Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements
Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements
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DOI：
10.1016/s0166-6851(96)02814-9
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发表时间：
1997-03-01
影响因子：
1.5
通讯作者：
Gross, U
中科院分区：
文献类型：
--
作者：
Bohne, W;Wirsing, A;Gross, U
BAG1 是弓形虫的一种小型热休克蛋白，在寄生虫的包囊形成缓殖子阶段特异性表达。 BAG1 mRNA 的上调发生在从速殖子到缓殖子分化途径的早期。为了定义涉及缓殖子特异性基因调控的基因组元件，使用 BAG1 基因的 5' 侧翼序列进行了氯霉素乙酰转移酶 (CAT) 报告基因研究。用 BAG1/cat 构建体瞬时转染的速殖子表现出非常低的 CAT 活性（比用微管蛋白启动子/cat 构建体转染的寄生虫低 200 倍）。然而，在通过碱性 pH 变化诱导缓殖子分化后，CAT 活性增加了 50 倍，表明 CAT 报告基因在 BAG1 5' 侧翼序列的控制下进行缓殖子特异性表达。 BAG1/CAT 的阶段特异性调节与 3' 侧翼区域无关，因为含有速殖子特异性 SAG1 基因 3' 侧翼序列的构建体显示出与含有 BAG1 3' 侧翼区域的构建体相同的调节。稳定转染寄生虫中 BAG1/CAT 诱导的动力学与内源 BAG1 表达的动力学相似：在碱性 pH 变化（20 倍）后的第 3 天首次检测到 CAT 活性增加，并在第 4 天急剧上调 250 倍。 BAG1 5'侧翼序列中的一系列删除表明，从 BAG1 转录起始上游 60 nt 处开始的 324 核苷酸 (nt) 片段是足以对 CAT 报告者进行特定阶段的监管。这些缺失分析表明，异源报告基因的缓殖子特异性表达仅需要最少的基因组序列。 (C) 1997 Elsevier Science B.V.
BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order to define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5' flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5' flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3'-flanking region, since constructs containing 3'-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3'-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5'-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences. (C) 1997 Elsevier Science B.V.