EXPRESSION OF THE UBIQUITIN GENES OF TRYPANOSOMA CRUZI

克氏锥虫是人类恰加斯病的病原体，是一种 这种疾病在南美洲和中美洲折磨着2000多万人。 尽管这种疾病在北美基本上不存在，但克氏锥虫 原产于美国西南部的部分地区。与之形成鲜明对比的是 关于非洲的相当多的分子遗传学研究 锥体，对转录和转录知之甚少 克鲁兹毛滴虫蛋白质编码基因的调控。此应用程序 描述侧重于更好地理解以下内容的实验 在克氏锥虫中的转录调控 泛素及其相关基因的表达分析。 我们对日本血吸虫的2.65和2.8个钙调蛋白-泛素基因座的研究有： 研究表明，这些基因座由重复钙调素(CaL)基因组成 然后是钙调蛋白-泛素相关(CUB)基因的单拷贝 接下来是一个FUS基因和一个或多个酒吧 基因。尽管DNA序列分析表明这些基因是 在相同的DNA链上编码，核连续分析-表明 钙调蛋白基因是独立转录单位的一部分。因此，它 似乎很可能每个钙调蛋白-泛素基因座包含至少两个 转录单位，一个终止，另一个在 5‘-侧翼区域。 为了进一步分析CUB2.65、FUS1和PUB12.5的表达 基因我们已经利用每个基因的5‘-扇区来开发有效的 暂态和稳定的变换系统1。克鲁兹。与其他产品一样 具有类似转化系统的锥虫物种 发展了我们将DNA导入克氏假单胞菌并研究其 Expression将继续为我们的调查提供便利。这些实验 本提案中所描述的使用遗传方法来识别 转录和反式剪接所需的序列 排列了CUB2.65、FUSI和PUB12.5基因。

Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, a disease that afflicts over 20 million people in South and Central America. Although the disease is essentially absent from North America, T. cruzi is indigenous to portions of the southwestern United States. In contrast to the considerable body of molecular genetic research on African trypanosomes, little is known about transcription and transcriptional regulation of protein encoding genes in T. cruzi. This application describes experiments that focus on developing a better understanding of transcriptional regulation in T. cruzi through a continuation of our analysis of expression of the ubiquitin and ubiquitin associated genes. Our studies of the 2.65 and 2.8 calmodulin-ubiquitin loci of I. cruzi have- shown that these loci consists of tandemly repeated calmodulin (Cal) genes followed by a single copy of the calmodulin-ubiquitin associated (CUB) gene which is in turn followed by one of the FUS genes and one or more PUB genes. Although DNA sequence analysis has shown that these genes are encoded on the same DNA strand, nuclear run-on analysis-suggests that the calmodulin genes are a part of a separate transcription unit. Therefore it seems probable that each calmodulin-ubiquitin locus contains at least two transcription units, one terminating and the other initiating within the 5'-flanking region. To further our analysis of the expression of the CUB2.65, FUS1 and PUB12.5 genes we have used the 5'-fanking regions of each gene to develop efficient transient and stable transformation systems for 1. cruzi. As with other trypanosome species in which analogous transformation systems have been developed our ability to introduce DNA into I. cruzi and study its expression will continue to facilitate our investigations. The experiments described in this proposal use genetic methodology to identify the sequences required for transcription and trans-splicing of the tandemly arrayed CUB2.65, FUSI and PUB12.5 genes.