Araújo Patrícia R, Burle-Caldas Gabriela A, Silva-Pereira Rosiane A, Bartholomeu Daniella C, Darocha Wanderson D, Teixeira Santuza M R
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Parasitol Int. 2011 Jun;60(2):161-9. doi: 10.1016/j.parint.2011.01.006. Epub 2011 Jan 28.
In trypanosomatids, transcription is polycistronic and gene expression control occurs mainly at the post-transcriptional level. To investigate the role of sequences present in the 3'UTR of stage-specific mRNAs of Trypanosoma cruzi, we generated a new vector, named pTcDUALuc, containing the firefly and Renilla luciferase reporter genes. To test this vector, sequences derived from the 3'UTR plus intergenic regions of the alpha tubulin gene, which is up-regulated in epimastigotes, and amastin, which is up-regulated in amastigotes, were inserted downstream from the firefly reporter gene and luciferase activity was compared in transient and stable transfected parasites. As expected, increased luciferase activity was detected in epimastigotes transiently transfected with pTcDUALuc containing tubulin sequences. Using stable transfected cell lines that were allowed to differentiate into amastigotes, we observed increased luciferase activity and mRNA levels in amastigotes transfected with pTcDUALuc containing amastin sequences. We also showed that the spliced leader sequence and poly-A tail were inserted in the predicted sites of the firefly luciferase mRNA and that deletions in the alpha tubulin 3'UTR resulted in decreased luciferase expression because it affects polyadenylation. In contrast to the constructs containing 3'UTR sequences derived from tubulin and amastin genes, the presence of the 3'UTR from a trans-sialidase gene, whose expression is higher in trypomastigotes, resulted in increased luciferase activity in trypomastigotes without a corresponding increase in luciferase mRNA levels.
在锥虫中，转录是多顺反子的，基因表达控制主要发生在转录后水平。为了研究克氏锥虫阶段特异性mRNA的3'UTR中存在的序列的作用，我们构建了一个新的载体，命名为pTcDUALuc，它包含萤火虫荧光素酶和海肾荧光素酶报告基因。为了测试这个载体，将来自α微管蛋白基因（在无鞭毛体中上调）和无鞭毛体蛋白（在无鞭毛体中上调）的3'UTR加上基因间区域的序列插入萤火虫报告基因的下游，并在瞬时和稳定转染的寄生虫中比较荧光素酶活性。正如预期的那样，在用含有微管蛋白序列的pTcDUALuc瞬时转染的无鞭毛体中检测到荧光素酶活性增加。使用允许分化为无鞭毛体的稳定转染细胞系，我们观察到在用含有无鞭毛体蛋白序列的pTcDUALuc转染的无鞭毛体中荧光素酶活性和mRNA水平增加。我们还表明，剪接前导序列和聚腺苷酸尾被插入到萤火虫荧光素酶mRNA的预测位点，并且α微管蛋白3'UTR中的缺失导致荧光素酶表达降低，因为它影响多聚腺苷酸化。与含有来自微管蛋白和无鞭毛体蛋白基因的3'UTR序列的构建体相反，转唾液酸酶基因的3'UTR的存在（其在锥鞭毛体中的表达较高）导致锥鞭毛体中荧光素酶活性增加，而荧光素酶mRNA水平没有相应增加。
