Characterization of a bi-directional promoter in Giardia lamblia

• Main Authors: Li-Chin Huang, 黃麗瑾
• Other Authors: 戴榮湘
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/18121777745363988111

碩士 === 國立臺灣大學 === 微生物學研究所 === 88 === Giardia lamblia, which is one of the earliest-divergent lineage of eukaryotes, causes one of the most common parasitic gastrointestinal diseases throughout the world. Little information is available about the regulation of gene expression in this primitive eukaryote. Previous study revealed a contiguous 32-bp AT-rich sequence to be the ran gene promoter. The 32-bp sequence in reverse orientation also exhibited 1/10 of the original activity, suggesting that the ran promoter may regulate transcription bi-directionally. In the present study, an uncharacterized 1.9-kb gene (referred to as upr, upstream of the ran gene) positioned head to head relative to the adjacent ran gene was detected by Northern blotting and cDNA library screening experiments using a probe spanning 5’ untranslated region (UTR) of the ran gene. Major transcription start sites of upr mRNA were consistently mapped to -53 and -74 relative to putative translation start codon of the upr gene. The upr gene promoter was then analyzed by a luciferase (luc+) reporter system. Deletion mapping and scanning mutagenesis within the 5’-UTR of the upr gene revealed two regions to be important for promoter activity, -159/-103, which spans the inverted 32-bp ran promoter element, and -88/-48, which spans the transcription start sites of upr mRNA. Electrophoretic mobility shift assay revealed that ssDNA binding proteins interacting with ran promoter may also interact with the upr promoter. Further mutation of a region spanning -47/-42 resulted in very little luciferase activity without reduction in luc+ mRNA, indicating that -47/-42 is probably important in translation. This study demonstrates the first bi-directional promoter in G. lamblia, and suggests that upr gene expression may also be regulated at the translation level.