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The gene cassette was spliced into the promoterless plasmid such that when the construct was linearized, and were in a tail-to-tail arrangement with the ori and ampicillin resistance gene in between

The gene cassette was spliced into the promoterless plasmid such that when the construct was linearized, and were in a tail-to-tail arrangement with the ori and ampicillin resistance gene in between. during the tachyzoite and bradyzoite stages. The presumptive gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes. The apicomplexan parasite causes congenital infections leading to blindness, mental retardation, and hydrocephaly. Recently, attention has been focused on this parasite because of the LYN-1604 increased incidence of toxoplasmic encephalitis in patients immunocompromised by AIDS (22). Although has a sexual cycle within the feline intestinal epithelium (13), it is usually transmitted asexually by carnivorism and scavenging. The asexual cycle has two developmental stages: a rapidly replicating form called the tachyzoite and a slow-growing, quiescent stage called the bradyzoite. The tachyzoite resides within an intracellular vacuole which is not capable of acidification or fusion with any organelle of the hosts endocytic pathway (18, 35). In response to some as-yet-unknown cellular signal, tachyzoites differentiate into encysted bradyzoites which lie essentially dormant within host tissues for months or years, apparently hidden from the immune system and antimicrobial brokers. Among immunocompromised patients, it is thought that toxoplasmic encephalitis is due to recrudescence of a latent contamination of bradyzoites as a result of the loss of cellular immune surveillance (22). Determining how this transition occurs is crucial for understanding disease pathology. The physiological conditions which can trigger the tachyzoite-bradyzoite conversion in vitro have been examined (5, 37), but the molecular signals which induce this transition in vivo have not been identified. This work has been aided by the availability of antibodies against Rabbit polyclonal to CCNA2 stage-specific markers for bradyzoites (P36, LYN-1604 P34, P21, and P18) and tachyzoites (P30) (19, 37, 42). Induction of bradyzoite protein synthesis is thought to be complex since P21 is usually expressed later than the other bradyzoite-specific proteins during differentiation, and heterogeneous vacuoles made up of parasites with tachyzoite and bradyzoite antigens were observed (38). Physiological conditions which can induce bradyzoite formation are alkalizing the culture medium to pH 8, shifting the heat from 37 to 43C, and treating infected macrophages with gamma interferon. Use of these conditions has enhanced the study of the bradyzoite stage, including the isolation of three genes which are bradyzoite specific, i.e., the genes encoding lactate dehydrogenase 2 ([45]), surface antigen P18 ([26]), and a small heat shock protein ([4, 27]). Many molecular genetic tools have been developed for by insertional mutagenesis is usually that encoding hypoxanthine-xanthine-guanine phosphoribosyltransferase (had been shown to have phosphoribosyltransferase activity for hypoxanthine, xanthine, and LYN-1604 guanine presumably within the same protein, since mutants usually lack activity for all those three nucleotides (29). Mutants defective in HXGPRT were obtained by unfavorable selection with 6-thioxanthine (6-TX) and then back selected for wild-type HXGPRT by positive selection with mycophenolic acid and xanthine (MPA-X [29]). These results were confirmed in the cloning and characterization of the gene (11). In contrast, the xanthine-guanine phosphoribosyltransferase (GPT) has been a commonly used selectable marker for animal cell transformation (24) and recombinant computer virus construction (7, 12, 24), but it does not use 6-TX as a substrate. Thus, the negative selections with GPT have been limited to the use of 6-thioguanine or 8-azaguanine in HGPRT-negative cell lines. The goal of this study was to combine insertional mutagenesis with the strong selectivity of the LYN-1604 HXGPRT system to isolate genes whose expression was up-regulated early in the tachyzoite-to-bradyzoite switch. For this bradyzoite-specific gene trap, promoterless was inserted throughout the genome and then unfavorable selection with 6-TX was performed to inhibit the growth of all recombinants with HXGPRT fused to tachyzoite-specific or constitutive genes. After alkalizing the culture medium to pH 8.1, which causes tachyzoites to convert to bradyzoites, positive selection with MPA-X was used to select recombinants that were expressing HXGPRT in.