EXPRESSION OF THE UBIQUITIN GENES OF TRYPANOSOMA CRUZI

克氏锥虫是人类恰加斯病的病原体， 这种疾病折磨着南美洲和中美洲的两千万人。 虽然这种疾病在北美基本上是不存在的，但T。克鲁济群岛 原产于美国西南部的部分地区。 相比 大量关于非洲人的分子遗传学研究 锥虫，对转录和转录 调节T.克鲁兹 本申请 描述了专注于更好地理解 T. cruzi通过延续我们的 泛蛋白和泛蛋白相关基因的表达分析。 我们对I.克鲁兹有- 表明这些位点由串联重复的钙调素（Cal）基因组成 随后是钙调素-泛素相关（CUB）基因的单拷贝 随后是一个FUS基因和一个或多个PUB 基因.尽管DNA序列分析表明这些基因是 编码在同一条DNA链上，核连续分析表明， 钙调蛋白基因是单独转录单位的一部分。 因此 每个钙调蛋白-遍在蛋白位点似乎可能包含至少两个 转录单位，一个终止，另一个在内部启动。 5 '-侧翼区。 为了进一步分析CUB 2.65、FUS 1和PUB 12.5在大肠癌中的表达， 我们已经使用每个基因的5 '-fanking区域来开发高效的基因， 瞬态和稳定的转换系统1。克鲁兹 与其他 锥虫物种，其中类似的转化系统已被 发展了我们将DNA引入I的能力。cruzi和研究其 我们会继续协助调查工作。 实验 本建议中所述的使用遗传方法来确定 转录和反式剪接所需的串联序列 CUB 2.65、FUSI和PUB 12.5基因排列。

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.