臺灣博碩士論文加值系統

出血性大腸桿菌O157:H7為一種致病性大腸桿菌，其所攜帶的LEE致病基因島嶼是形成第三型分泌系統的重要致病基因，主要由五個操縱子所構成。位於其中lee3操縱子的第一個基因位置的mpc，在先前的研究指出當其表現過量時具有負向影響第三型分泌系統的能力；此外，其本身的轉譯過程對其他基因有重要的調控功能，惟其中的機制尚待瞭解。在本研究中，我們分析了lee3操縱子以及其轉譯後的產物，我們發現mpc的轉譯對其緊鄰的下游基因escV有決定性的影響，而escV又能接著影響下游的escN基因，escN再接續影響了更下游基因的表現。我們也更進一步分析了mpc的開放閱讀框架，並發現其中第1至100的核苷酸對於mpc影響下游基因表現的現象扮演最關鍵的角色。另外，藉由即時定量聚合酶連鎖反應的分析，我們發現mpc的RNA量遠低於其他同樣位於lee3操縱子的基因。綜合來看，我們認為當LEE致病基因島嶼的表現被啟動時，mpc的表現將在RNA層次受到嚴格的調控，使其轉譯的量足以讓下游基因表現，同時又無法過量表現，以達到第三型分泌系統的正常運作。

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) carries a pathogenic island LEE that is consisted mainly of five polycistronic operons. In the lee3 operon, mpc is the first gene and has been reported to down regulate the type-3 secretion system of EHEC when its gene product is over-expressed. Furthermore, mpc has been suggested to have a regulation function via translation but the mechanism remains unclear. To clarify this hypothesis, we dissected the polycistron and examined the translated products. We conclude that translation of mpc detrimentally governs the translation of the second gene, escV, which in turn affects the translation of the third gene, escN. Then sequentially, escN affects the expression of the downstream genes. Furthermore, we located a critical cis element within the mpc open-reading frame that plays a negative role in the translation-dependent regulation of lee3. Using qRT-PCR, we found that the amount of mpc RNA transcript present in EHEC was relatively limited when compared to any other genes within lee3. Taken together, when the transcription of LEE is activated, expression of mpc is tightly controlled by a restriction of the RNA transcript of mpc, translation of which is then critical for the efficient production of the operon’s downstream gene products.

Content i
Abstract 1
中文摘要 2
Introduction 3
Materials and methods 8
BACTERIAL STRAINS AND CULTURE CONDITIONS 8
PRIMERS AND EXPRESSION PLASMIDS 8
CONSTRUCTING THE PM-V-N VARIANTS 9
PROTEINS EXPRESSION AND WESTERN BLOTTING ANALYSIS 10
RNA ISOLATION AND REAL-TIME QUANTITATIVE RT-PCR (QRT-PCR) 12
Results 13
TRANSLATION BLOCKAGE OF MPC IS DETRIMENTAL TO EXPRESSION OF DOWNSTREAM ESCV AND ESCN 13
ESCA, THE THIRD GENE ON LEE3 OPERON, IS AFFECTED BY THE TRANSLATION INITIATION OF MPC 14
TRANSLATION INITIATION OF MPC IS CRUCIAL FOR EFFICIENT PRODUCTION OF THE REST OF LEE3 GENES 15
VICINITY EFFECTS IN LEE3 OPERON VARIES IN DEGREE 17
ESCN IS MORE PIVOTAL TO SUCCEEDING GENE EXPRESSION THAN ESCA AND ESCP 20
A CIS ELEMENT WITHIN MPC REGULATES DOWNSTREAM GENE EXPRESSION. 22
TRANSCRIPTION OF THE MRNA CODING FOR MPC IS AT THE LOWEST OF ALL LEE3-ORFS DURING T3SS ACTIVATION. 27
Discussion 29
Table 36
TABLE 1. PRIMERS USED AND THEIR SEQUENCES. 36
References 39
Figure 44
FIG. 1. TRANSLATION INITIATION OF MPC IS REQUIRED FOR EFFICIENT EXPRESSION OF ESCV AND ESCN. 44
FIG. 2. EXPRESSION OF ESCA IS AFFECTED BY THE TRANSLATION INITIATION OF MPC. 46
FIG. 3. TRANSLATION INITIATION OF MPC GOVERN THE EXPRESSION OF DOWNSTREAM ESCP, SEPQ, AND ESPH. 47
FIG. 4. VICINITY EFFECTS WITH DIFFERENTIAL EXTENT WERE OBSERVED BETWEEN LEE3 GENE CISTRONS. 50
FIG. 5. TRANSLATION BLOCKAGE OF ESCN IN THE MIDDLE OF A TRANSCRIPT REDUCED THE EXPRESSION OF ESCN AND THE GENES DOWNSTREAM. 51
FIG. 6. TRANSLATION OBSTRUCTION OF ESCA AND ESCP IN THE MIDDLE OF A POLYCISTRONIC TRANSCRIPT IS LESS INFLUENTIAL TO DOWNSTREAM GENES THAN ESCN. 53
FIG. 7. CIS ELEMENT WITHIN NUCLEOTIDES 1-100 OF MPC IS CRITICAL FOR AFFECTING THE EXPRESSION OF DOWNSTREAM ESCN. 54
FIG. 8. MRNA OF ESCN REDUCED WHEN TRANSLATION OF UPSTREAM MPC WAS OBSTRUCTED. 56
FIG. 9. CIS ELEMENT IN THE 5’-END OF ESCV CONTRIBUTES TO THE VICINITY EFFECT ON ESCA. 57
FIG. 10. NO OBVIOUS CIS ELEMENT IN ESCN WAS FOUND IMPORTANT TO EXERT VICINITY EFFECT ON DOWNSTREAM ESCA. 59
FIG. 11. MRNA RESPONSIBLE FOR MPC WAS THE MOST LIMITED ONE AMONG LEE3 GENES. 61
FIG. 12. DIAGRAM OF THE LEE3 OPERON AND THE FEATURES FOUND THEREIN. 62
FIG. 13. MODEL OF MPC TRANSLATION INITIATION EXERTING EFFECTS ON EXPRESSION OF DOWNSTREAM LEE3 GENES. 64
Appendix 64
APPENDIX 1. VIRULENCE FACTORS OF EHEC AND LOCATIONS OF THEIR GENETIC ELEMENTS. 65
APPENDIX 2. SCHEMATIC DISPLAY OF EHEC T3SS COMPLEX. 65
APPENDIX 3. SCHEMATIC REPRESENTATION OF GENE ARRANGEMENT ON LEE ISLAND. 66

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