunits and Tg-p43 knockout To construct the vectors: pLIC-p43-Myc-FLAG, pLICp43DCterm-HA-FLAG, pLIC-p43-HA-FLAG, pLIC-YRS-HAFLAG, and pLIC-MRS-HA-FLAG, coding sequences of Tgp43, Tg-YRS and Tg-MRS were amplified from genomic DNA of a RH DKu80 strain using primers. The resulting PCR products were cloned into pLIC-MF-dhfr and pLIC-HF-dhfr vectors using the LIC cloning method as described previously. Plasmids were transfected into freshly isolated tachyzoites by electroporation as described previously PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632393 and transfected parasites were selected for resistance to pyrimethamine and cloned by limiting dilution. To generate a Tg-p43 knockout, the Multisite Gateway Pro 3fragment Recombination system was used to clone the dhfr cassette or the hxgprt cassette flanked by the 59 and 39 surrounding regions of the Tg-p43 coding sequence of RHDKu80 and Pru DKu80 genomic DNA as described previously using primers attB1-066670, attB4-066670, attB3-066670 and attB2-066670. The final plasmids were used as templates to amplify the sequences for transfection, using primers attB1066670 and attB2066670. Purified DNA fragments were transfected as above and clones selected for by incorporation of both xanthine and mycophenolic acid. Materials and Methods Chemicals and Cell culture methods All chemicals were sourced through Sigma-Aldrich and cell culture media from Life technologies unless otherwise specified. Human foreskin fibroblast and Human Embryonic Kidney 293 cells cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES buffer, 4 mM glutamine, and 50 mg/mL each of penicillin and streptomycin. Cells were incubated at 37uC with 5% CO2 in humidified air. All Toxoplasma strains were maintained by serial passage