陸、 、 、參考文 、 參考文 參考文獻 參考文 獻 獻 獻
E. coli. expression system
pBAD24 ori (pBR322) araC PBAD ampr Guzman et al.(1995)
pBAD33 ori (pACYC184) araC PBAD cmr Guzman et al.(1995)
pTH18kr ori (pSC101) Plac kanr Hashimoto-Gotoh et
al.(2000)
Yeast two-hybrid system
p8op-lacZ lexAop(x8)-lacZ URA3 ampr Clontech
pGilda PGAL1 HIS3 ampr; expresses LexA DNA binding domain (BD) Clontech pB42AD PGAL1 TRP1 ampr; expresses B42 polypeptide activation domain (AD), HA
epitope tag
Clontech
Others
pCP20 FLP+ λcI857+ λPR Repts ampr cmr Cherepanov et al.(1995) pKD46 exo, bet, gam ParaB repA101ts oriR101 ampr Datsenko et al.(2000) pSUB11 Epitope tagging plasmid carrying 3x FLAG tag and kanamycin resistance cassette Uzzau et al.(2001)
表三、本論文所使用的引子對 Table 3. Primers used in this study.
Primers DNA sequences (5' 3') Notes
sulA-3xFLAG_Fw ACAACTTTCCGGGCTAAAAATTCACTCTAATTTGTATCATGACTACAAAGACCATGACGG a.
sulA-3xFLAG_Rv CAAAAAAAGTTCCAGGATTAATCCTAAATTTACTTAATGACATATGAATATCCTCCTTAG a.
sulA(Fw) CCAACGTTGCCAGGTATTCT Colony PCR
sulA(Rv) ATCCTTCACCGGGGGATCT Colony PCR
5' sulA EcoRI CGGAATTCGAATTCATGTACACTTCAGGCTATGCACATC b.
3' sulA BamHI GGGGGATCCGGATCCTTAATGATACAAATTAGAGTG b.
3' sulA delete10 GGGGGATCCGGATCCTTAGGAAAGTTGTCTCGTGGC b.
3' sulA delete20 GGGGGATCCGGATCCTTAGCTTACCGGACGCATAAT b.
3' sulA delete30 GGGGGATCCGGATCCTTAGTTACCTTCATTTGCCGC b.
3' sulA delete45 GGGGGATCCGGATCCTTAAGTCAAATCATCTGCCAA b.
3' sulA delete20-45 AGAGGATGCAGTCAAATCATCTGCCAACCAACCGAT
5' sulA delete20-45 GGTTGGTTGGCAGATGATTTGACTGCATCCTCTCACGCCACGAGACAACTTTCC 3' sulA delete20-30 AGAGGATGCGTTACCTTCATTTGCCGCATCAACAAG
5' sulA delete20-30 GTTGATGCGGCAAATGAAGGTAACGCATCCTCTCACGCCACGAGACAACTTTCC 5' sulA delete30-45 GTTGGCAGATGATTTGACTGCTATGGGGTTTATTATGCGTCCGGTAAGC
3' sulA delete30-45 ATAATAAACCCCATAGCAGTCAAATCATCTGCCAACCAACCGATCAC
Continued on following page
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表三、本論文所使用的引子對 (接續上頁) Table 3. Primers used in this study. (Continued)
Primers DNA sequences (5' 3') Notes
sulA F143Y fw TATGGGGTATATTATGCGTCCGGTAAGCGCATC c.
sulA F143Y rv GGACGCATAATATACCCCATAGCGTTACCTTCATTT
sulA F143A fw TATGGGGGCTATTATGCGTCCGGTAAGCG c.
sulA F143A rv GACGCATAATAGCCCCCATAGCGTTACCTTCATTT
sulA I144N fw TATGGGGTTTAATATGCGTCCGGTAAGCGCAT c.
sulA I144N rv CGGACGCATATTAAACCCCATAGCGTTACCTTCAT
sulA M145I fw GGTTTATTATTCGTCCGGTAAGCGCATCCTCTCAC c.
sulA M145I rv CTTACCGGACGAATAATAAACCCCATAGCGTTACC
sulA R146L fw GGTTTATTATGCTTCCGGTAAGCGCATCCTCTCAC c.
sulA R146L rv CTTACCGGAAGCATAATAAACCCCATAGCGTTACC
sulA R156L fw CCACGCTACAACTTTCCGGGCTAAAAATTCA c.
sulA R156L rv AAAGTTGTAGCGTGGCGTGAGAGGATGCGCTTA
sulA N136LN139L fw TGCGGCACTTGAAGGTCTCGCTATGGGGTTTATTATGCG c.
sulA N136LN139L rv CCATAGCGAGACCTTCAAGTGCCGCATCAACAAGTTCA
a. Primers were designed to anneal to the beginning of the 3xFLAG-coding sequence (fw) and on the opposite side from the KnR cassette (rv) of template plasmid pSUB11 (in italics), and carry extensions homologous to the last 43 nts of the sulA gene coding sequence (fw) and to a region downstream from it (rv). fw, forward; rv, reverse.
b. Specific restriction sites are underlined.
c. The positions of mutantions are shown in bold.
表四、在酵母菌雙雜交系統中 SulA 突變蛋白與 ClpY 之交互作用情形 Table 4. Interaction between SulA mutants and ClpY in yeast two-hybrid system.
pB42ADa- SulA mutants
pGildab-clpY*M187I
β-galactosidase unitsd (U) X-gal teste LEU2f Control
c. It was just AD domain as the negative control.
d. β-galactosidase activity presented as Miller units (Miller, 1972).
e. LacZ expression with colony color was evaluated on Gal/Raf/-Ura/-His/-Trp plates containing X-gal over 4 days.
f. LEU2 expression was evaluated on Gal/Raf/-Ura/-His/-Trp/-Leu plates over 4 days. +, the degree of growth compared to positive control. -, no growth.
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表五、SulA 突變蛋白之活性測試及受 ClpYQ 蛋白酶降解之情形
Table 5. The activation assay of the SulA mutants and the degradation by ClpYQ.
pTh18kra- SulA mutants
Activation assaye Degradation by ClpYQf AC3112 strainc YT10010 straind
Control
a. Plasmid pTH18kr that carried sulA mutant gene was transformed into AC3112 or YT10010 strain.
b. There was no sulA gene in plasmid pTH18kr as the negative control.
c. AC3112 strain had the sulA gene in the chromosome.
d. YT10010 strain had no the sulA gene in the chromosome.
e. Activation assay was evaluated on LB plates plus 1 mM IPTG and appropriate kanamycin. Serial dilutions of the bacterial cultures were spotted on the plates.
+, activity and no cell growth. -, no activity and cell growth.
±, incomplete inactivity and cell growth incomplete.
f. Degradation was experimented by Western blotting.
+, can be degraded. -, cannot be degraded. ±,cannot be degraded completely.
A A
B B
Arg156 Arg146
Met145
Ile144 Phe143
Asn136
Asn139
圖一、大腸桿菌之 SulA 結構圖
Figure 1. The structure of SulA from Escherichia coli.
以 PyMol 軟體繪製大腸桿菌之 SulA 結構。A. 紅色為 C-端末 10 個胺基酸，黃色 為 C-端 10 - 20 個胺基酸，綠色 β-strand 結構為 C-端 20 - 30 個胺基酸，藍色 α-helix 結構為 C-端 30 - 45 個胺基酸。B. SulA 點突變蛋白於結構上之相關位置。
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Figure 2. Interaction between SulA ∆C10 / SulA ∆C20 mutants and ClpY in yeast two-hybrid system.
pGilda 帶有 BD domain 與 ClpY 形成融合蛋白，pB42AD 帶有 AD domain 與 SulA 及其各種缺失突變形成融合蛋白，於酵母菌 EGY48 [p8op-lacZ]中進行分析。A.
LacZ expression：β-galactosidase 活性分析。B. LacZ expression：X-gal 測試。C. LEU2 expression：生長測試。SulA ∆C10 和 SulA ∆C20 與 ClpY 均有明顯的交互作用，在 β-galactosidase 活性測試中，SulA ∆C10 的活性增加近一倍，SulA ∆C20 的活性則 提高至六倍；在 X-gal 培養基上均呈現藍色菌落，在缺乏 leucine 的培養基中均可 正常生長。D. 在西方墨點法中以 anti-HA 抗體，偵測酵母菌中 AD domain 與 SulA 形成之融合蛋白表現情形，各種 SulA 缺失突變蛋白表現量均相一致。
A
B
C
0.0 50.0 100.0 150.0 200.0 250.0
pB42AD pB-sulA ΔC30 ΔC45 ΔC20-30 ΔC30-45 ΔC20-45
ββββ-galactosidase unit
pGilda-Y*M187I
pGilda-ClpY*M187I
pGilda-ClpY*M187I
D
SulA
圖三、以酵母菌雙雜交系統測試二級結構缺失之 SulA 突變蛋白與 ClpY 交互作用 情形
Figure 3. Interaction between SulA deletion mutants and ClpY in yeast two-hybrid system.
pGilda 帶有 BD domain 與 ClpY 形成融合蛋白，pB42AD 帶有 AD domain 與 SulA 及其各種缺失突變形成融合蛋白，於酵母菌 EGY48 [p8op-lacZ]中進行分析。A.
LacZ expression：β-galactosidase 活性分析。B. LacZ expression：X-gal 測試。C. LEU2 expression：生長測試。不論何種 SulA 突變蛋白與 ClpY 之間皆不具交互作用，在 β-galactosidase 活性測試中沒有活性表現，在 X-gal 培養基上均呈現白色菌落，在 缺乏 leucine 的培養基中無法正常生長。D. 在西方墨點法中以 anti-HA 抗體，偵測 酵母菌中 AD domain 與 SulA 形成之融合蛋白表現情形，各種 SulA 缺失突變蛋白 表現量均相一致。
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A A
EEEHAELVDAANEGNAMGFIMRPVSASSHATRQLSGLKIHSNLYH
40 30 20 10
13 hydrophobic aa 7 hydrophobic aa
B B
amino-acid position
surface-burial score
SulA 20
圖四、大腸桿菌 SulA 之 C 末端序列分析
Figure 4. Analysis of the sequence of SulA C-terminal region from E. coli.
A. SulA 之 C 末端 45 個胺基酸序列，底線表示為非極性胺基酸。
B. 以軟體計算 SulA 之 surface-burial scores，值愈高表示具有較高的疏水性質。陰