臺灣博碩士論文加值系統

梨形鞭毛蟲是一種寄生於腸道之原蟲，可引起流行性的腹瀉疾病，它也代表演化上極古老的真核生物，具有特殊且有趣的生物特性。為了解此原始生物的轉錄調控，此研究中我已針對其 ran 基因啟動子做分析。另外，我建立了兩套工具，持續 DNA 轉染系統，和可誘導之基因表現系統，以供研究梨形鞭毛蟲基因功能之用。
首先我著手建立梨形鞭毛蟲之持續 DNA 轉染系統，將對抗 neomycin 基因置於 ran 基因啟動子之調控下以構築出質體 pRANneo。此質體經電脈衝法轉染至細胞後，持續轉染細胞便可用 G418 篩選出來，而這些持續轉染細胞經測試後發現含有高分子數的游離 pRANneo 質體。我進一步將 gdh 基因啟動子所調控之螢火蟲發光基因插入 pRANneo 而構築出另一質體 pRANneo/GDHluc，經測試後發現此質體亦以游離方式維持於持續轉染細胞中，而 NEO 蛋白質和發光蛋白質二者皆在細胞中有高度的表現，且質體分子數與發光蛋白質表現量皆隨著篩選用之 G418 濃度增加而增加。此系統將是梨形鞭毛蟲研究領域中一個有用的工具，可用來研究某些有興趣的基因之功能。
其次我針對梨形鞭毛蟲 ran 基因啟動子做分析，發現 ran 基因之 5 端和 3 端未轉譯區域接於發光基因時，在短期轉染實驗可促使發光基因大量表現。進一步做刪除及突變分析時，發現位於 ran 基因 5 端未轉譯區域之 -51/-20 多 AT 區域為啟動子活性所必需之處。如插入此 32 鹼基對序列至無啟動子之發光基因前，可活化其表現，因此可知此序列作用如同啟動子，而當插入此序列之10 鹼基對短片段時，則只有較低表現。電泳位移實驗的結果，顯示此 32 鹼基對序列是以單股而非雙股形式來專一地結合某些細胞核蛋白質。用引子延伸法來研究時，發現此 32 鹼基對序列可決定轉錄起始點之位置。這些結果顯示此多 AT 之 32 鹼基對序列在梨形鞭毛蟲之轉錄調控方面扮演極重要的角色。
我第三個研究方向是建立梨形鞭毛蟲之可誘導基因表現系統，此目的在改善前述之持續 DNA 轉染系統，方法是將此系統與細菌之 tet 抑制子及操縱子系統合併。首先將 1 個，2 個，或 3 個 tet 操縱子插入 32 鹼基對 ran 啟動子區域之不同位置來調控發光基因的表現，並以梨形鞭毛蟲中不同的基因啟動子來調控 tet 操縱子的表現。然後這些重要的元素再進一步放入 pRANneo 以構築出不同的質體，並建立這些質體的持續轉染細胞。再進一步分析加入或不加入誘導子四環黴素時，比較發光蛋白質的活性表現，結果發現最佳的誘導效果是由某一質體達成，其含有 2 個 tet 操縱子於 ran 啟動子之下游及 a-giardin 啟動子所調控之 tet 抑制子基因。而發光蛋白質表現的誘導效果，視所加之四環黴素的量與作用時間而定，測試所得之最佳的誘導比率為 50，在加入 10 mg/ml 的四環黴素作用 10 小時可達成。此系統為一改良工具以供梨形鞭毛蟲之研究，可針對欲研究的基因調整其表現，因而可避免過量表現時所造成對細胞的毒害。
這些研究成果，奠定了未來研究轉錄機制的基礎，也提供了做分子研究的重要工具，對梨形鞭毛蟲的領域幫助很大。

Giardia lamblia is an enteric protozoan parasite causing infectious diarrheal diseases worldwide. It also represents an early-diverging eukaryote and exhibits an unusual and fascinating biology. To understand the transcriptional regulation in this primitive organism, I have characterized the promoter region of the ras-related nuclear protein (ran) gene of G. lamblia. In addition, two molecular tools, a stable DNA transfection system and an inducible gene expression system, have also been developed for studies of gene functions in G. lamblia.
I started the study by developing a stable DNA transfection system in G. lamblia. Plasmid pRANneo was constructed by placing a neomycin resistant gene under the control of the ran promoter. After transfection with this plasmid by electroporation, stable transfectants were established under G418 selection. The stable transfectants were found to contain episomal pRANneo molecules in high copy numbers. A luciferase gene directed by the glutamate dehydrogenase (gdh) promoter was inserted into pRANneo to generate another construct, pRANneo/GDHluc. Results showed that this plasmid was maintained episomally and the NEO and luciferase proteins were both highly expressed in the stable transfectants. The plasmid copy numbers and luciferase expression levels were both increased with increasing concentrations of G418. This system will be a useful tool for studying the specific effects of candidate genes in G. lamblia.
I then proceeded to characterize the G. lamblia ran gene promoter. The 5''- and 3''-untranslated regions (UTRs) of the ran gene were found to confer significant activity when linked to a luciferase reporter gene and transiently transfected into G. lamblia. Deletion and mutation analyses revealed that an AT-rich region spanning -51/-20 of the ran 5''-UTR gene was required for promoter activity. This 32-bp element was also sufficient for promoter function when placed upstream of a promoterless luciferase gene, and low levels of expression remained when it was dissected into 10-bp smaller fragments. Electrophoretic mobility shift assays showed that the 32-bp element bound specifically to nuclear proteins in single-stranded but not double-stranded configurations. Results from primer extension studies revealed that the 32-bp element was able to determine the transcription start sites. These data suggest that the 32-bp AT-rich region plays a major role in the transcriptional regulation of the ran gene in G. lamblia.
In order to improve the stable DNA transfection system, I have developed an inducible gene expression system by adapting the bacterial tet repressor-operator system into G. lamblia. One, two, or three tet operators were inserted into the 32-bp ran promoter context at various positions to drive the expression of luciferase reporter gene. Different G. lamblia promoters were also tested to drive the expression of the tet repressor gene. These regulatory elements were incorporated into the pRANneo stable transfection vector and the stable transfectants of the resulting constructs were established. By assaying the luciferase activity in the presence and absence of the inducer tetracycline, it was found that the best inducibility was mediated by the construct containing two tet operators downstream of the ran promoter and the tet repressor gene directed by the a-giardin promoter. The induction of luciferase expression was tetracycline dose- and time-dependent. The optimal induction ratio, ~50, was achieved after 10 hr upon addition of 10 mg/ml tetracycline. This system could be an improved tool for studies in G. lamblia since it allows adjustable expression of a target gene and thereby avoids the potential toxic effects of overexpression.
These studies may form a basis for future investigation of the transcriptional mechanisms and provide valuable tools for molecular studies in G. lamblia.

COVER
Contents
List of Tables
List of Figures
List of Abbreviations
Chinese Abstract
English Abstract
Introduction
G. lamblia is an Important Human Pathogen
G. lambliais an Ancient Eukaryote
Unique Features ofG. lamblia
Life Cycle ofG. lamblia
Objectives
Chapter I Development of a Stable DNA Transfection System
Abstract
Introduction
Materials and Methods
Results
Discussion
Chapter 2 Identification of & ran Gene Promoter
Abstract
Introduction
Materials and Methods
Results
Discussion
Chapter 3 Development of an Inducible Gene Expression System.
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Appendixes

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