«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»
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coli and the At2g30210 gene. Codon usage is expressed as the fraction of all possible codons for a given amino acid. “All genes” is the fraction represented in all 4,290 coding sequences in the E. coli genome. “Class II” is the fraction represented in 195 genes highly and continuously expressed during exponential growth. Table is modified from Novy et al. (2001).
76 Figure 2.1. Expression profile of LMCOs in Arabidopsis thaliana.
LMCO gene expression data in selected Arabidopsis tissues obtained using from massively parallel signature sequencing (MPSS) was collected from the Arabidopsis MPSS website (http://mpss.udel.edu/at/). Values represent the pooled number of times the predominant signature tag for each Arabidopsis LMCO gene was seen in MPSS libraries from various Arabidopsis tissues.
77 Figure 2.2. Structure of the Arabidopsis At2g30210 gene. The Genomic DNA sequence of At2g30210 is shown (A). Introns (gray) and the untranslated regions (blue and underlined) are represented in lowercase letters. A putative TATA box is shown in boldface. Intron positions were based on cDNA sequence compared with genomic sequence. Relative locations of the signal peptide (red) and copper-binding domains (yellow) in the At2g30210 gene are shown (B). Exon color indicates featured domains.
1287044 aataaattgt ttaattgcta acgaattcac gaagacctct gttgtttccc gcttagtgtg 1286984 actccttcct tttctccaaa ctcatctcac tataaatacc aaatctccaa cacagagagc
ATGGAGTCTT TTCGGCGATT CTCCTTGCTA TCCTTCATTG1286924 ttctcacagt gaagcaaaca
1286864 CCCTACTTGC CTACTTCGCT TTCCTCGCTT CTGCTGAACA TCACGTCCAT CAATTCGTGg1286804 taagctagct actaatgtta cattaaagct ttttggtatg ttcttacgtt tatagaattc 1286744 ccttaattta agtttaaacg acgatgactt tagATCACAC CGACACCAGT GAAGAGGCTG
1286684 TGCAGAACTC ACCAAAGCAT CACTGTGAAT GGTCAGTACC CTGGTCCAAC GCTTGTGGTC
1286624 AGGAACGGTG ACTCTCTCGC AATCACTGTC ATCAACAGAG CCCGTTACAA CATTAGTATT1286564 CATTGgtaaa atatttgacc atcaaattca acaaacatat gttatgttat gttctctgtt 1286504 ttcatacttt gttttttgct ctgttttttg ggatagGCAT GGAATCAGAC AGCTGCGGAA
1286444 TCCGTGGGCC GATGGTCCAG AGTATATAAC ACAATGTCCG ATCCGTCCAG GACAAACCTA
1286384 CACTTACAGA TTCAAAATCG AGGATCAAGA GGGTACGCTT TGGTGGCACG CTCATAGCCG
1286324 CTGGCTCAGA GCCACGGTCT ATGGTGCTCT CATCATTTAC CCTCGTCTTG GTTCTCCTTA1286264 TCCCTTCTCT ATGCCCAAAC GTGACATTCC AATTCTTCTT Ggtaaacaac taaacatttt 1286204 gtaacttcta atcacttcaa attatatttt catgatcaca actaataata cttactaaaa 1286144 tagGGGAATG GTGGGATAGA AACCCAATGG ATGTTTTGAA GCAAGCACAA TTTACGGGAG
1286084 CAGCAGCTAA TGTCTCTGAC GCTTACACAA TCAACGGTCA ACCAGGCGAT CTTTACCGCT
1286024 GCTCGCGGGC TGGGACAATC CGTTTTCCAA TTTTCCCCGG GGAGACGGTG CAACTCCGTG
1285964 TCATCAACGC TGGTATGAAC CAAGAGCTCT TCTTCTCAGT CGCCAACCAC CAGTTCACAG
1285904 TTGTAGAAAC TGATTCCGCC TACACGAAAC CATTCACCAC AAATGTCATC ATGATCGGTC
1285844 CTGGCCAAAC CACTAACGTC CTCCTCACGG CAAACCAGAG ACCAGGCCGC TACTACATGG
1285784 CAGCTCGAGC CTACAACAGC GCAAACGCCC CGTTCGACAA CACAACCACT ACTGCTATCT
1285724 TACAATACGT CAACGCTCCA ACAAGACGTG GCCGTGGTCG TGGTCAAATC GCTCCTGTTT
1285664 TCCCAGTCCT CCCCGGGTTC AACGACACCG CAACCGCAAC TGCTTTCACC AACCGTCTCC
1285604 GATACTGGAA ACGAGCTCCA GTACCACAAC AAGTCGACGA GAACCTCTTT TTCACCGTCG
1285544 GATTAGGGCT AATCAACTGT GCCAACCCAA ACAGTCCCCG TTGCCAAGGT CCTAACGGGA
1285484 CCCGATTCGC AGCAAGCATG AACAACATGT CCTTCGTGCT ACCACGAAGT AACTCCGTCA
1285424 TGCAAGCATA TTACCAAGGC ACCCCAGGAA TCTTCACAAC GGATTTTCCG CCCGTTCCAC
1285364 CGGTGCAATT CGATTACACA GGTAACGTTA GCCGCGGGTT ATGGCAGCCC ATAAAAGGAA
1285304 CCAAAGCTTA CAAGCTTAAG TACAAATCTA ATGTTCAGAT TGTGTTACAA GACACTAGCA
1285244 TTGTCACGCC AGAGAATCAT CCCATGCATC TACACGGGTA CCAATTCTAC GTGGTCGGGT
1285184 CAGGTTTCGG TAATTTCAAC CCGAGAACAG ACCCGGCTAG GTTTAACTTA TTTGACCCAC
1285124 CAGAGAGGAA CACCATTGGA ACACCTCCAG GTGGTTGGGT GGCAATTCGG TTCGTCGCTG1285064 ATAATCCAGg ttagatgatc ttattgactc gaaagcattt gatactttgt aaccataggt 1285004 taaatgatct tattgactca aaaatgaaaa acatattttg atatacttgt ggtgtaatgc
1284944 agGAGCATGG TTTATGCATT GTCACATTGA TTCACATTTG GGATGGGGTT TGGCTATGGT
1284884 TTTCTTGGTA GAGAACGGTC GTGGACAGTT GCAATCGGTG CAGGCTCCAC CATTGGATCT1284824 TCCAAGATGC TAAtaaaaaa cttctaccgt tggatctaaa gaatttatgg ggtttggttt 1284764 tttgttcttg ttttttaatc aaaaaaagac ttcgtgagtc ttgatatgtt aaagatggac 1284704 aactgctcta ttgaaagtgg ttcaaccttt gttttcgatg tacttttgtt attttcctcg 1284644 taatagagtg agtgttttca tttttccgat taaaaattta ccaaataaaa gctgtgttag ttagttatta acttaatatg 1284584 ttaatcttct ttttctagct gactttccca aacaacttta 1284444 aacccacaag atacttttaa ttacattatc atacataaac ttttcttgca aaaatagcat B.
5’ 3’ 79 Figure 2.3. Possible secondary structure in the At2g30210 LMCO 5’UTR. An optimal secondary structure based on the minimum free energy was predicted using RNAfold. The 5’UTR sequence was directly submitted to the RNAfold program at a web interface server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (Hofacker et al. 1994).
80 Figure 2.4. Heterologous expression of At2g30210 in E. coli.
Aliquots (10µl) of crude sample lysates and different purification steps were loaded onto gels for SDS-PAGE and immunoblot analysis, respectively. A. Crude extracts from transformed (Lane 1) and control cells (Lane 2) after four hours of induction were analyzed by 10% SDS-PAGE (left panel) and immunoblot with an antibody against XpressTM tag (right panel).
B. Purification was performed with an affinity purification column under denaturing conditions using 8M urea buffer. Each step of the purification was collected and analyzed by 10% SDSPAGE (left panel) and immunoblot (right panel). Lane 1: crude
extract: Lane 2: flow through of binding buffer, pH 7.8; Lane 3:
flow through of wash buffer, pH 6.0; Lane 4: flow through of wash buffer, pH 5.3; Lane 5~10: a series of fractions eluted using elution buffer, pH 4.0. Samples were analyzed by SDS-PAGE (left). C. Lane 1: molecular weight marker; Lane 2: crude lysate of induced cells; Lane 3: unbound fraction of sample through affinity column; Lane 4: pooled final elution using wash buffer (pH 4.0); Lane 5: sample after dialysis against 10mM Tris, pH 8.0, 0.1% triton X-100 overnight at 4 oC; Lane 6: insoluble pellet after dialysis; Lane 7: 1 µg BSA: Lane 8: 5 µg BSA.
Enzyme activity analysis of the At2g30210 LMCO expressed in tobacco cells. A. Phenoloxidase activity was demonstrated in three kanamycin-resistant cell lines by testing the ability to oxidize the laccase substrate, ABTS, in liquid assays. B. Activity staining of multicopper oxidases separated using SDS-PAGE. Crude protein extracts from three cell lines (2,3,5) transformed with the AT2g30210 gene and a vector control cell line (C) were stained with 1,8-diaminonaphthalene (DAN) for phenoloxidase activities.
83 Figure 2.6. Testing the At2g30210 protein for ferroxidase activity. A. Partially purified protein (20 µg) from one resistant cell line (#2) was used for phenoloxidase staining with DAN (left panel). For detecting ferroxidase activity (right panel), the amount loaded for the sample was 10x that loaded (200 µg, Lane #2) for phenoloxidase staining and an equivalent amount of ABTS-oxidizing activity from the yacK protein (Lane yacK) was used as a positive control (Kim et al. 2001). Cleared zones representing ferroxidase activity were visualized by applying 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4triazine (ferrozine). B. Solution assay for ferroxidase activity was determined using ferrous sulfate as the electron donor and ferrozine as a specific chelator to bind ferrous iron remaining at the end of the reaction. Aliquots of 3 µg protein from tobacco cells transformed with At2g3210 cDNA (#2) or vector alone (Vec) were added to a total 1 mL reaction. Reaction buffer (Buffer) and a boiled sample (Boiled #2) were also included for negative control. Another set of samples were also analyzed in the same buffer with addition of 1 µM apo-transferrin (+Tf), as a Fe3+ acceptor to promote reactions.
0.5 0 0 50 100 150 200 250 300
85 Figure 2.7. Expression profile of the At2g30210 gene in various tissues at different developmental stages. A. Messenger RNA isolated from the identified tissues was reverse transcribed, and diluted cDNA product was used as template for each PCR reaction. QUB10 was included to control for equal cDNA input between tissue samples. fl: flowers, sil: siliques, lf: leaves, px: inflorescence, rt: roots, sdl: two weeks old seedling. B.
Gene expression for the At2g30210 gene in selected Arabidopsis tissues as determined using massively parallel signature sequencing (MPSS), as described in Figure 1. C. Affymetrix expression data for the At2g30210 gene represented on the ATH1 whole-genome chip. Microarray data for selected tissues were collected from the public database (https://www.genevestigator.ethz.ch) (Zimmermann et al. 2004)