Knoll L J, Boothroyd J C
Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305-5124, USA.
Mol Cell Biol. 1998 Feb;18(2):807-14. doi: 10.1128/MCB.18.2.807.
Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
在中间宿主体内，刚地弓形虫可在两种形态之间转换：快速增殖的速殖子和包囊化的缓殖子。缓殖子在宿主体内终生存在，对抗菌剂和免疫系统具有隐匿性。介导这种转换的信号目前了解甚少。利用基因捕获技术来分离那些在缓殖子转换早期通过阴性和阳性选择标记次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶（HXGPRT）上调表达的基因。用无启动子的HXGPRT转染刚地弓形虫，并用6-硫代黄嘌呤进行阴性选择，以抑制表达HXGPRT的速殖子的生长。然后诱导存活的速殖子在体外形成缓殖子，并用霉酚酸和黄嘌呤进行阳性选择，以筛选出构建体已整合到缓殖子特异性基因下游的寄生虫。通过使用双花扁豆凝集素（一种缓殖子特异性凝集素）和抗P36（一种缓殖子特异性表面抗原）的单克隆抗体来检查各菌株的分化能力。分化后，所有基因捕获克隆均有双花扁豆免疫荧光，除一个克隆外均表达P36。该P36阴性菌株插入位点两侧的序列与弓形虫表面抗原家族同源，强烈提示P36由该破坏基因编码。对P36阴性菌株进行遗传定位和互补分析进一步表明，被破坏的基因是P36。利用逆转录酶PCR和S1核酸酶消化来比较速殖子和缓殖子阶段的mRNA水平。推测的P36基因在两个阶段之间似乎并未调节其mRNA水平，这表明早期缓殖子特异性基因存在转录后调控机制。
