Yang S, Parmley S F
Department of Medicine, Stanford University School of Medicine, Palo Alto, CA 94301, USA.
Gene. 1997 Jan 3;184(1):1-12. doi: 10.1016/s0378-1119(96)00566-5.
Two Toxoplasma gondii genes were characterized that are differentially expressed during the parasite's life cycle. The genes named LDH1 and LDH2, respectively, encode polypeptides similar to the enzyme lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from a variety of organisms. They show 64.0% nucleotide identity in the coding region and both have an intron at the same relative position. The deduced amino acid sequences of LDH1 and LDH2 share 71.1% identity. LDH1 and LDH2 are most similar to an LDH of Plasmodium falciparum (46.5% and 48.5% amino acid identities, respectively). The mRNA of LDH2 was only detected in the bradyzoite stage, while the mRNA of LDH1 was detected in both the bradyzoite and tachyzoite stages. However, by isoelectric focusing and immunoblot analysis, only one LDH isoform was found to be expressed in each stage. Furthermore, the expression of a reporter gene carrying chloramphenicol acetyltransferase (CAT) coding sequence and the putative LDH2 promoter sequence was significantly up-regulated by growing parasites in tissue culture in media with alkaline pH (pH 8.2, a condition known to induce the expression of bradyzoite-specific antigens), while the expression of a CAT reporter construct carrying the putative LDH1 promoter sequence was down-regulated by similar treatment. These results indicate that LDH expression is developmentally regulated in T. gondii and suggest a possible correlation between stage conversion and alteration in carbohydrate or energy metabolism in this parasite.
对两个刚地弓形虫基因进行了表征，它们在寄生虫的生命周期中差异表达。这两个分别名为LDH1和LDH2的基因编码的多肽与多种生物的乳酸脱氢酶（LDH；L-乳酸：NAD+氧化还原酶，EC 1.1.1.27）相似。它们在编码区的核苷酸同一性为64.0%，并且在相同的相对位置都有一个内含子。LDH1和LDH2推导的氨基酸序列同一性为71.1%。LDH1和LDH2与恶性疟原虫的一种LDH最为相似（氨基酸同一性分别为46.5%和48.5%）。仅在缓殖子阶段检测到LDH2的mRNA，而在缓殖子和速殖子阶段均检测到LDH1的mRNA。然而，通过等电聚焦和免疫印迹分析，发现在每个阶段仅表达一种LDH同工型。此外，携带氯霉素乙酰转移酶（CAT）编码序列和假定的LDH2启动子序列的报告基因的表达，在碱性pH（pH 8.2，一种已知可诱导缓殖子特异性抗原表达的条件）的培养基中进行组织培养时，随着寄生虫生长而显著上调，而携带假定的LDH1启动子序列的CAT报告构建体的表达则因类似处理而下调。这些结果表明，刚地弓形虫中LDH的表达受到发育调控，并提示该寄生虫的阶段转换与碳水化合物或能量代谢改变之间可能存在关联。
