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Unraveling the stepwise activation mechanism of HacA, the key regulator of the unfolded protein response in Aspergillus niger

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(1)Unraveling the stepwise activation mechanism of HacA, the key regulator of the unfolded protein response in Aspergillus niger Mulder, H.J.. Citation Mulder, H. J. (2010, February 2). Unraveling the stepwise activation mechanism of HacA, the key regulator of the unfolded protein response in Aspergillus niger. Retrieved from https://hdl.handle.net/1887/14647 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/14647. Note: To cite this publication please use the final published version (if applicable)..

(2)  Chapter3     HacA,theTranscriptionalActivatorof theUnfoldedProteinResponsein Aspergillusniger,BindstoPartly PalindromicUPRElements oftheConsensusSequence 5'CAN(G/A)NTGT/GCCT3'    ƒ” Ǥ—Ž†‡”ǡ

(3) ‰‘”‹‘Žƒ‡˜ǡ—•ƒǤƒ†”‹†          —„Ž‹•Š‡†‹ —‰ƒŽ ‡‡–‹ •ƒ†‹‘Ž‘‰›ǤʹͲͲ͸ǡͶ͵ǣͷ͸ͲǦͷ͹ʹ.

(4) Chapter3. Abstract  Š‡’”‘‘–‡”•‘ˆ–ƒ”‰‡–‰‡‡• ‘–ƒ‹ƒ—ˆ‘Ž†‡†’”‘–‡‹”‡•’‘•‡‡Ž‡‡–ȋȌǡ ™Š‹ Š ‘ˆ‡”•–Š‡•–”‡••‹†— ‹„‹Ž‹–›–‘–Š‡‰‡‡ǡ˜‹ƒƒ‹–‡”ƒ –‹‘™‹–Š–Š‡–”ƒ• ”‹’–‹‘ ƒ –‹˜ƒ–‘” Ǥ

(5) –Š‡’”‘‘–‡”•‘ˆ–Š‡AspergillusǦ•–”‡••”‡•’‘•‹˜‡‰‡‡•bipA,cypB, pdiA,prpA,tigA,andhacA,ƒ ‘•‡•—••‡“—‡ ‡™ƒ•‹†‡–‹ˆ‹‡†ǡ™Š‹ Š™ƒ•Ž‘ ƒ–‡† Ž‘•‡–‘ –Š‡ –”ƒ• ”‹’–‹‘ •–ƒ”– •‹–‡ ‘ˆ –Š‡ ‰‡‡ ȋδ ͺͳ „’Ȍǡ ƒ† ‘””‡•’‘†• –‘ –Š‡ •‡“—‡ ‡ ȋ ȀȌ T/GCCTǤ Š‡  ‹• ƒ ’ƒ”–Ž› ’ƒŽ‹†”‘‹  •‡“—‡ ‡ ƒ”‘—† ƒ †‹•’‡•ƒ„Ž‡ •’ƒ ‡” — Ž‡‘–‹†‡ǡ ˆ‘ŽŽ‘™‡† „› ˆ‘—” Š‹‰ŠŽ› ‘•‡”˜‡† „ƒ•‡•Ǥ › ƒ in vitro •‡Ž‡ –‹‘ ’”‘ ‡†—”‡ǡ ƒ ‘’–‹ƒŽ „‹†‹‰ •‹–‡ ˆ‘” ƒ  ™ƒ• ‹•‘Žƒ–‡†Ǥ Š‹• •‡“—‡ ‡ǡ   ǡ ”‡•‡„Ž‡•–Š‡„—–Žƒ •–Š‡•’ƒ ‡”— Ž‡‘–‹†‡Ǥ

(6) –Šƒ•ƒ— ŠŠ‹‰Š‡”„‹†‹‰ƒˆˆ‹‹–› –Šƒ–Š‡‹†‡–‹ˆ‹‡†•ǡƒ†invivo‹–„‡Šƒ˜‡•ƒ•ƒ‘”‡’‘™‡”ˆ—ŽcisǦƒ –‹‰‡Ž‡‡–Ǥ  . Introduction  ‹Žƒ‡–‘—• ˆ—‰‹ǡ •— Š ƒ• Aspergillus ƒ† Trichoderma •’‡ ‹‡• ƒ”‡ ‹’‘”–ƒ– ‹†—•–”‹ƒŽ ’”‘†— –‹‘ ‘”‰ƒ‹•• †—‡ –‘ –Š‡‹” ƒ„‹Ž‹–› –‘ •‡ ”‡–‡ Žƒ”‰‡ ƒ‘—–• ‘ˆ ƒ–‹˜‡ ’”‘–‡‹•Ǥ š’”‡••‹‘‘ˆŠ‡–‡”‘Ž‘‰‘—•’”‘–‡‹•‹•Š‘™‡˜‡”‘ˆ–‡Šƒ’‡”‡†„›Ž‘™‡š’”‡••‹‘›‹‡Ž†• Ͷ͹ǤŽ–Š‘—‰Š•‡˜‡”ƒŽ•–”ƒ–‡‰‹‡••— Šƒ•‰‡‡ˆ—•‹‘•ǡ–Š‡†‡˜‡Ž‘’‡–‘ˆ’”‘–‡ƒ•‡†‡ˆ‹ ‹‡–. Š‘•– •–”ƒ‹•ǡ ƒ† ‘Ǧ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ˆ ˆ‘Ž†ƒ•‡• Šƒ˜‡ •‘‡–‹‡• ‹’”‘˜‡† –Š‡ ›‹‡Ž†• ‘ˆ •‘‡Š‡–‡”‘Ž‘‰‘—•’”‘–‡‹•ǡ‹‘–Š‡” ƒ•‡•‘‹’”‘˜‡‡–™ƒ•ˆ‘—† ͳͺǤ‘”‡†‡–ƒ‹Ž‡† ‹•‹‰Š– ‹ –Š‡ ˆ—‰ƒŽ •‡ ”‡–‘”› ’ƒ–Š™ƒ› ƒ› Ž‡ƒ† –‘ –Š‡ ‹†‡–‹ˆ‹ ƒ–‹‘ ‘ˆ „‘––Ž‡‡ • ƒ† ’”‘„Ž‡•‹Š‡–‡”‘Ž‘‰‘—•’”‘–‡‹’”‘†— –‹‘Ǥ

(7)  ‡—ƒ”›‘–‹  ‡ŽŽ•ǡ ‡™Ž› •›–Š‡•‹•‡† •‡ ”‡–‘”› ƒ† –”ƒ•‡„”ƒ‡ ’”‘–‡‹• ƒ”‡ ˆ‘Ž†‡† ƒ† ƒ••‡„Ž‡† ‹ –Š‡ ‡†‘’Žƒ•‹  ”‡–‹ —Ž— ȋȌǤ Š‡  ’”‘˜‹†‡• ƒ ‘š‹†‹•‹‰ ‡˜‹”‘‡–‹™Š‹ Š’”‘–‡‹ˆ‘Ž†‹‰‹•ƒ••‹•–‡†„›ƒ—„‡”‘ˆ‘Ž‡ —Žƒ” Šƒ’‡”‘‡•ƒ† ˆ‘Ž†‹‰‡œ›‡•͵ͻǡͷ͸Ǥ”‘–‡‹ˆ‘Ž†‹‰‹–Š‡ ƒ„‡ ‘’”‘‹•‡†„›•‡˜‡”ƒŽ‡†‘‰‡‘—• ƒ† ‡š‘‰‡‘—• ˆƒ –‘”• Ž‹‡ Šƒ‰‹‰ ‡˜‹”‘‡–ƒŽ ‘†‹–‹‘•ǡ ‡ŽŽ †‹ˆˆ‡”‡–‹ƒ–‹‘ǡ Š‡‹ ƒŽ ‹•—Ž–ǡ ’ƒ–Š‘‰‡‹  ‹ˆ‡ –‹‘•ǡ ƒ† ‡š’”‡••‹‘ ‘ˆ Š‡–‡”‘Ž‘‰‘—• ’”‘–‡‹• ͳ͵ͺǤ Š‹• —Ž–‹ƒ–‡Ž› ”‡•—Ž–• ‹ –Š‡ ƒ ——Žƒ–‹‘ ‘ˆ —ˆ‘Ž†‡† ’”‘–‡‹• ™‹–Š‹ –Š‡ ǡ •‹ ‡ ‘Ž› ‘””‡ –Ž›ˆ‘Ž†‡†’”‘–‡‹•–”ƒ•‹––Š‡•‡ ”‡–‘”›’ƒ–Š™ƒ› ʹͺǤŠ‡‡—ƒ”›‘–‹  ‡ŽŽ”‡ƒ –•–‘–Š‡ ƒ ——Žƒ–‹‘‘ˆ—ˆ‘Ž†‡†’”‘–‡‹•‹–Š‡„›‹†— ‹‰ƒ’ƒ–Š™ƒ›‘™ƒ•–Š‡—ˆ‘Ž†‡† ’”‘–‡‹”‡•’‘•‡ȋȌ ͸ͶǡͻͶǡͳͷͲǡͳ͹ͻǤŠ‡‹•ƒ˜‡”•ƒ–‹Ž‡”‡‰—Žƒ–‘”›•›•–‡–Šƒ–•‡”˜‡•–‘ ƒ‹–ƒ‹Š‘‡‘•–ƒ•‹•‘ˆˆ— –‹‘•—†‡”•–”‡•• ‘†‹–‹‘•Ǥ . 60CharacterisationoftheUnfoldedProteinResponseElement.

(8) Chapter3 Š‡  ’ƒ–Š™ƒ› Šƒ• „‡‡ ™‡ŽŽ Šƒ”ƒ –‡”‹•‡† ‹ Saccharomyces cerevisiaeǤ Š”‡‡ ‡› ‘’‘‡–• ‘ˆ –Š‹• •‹‰ƒŽ –”ƒ•†— –‹‘ ’ƒ–Š™ƒ› ‹ S. cerevisiae ƒ”‡ǣ ȋͳȌ

(9) ”‡ͳ’ǡ ƒ •‡”‹‡Ȁ–Š”‡‘‹‡’”‘–‡‹‹ƒ•‡™‹–Šƒƒ•‡Ž‹‡‡†‘— Ž‡ƒ•‡†‘ƒ‹Ǥ

(10) ”‡ͳ’ ”‡•‹†‡• ‹–Š‡‡„”ƒ‡ǡƒ†™ƒ•‹†‡–‹ˆ‹‡†ƒ•–Š‡’”‘š‹ƒŽ•‡•‘”‘ˆ–Š‡ ʹͳǡͻ͸ǤȋʹȌ ƒ ͳ’ǡ ƒ„ƒ•‹ Ž‡— ‹‡œ‹’’‡”ȋ„

(11) Ȍ–›’‡‘ˆ–”ƒ• ”‹’–‹‘ˆƒ –‘”–Šƒ–—’Ǧ”‡‰—Žƒ–‡•–Š‡–”ƒ• ”‹’–‹‘ ‘ˆ ˜ƒ”‹‘—• –ƒ”‰‡–Ǧ‰‡‡• ‘ˆ –Š‡  ’ƒ–Š™ƒ›. ʹʹǡͻͷǤ. ȋ͵Ȍ Š‡ —ˆ‘Ž†‡† ’”‘–‡‹ ”‡•’‘•‡. ‡Ž‡‡–ȋȌǡƒcisǦƒ –‹‰‡Ž‡‡–’”‡•‡–‹–Š‡’”‘‘–‡”•‘ˆǦ–ƒ”‰‡–‰‡‡•ǡƒ†ƒ –ƒ”‰‡–ˆ‘” ƒ ͳ’ͻ͹Ǥ

(12) ”‡ͳ’•‡•‡•–Š‡ƒ ——Žƒ–‹‘‘ˆ—ˆ‘Ž†‡†’”‘–‡‹•–Š”‘—‰Šƒ†›ƒ‹ ‹–‡”ƒ –‹‘ ™‹–Š –Š‡ ‘Ž‡ —Žƒ” Šƒ’‡”‘‡ ‹ ͺǡͳͳͷǤ ‡Ž‡ƒ•‡ ‘ˆ ‹ –”‹‰‰‡”• –Š‡ †‹‡”‹•ƒ–‹‘ ƒ† •—„•‡“—‡– –”ƒ•Ǧ’Š‘•’Š‘”›Žƒ–‹‘ ‘ˆ

(13) ”‡ͳ’ǡ –Š‡”‡„› ƒ –‹˜ƒ–‹‰ ‹–• ƒ•‡ Ž‹‡ ‡†‘— Ž‡ƒ•‡ †‘ƒ‹Ǥ Š‹• —Ž–‹ƒ–‡Ž› ”‡•—Ž–• ‹ –Š‡ ƒ –‹˜ƒ–‹‘ ‘ˆ HAC1 ǡ „› –Š‡ •’Ž‹ ‹‰‘ˆƒ— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘–Š‡‡••‡‰‡” Ͷ͵ǡͳͷʹǤŠ‡•’Ž‹ ‡†HAC1‹• ‡ˆˆ‹ ‹‡–Ž›–”ƒ•Žƒ–‡†ǡ”‡•—Ž–‹‰‹–Š‡ƒ–—”‡ˆ‘”‘ˆ ƒ ͳ’ǡ–Š‡–”ƒ• ”‹’–‹‘ƒŽƒ –‹˜ƒ–‘”‘ˆ –Š‡ͳͶǡ͸͸ǡͳ͵͹Ǥ Ž–Š‘—‰Š –Š‡ ’ƒ–Š™ƒ› ‹• — Š ‘”‡ ‡Žƒ„‘”ƒ–‡ ‹ Š‹‰Š‡” ‡—ƒ”›‘–‡•ǡ –Š‡ ‡–”ƒŽ ƒ –‹˜ƒ–‹‘•–‡’‘ˆ–Š‡–”ƒ• ”‹’–‹‘ƒŽƒ –‹˜ƒ–‘”‘ˆ–Š‡„›— ‘˜‡–‹‘ƒŽ•’Ž‹ ‹‰‘ˆ‹–•  Šƒ• „‡‡ ‘•‡”˜‡† ͳͻͳǤ Ž•‘ ‹ –Š‡ ˆ‹Žƒ‡–‘—• ˆ—‰‹ Aspergillus nigerǡ Aspergillus nidulans ƒ† Trichoderma reeseiǡ ƒ –‹˜ƒ–‹‘ ‘ˆ –Š‡  ‹˜‘Ž˜‡• — ‘˜‡–‹‘ƒŽ ‹–”‘ •’Ž‹ ‹‰ ˆ”‘ –Š‡ hacA/hac1 Ǥ ‘™‡˜‡”ǡ ‹ ƒ††‹–‹‘ –‘ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ •’Ž‹ ‹‰ǡ –Š‡ ͷ̵Ǧ—–”ƒ•Žƒ–‡† ”‡‰‹‘ ȋͷǯȌ ‘ˆ –Š‡ –”ƒ• ”‹’– ‹• –”— ƒ–‡† —’‘  •–”‡•• ͳͲͳǡͳͶͲǤŠ‹• Šƒ”ƒ –‡”‹•–‹ Šƒ•‘–„‡‡”‡’‘”–‡†ˆ‘”–Š‡S.cerevisiaeHAC1ƒ†ƒƒŽ‹ƒ. XBP1Š‘‘Ž‘‰—‡• ͳʹǡ͸͹ǤŠ‡‡ Šƒ‹•„‡Š‹†–Š‡–”— ƒ–‹‘‘ˆ–Š‡ͷǯ‘ˆˆ‹Žƒ‡–‘—• ˆ—‰ƒŽhacA/hac1‹••–‹ŽŽ—‘™ǡ„—––Š‹• ‘—Ž†‘ˆˆ‡”–Š‡ˆ—‰—•ƒ‡š–”ƒ ‘–”‘Ž ’‘‹–‹–Š‡ƒ –‹˜ƒ–‹‘‘ˆ–Š‡ͳͲͳǡͳͶͲǤ ƒŽ›•‹•‘ˆ–Š‡’”‘‘–‡””‡‰‹‘•‘ˆ•‡˜‡”ƒŽ–ƒ”‰‡–‰‡‡•Šƒ•”‡˜‡ƒŽ‡††‹ˆˆ‡”‡– cisǦƒ –‹‰ ‡Ž‡‡–• ‹ S. cerevisiae ƒ† ƒƒŽ‹ƒ ‡ŽŽ•Ǥ

(14)  S. cerevisiaeǡ –Š‡ KAR2  ‘””‡•’‘†•–‘   ǡƒ’ƒ”–Ž›’ƒŽ‹†”‘‹ •‡“—‡ ‡•‡’ƒ”ƒ–‡†„›ƒ•’ƒ ‡”— Ž‡‘–‹†‡ ͻ͹Ǥ

(15) ƒƒŽ•ǡ•‡˜‡”ƒŽ†‹ˆˆ‡”‡–cisǦƒ –‹‰‡Ž‡‡–•™‡”‡‹†‡–‹ˆ‹‡†Ǥ ‘–ƒ‹‹‰. –Š‡ ‘•‡•—••‡“—‡ ‡  . Ȁǡ‹•”‡ ‘‰‹•‡†„› ͸ ͳ͹͸Ǥ™‘•–”‡••”‡•’‘•‡ ‡Ž‡‡–•ǡ ͳ ‘””‡•’‘†‹‰ –‘ ǦͻǦ  ͳ͵͸ǡͳͺͷǡ ƒ† ʹ ‘””‡•’‘†‹‰ –‘ . ǦͻǦ  ͹Ͷ ƒ”‡ –ƒ”‰‡–• ˆ‘” „‘–Š  ͸ ƒ† ͳǤ

(16)  Aspergilli, ƒ ǦŽ‹‡ ‘•‡•—••‡“—‡ ‡Šƒ•‘–„‡‡ Šƒ”ƒ –‡”‹•‡†–Š—•ˆƒ”ǡ„—–•‡˜‡”ƒŽ”‡•’‘•‹˜‡‰‡‡• Šƒ˜‡ „‡‡ Ž‘‡†ǡ ‘•–Ž› ‡ ‘†‹‰  Ž‘ ƒŽ‹•‡† ’”‘–‡‹ ˆ‘Ž†ƒ•‡•Ǥ Š‡ –”ƒ• ”‹’–‹‘ƒŽ „‡Šƒ˜‹‘—” ‘ˆ –Š‡•‡ ‰‡‡• —’‘  •–”‡•• ‹†‹ ƒ–‡• –Š‡ ’”‡•‡ ‡ ‘ˆ ƒ  ‹ –Š‡‹” ’”‘‘–‡”•Ǥ‘‰–Š‡ƒ”‡–Š‡A.niger‰‡‡•bipAǡ‡ ‘†‹‰ƒ‘Ž‡ —Žƒ” Šƒ’‡”‘‡ǡcypB ‡ ‘†‹‰ ƒ ’”‘Ž›ŽǦcistransǦ‹•‘‡”ƒ•‡ǡ ƒ† –Š”‡‡ ‰‡‡• ‡ ‘†‹‰ –Š‡ ’”‘–‡‹ †‹•—Ž’Š‹†‡ ‹•‘‡”ƒ•‡•pdiAǡprpAƒ†tigAʹ͹ǡ͸ʹǡͳͲͶǡͳ͸ͺǡͳ͹ͶǤ CharacterisationoftheUnfoldedProteinResponseElement61.

(17) Chapter3. ‡‘‹ ƒƒŽ›•‡•‹S.cerevisiae,Arabidopsisthalianaƒ†A.nidulansŠƒ˜‡Ž‹‡†ƒŽƒ”‰‡ —„‡” ‘ˆ ‰‡‡• –‘ –Š‡ ǡ ƒˆˆ‡ –‹‰ ˜‹”–—ƒŽŽ› ‡˜‡”› •–ƒ‰‡ ‘ˆ –Š‡ •‡ ”‡–‘”› ’ƒ–Š™ƒ› ͺͻǡͳͷͶǡͳ͸ͲǤ Š‡ „”‡ƒ†–Š ‘ˆ –Š‡  ‡’Šƒ•‹•‡• –Š‡ ‡ ‡••‹–› –‘ —†‡”•–ƒ† –Š‡ ’ƒ–Š™ƒ› ‹. †‡–ƒ‹Žǡ ‹ ‘”†‡” –‘ „‡ ƒ„Ž‡ –‘ ‘’–‹‹•‡ –Š‡ ˆ—‰ƒŽ •‡ ”‡–‘”› ’ƒ–Š™ƒ› ˆ‘” Š‡–‡”‘Ž‘‰‘—• ’”‘–‡‹’”‘†— –‹‘Ǥ

(18)  –Š‹• ’ƒ’‡”ǡ ™‡ †‡• ”‹„‡ –Š‡ ‹†‡–‹ˆ‹ ƒ–‹‘ ƒ† Šƒ”ƒ –‡”‹•ƒ–‹‘ ‘ˆ Aspergillus • ‹ –Š‡ ’”‘‘–‡”• ‘ˆ ˆ‹˜‡  ”‡•‹†‡– Šƒ’‡”‘‡•Ȁˆ‘Ž†ƒ•‡• ƒ† ‹ –Š‡ ’”‘‘–‡” ‘ˆ hacA ‹–•‡ŽˆǤ —”–Š‡”‘”‡ǡ ™‡ †‡‘•–”ƒ–‡† „› —–ƒ–‹‘ ƒƒŽ›•‹• –Š‡ ‹’‘”–ƒ ‡ ‘ˆ –Š‡ Ž‡— ‹‡œ‹’’‡”†‘ƒ‹‘ˆ ƒ ‹„‹†‹‰–Š‡•Ǥ  . MaterialandMethods  StrainsandTransformations A.nigerͶǤͳȋcspA1,pyrGȌ™ƒ•‰”‘™‘‹‹ƒŽ‡†‹ƒ’Žƒ–‡• ‘–ƒ‹‹‰’‡”Ž‹–”‡ǣ͸‰ ƒ͵ǡͳǤͷ‰ ʹͶǡͲǤͷ‰‰Ͷ˜͹ ʹǡͲǤͷ‰Žǡ–”ƒ ‡‡Ž‡‡–•ͳ͹ʹǡͳǤͷΨƒ‰ƒ”ǡƒ†ʹͲ‰ ˆ”— –‘•‡ƒ•ƒ ƒ”„‘•‘—” ‡ȋ’ ͸ǤͲȌǤ”‘–‘’Žƒ•–•™‡”‡–”ƒ•ˆ‘”‡†ƒ ‘”†‹‰–‘•–ƒ†ƒ”† ’”‘ ‡†—”‡• ͹͸ǤEscherichiacoli•–”ƒ‹ͳͲȋ

(19) ˜‹–”‘‰‡Ȍ™ƒ•—•‡†ƒ•ƒŠ‘•––‘’”‘’ƒ‰ƒ–‡ ’Žƒ•‹†•ǡƒ†E.coliʹͳȋ͵Ȍ’›•ȋ‘˜ƒ‰‡Ȍˆ‘”‡š’”‡••‹‘‘ˆ ƒ .  •‡“—‡ ‹‰ ”‡ƒ –‹‘• ™‡”‡ ’‡”ˆ‘”‡† ™‹–Š –Š‡ ‹‰†›‡ •‡“—‡ ‹‰ ‹– ȋ’’Ž‹‡† ‹‘•›•–‡•Ȍƒ†ƒƒŽ›•‡†™‹–Š–Š‡

(20) ”‹•͵ͳͲͲ ‡‡–‹ ƒŽ›•‡”ȋ’’Ž‹‡†‹‘•›•–‡•ȌǤ   Table1.‘Ž‹‰‘— Ž‡‘–‹†‡•—•‡†‹–Š‹••–—†› Oligo ͳ ʹ ͵ Ͷ ͷ ͸ ͹ ͺ. Sequence ͷ̵Ǧ.  .     ͷ̵Ǧ     .  ͷ̵Ǧ. .    ͷ̵Ǧ . .  .   ͷ̵Ǧ.      ͷ̵Ǧ G.     ͷ̵ǦGGGATCCTAGTGGTGGTGGTGGTGGTG .      ͷ̵Ǧ.   .   ǦȋȌʹ͸Ǧ. .       ͻ ͷ̵Ǧ.   .   ǦȋȌͺǦǦȋȌͶǦ ǦȋȌͷǦ. .       ͳͲ ͷ̵Ǧ.   .    ͳͳ ͷ̵Ǧ       ͳʹ ͷ̵Ǧ   C.  ͳ͵ ͷ̵Ǧ   ACCG T   Šƒ‰‡• –‘ –Š‡ ™‹Ž†Ǧ–›’‡ •‹–—ƒ–‹‘ ƒ”‡ •Š‘™ ‹ „‘Ž† ƒ† ‹–”‘†— ‡† ”‡•–”‹ –‹‘ •‹–‡• ƒ”‡ —†‡”Ž‹‡†Ǥ. 62CharacterisationoftheUnfoldedProteinResponseElement.

(21) Chapter3 5’RandomAmplificationofcDNAEnds(5’RACE) ͷǯ™ƒ•’‡”ˆ‘”‡†‘ͳɊ‰‘ˆ–‘–ƒŽ—•‹‰–Š‡ ’Ž‹ˆ‹ ƒ–‹‘‹– ȋŽ‘‡–‡ ŠȌǡƒ ‘”†‹‰–‘–Š‡”‡ ‘‡†ƒ–‹‘•‘ˆ–Š‡•—’’Ž‹‡”Ǥ”‹‡”•ͳǡʹǡ͵ǡͶǡƒ† ͷȋƒ„Ž‡ͳȌ™‡”‡—•‡†–‘ƒ’Ž‹ˆ›bipA,cypB,pdiA,prpA,ƒ†tigA ”‡•’‡ –‹˜‡Ž›Ǥ  TruncationofHacA  ˆ”ƒ‰‡– ‡ ‘†‹‰ –Š‡ ˆ‹”•– ʹͳͶ ƒ‹‘ ƒ ‹†• ‘ˆ ƒ  ȋ ƒ ȋͳǦʹͳͶȌȌ ™ƒ•  ƒ’Ž‹ˆ‹‡† ˆ”‘ hacAi  —•‹‰ ’”‹‡”• ͸ ƒ† ͹ ȋhacAi ”‡ˆ‡”• –‘ –Š‡ ‹†— ‡† ˆ‘” ‘ˆ –Š‡ hacA –”ƒ• ”‹’–ǡ™Š‹ ŠŽƒ •–Š‡ʹͲ–— ‘˜‡–‹‘ƒŽ‹–”‘ǤhacAu”‡ˆ‡”•–‘–Š‡—•’Ž‹ ‡†ǡƒ† –Š‡”‡ˆ‘”‡–Š‡—‹†— ‡†ˆ‘”‘ˆ–Š‡hacAȌǤǦ–‡”‹ƒŽȋŠ‹•Ȍ͸Ǧ–ƒ‰™ƒ•‹ Ž—†‡†ˆ‘” ’—”‹ˆ‹ ƒ–‹‘ ’—”’‘•‡•Ǥ ˆ–‡” •‡“—‡ ‹‰ ‘ˆ –Š‡ ‘•–”— –ǡ ƒ NcoIBamHI ˆ”ƒ‰‡– ™ƒ• Ž‹‰ƒ–‡†‹–‘’Žƒ•‹†’͵†ȋ‘˜ƒ‰‡ȌǤE.coliʹͳȋ͵Ȍ’›•ȋ‘˜ƒ‰‡Ȍ™ƒ•–”ƒ•ˆ‘”‡† ™‹–Š–Š‡”‡•—Ž–‹‰’Žƒ•‹†–‘‡š’”‡••–Š‡–”— ƒ–‡† ƒ ’”‘–‡‹ǤŠ‡’”‘–‡‹™ƒ•’—”‹ˆ‹‡† —†‡” ƒ–‹˜‡ ‘†‹–‹‘• „› ƒˆˆ‹‹–› Š”‘ƒ–‘‰”ƒ’Š› —•‹‰ ‹Ǧ ƒ‰ƒ”‘•‡ ‘Ž—• ȋ‡”•Šƒ Šƒ”ƒ ‹ƒ ‹‘–‡ ŠȌǤ ”‘–‡‹ ‘ ‡–”ƒ–‹‘• ™‡”‡ †‡–‡”‹‡† ƒ ‘”†‹‰ –‘ •–ƒ†ƒ”†’”‘ ‡†—”‡•ͻ—•‹‰„‘˜‹‡•‡”—ƒŽ„—‹ȋȌƒ•ƒ•–ƒ†ƒ”†Ǥ  ElectrophoreticMobilityShiftAssay(EMSA) Ž‹‰‘— Ž‡‘–‹†‡ ’”‹‡”• ™‡”‡ ƒ‡ƒŽ‡† ‹ ͳͲ  

(22) Ǧ Ž ȋ’  ͹ǤͷȌ ‘–ƒ‹‹‰ ͳͲͲ ƒŽǡ„›Š‡ƒ–‹‰–Š‡‹š–—”‡•–‘ͻͷ郐† ‘‘Ž‹‰–Š‡†‘™–‘”‘‘–‡’‡”ƒ–—”‡Ǥͳ’‘Ž ‘ˆ ƒ‡ƒŽ‡† ‘Ž‹‰‘ ™ƒ• ‡†ǦŽƒ„‡ŽŽ‡† ™‹–Š ɀǦ͵ʹ —•‹‰ Ͷ ‘Ž›— Ž‡‘–‹†‡ ‹ƒ•‡ ȋ‡™ ‰Žƒ†‹‘Žƒ„•Ȍǡƒ†’—”‹ˆ‹‡†‘‹‘‰‡Ž͵Ͳ•’‹ ‘Ž—•ȋ‹‘ƒ†ȌǤ‹†‹‰”‡ƒ –‹‘•ȋʹͲ ɊŽȌ ™‡”‡ ’‡”ˆ‘”‡† ‹ –Š‡ ˆ‘ŽŽ‘™‹‰ „‹†‹‰ „—ˆˆ‡”ǣ ʹͷ  

(23) Ǧ Ž ȋ’  ͺǤͲȌǡ ͷ  †‹–Š‹‘–Š”‡‹–‘Žǡ ͳͲͲ  Žǡ Ͷ  •’‡”‹†‹‡ǡ ͲǤͳ Ɋ‰ȀɊŽ ’‘Ž›ȏ†ȋ

(24) ǦȌȐǡ ͲǤʹͷΨ ǡ ͷΨ ‰Ž› ‡”‘Žǡƒ†‹ Ž—†‡†ͳŽƒ„‡ŽŽ‡†’”‘„‡ǡƒ†˜ƒ”‹ƒ„Ž‡ƒ‘—–•‘ˆ’—”‹ˆ‹‡† ƒ ’”‘–‡‹Ǥ ‹†‹‰ ™ƒ• ƒŽŽ‘™‡† –‘ ’”‘ ‡‡† ‘ ‹ ‡ ˆ‘” ͳͷ ‹Ǥ Š‡ ”‡ƒ –‹‘• ™‡”‡ Ž‘ƒ†‡† ‘ ƒ ͸Ψ ’‘Ž›ƒ ”›Žƒ‹†‡‰‡Žƒ†‡Ž‡ –”‘’Š‘”‡•‡†ƒ–ͳͲͲ‹ͲǤʹͷšȋͺͻ

(25) Ǧ ŽǡʹͲ ǡͺͻ‘”‹ ƒ ‹†ǡ’ ͺǤ͵ȌǤ  SelectionforOligonucleotideswithHighAffinityforHacA ‹†‹‰ ”‡ƒ –‹‘• ™‡”‡ ’‡”ˆ‘”‡† ƒ• †‡• ”‹„‡† „‡ˆ‘”‡ǡ ™‹–Š ”ƒ†‘ ‘Ž‹‰‘ ͺ ƒ† –Š‡ ’ƒ”–Ž› ”ƒ†‘ ‘Ž‹‰‘— Ž‡‘–‹†‡ ͻǤ Š‡ ‘’Ž‡‡–ƒ”›  •–”ƒ† ‘ „‘–Š ‘Ž‹‰‘• ™ƒ• ‰‡‡”ƒ–‡† ‹ ƒ Ž‡‘™ ”‡ƒ –‹‘ –‘‰‡–Š‡” ™‹–Š ‘Ž‹‰‘ ͳͳǤ ‘ ‡•—”‡ •–”‹‰‡– •‡Ž‡ –‹‘ ‘†‹–‹‘•ǡ ͳͲ  ‘ˆ –Š‡ cypB  ‘Ž‹‰‘ ™ƒ• ‹ Ž—†‡† ‹ –Š‡ „‹†‹‰ ”‡ƒ –‹‘Ǥ ˆ–‡” „‹†‹‰ǡ –Š‡ ƒ Ǧ‘Ž‹‰‘ ‘’Ž‡š ™ƒ• ’—”‹ˆ‹‡† „› ƒˆˆ‹‹–› Š”‘ƒ–‘‰”ƒ’Š› —•‹‰ ‹Ǧ ƒ‰ƒ”‘•‡‹‹ ‘Ž—•ǤŠ‡ ‘Ž—•™‡”‡™ƒ•Š‡†‡š–‡•‹˜‡Ž›™‹–Š„—ˆˆ‡” ‘–ƒ‹‹‰ʹͷ 

(26) Ǧ Ž ȋ’  ͺǤͲȌǡ ͷ  †‹–Š‹‘–Š”‡‹–‘Ž ȋȌǡ ͳͲͲ  Žǡ Ͷ  •’‡”‹†‹‡ǡ ƒ† ʹͲ  ‹‹†ƒœ‘Ž‡ǡƒ†‡Ž—–‡†™‹–Š„—ˆˆ‡” ‘–ƒ‹‹‰ͷͲƒ ʹͶȋ’ ͺǤͲȌǡ͵ͲͲƒŽǡƒ† CharacterisationoftheUnfoldedProteinResponseElement63.

(27) Chapter3 ʹͷͲ  ‹‹†ƒœ‘Ž‡Ǥ ‘—† ‘Ž‹‰‘— Ž‡‘–‹†‡• ™‡”‡ ƒ’Ž‹ˆ‹‡† „›  ™‹–Š ’”‹‡”• ͳͲ ƒ† ͳͳǤ Š‡ ƒ’Ž‹ˆ‹‡† ƒ–‡”‹ƒŽ ™ƒ• –Š‡ —•‡† ‹ ƒ ‡š– › Ž‡ ‘ˆ •‡Ž‡ –‹‘ ƒ† ƒ’Ž‹ˆ‹ ƒ–‹‘Ǥ ˆ–‡” ͺ › Ž‡•ǡ –Š‡ •‡Ž‡ –‡† ‘Ž‹‰‘— Ž‡‘–‹†‡• ™‡”‡ Ž‘‡† ‹–‘ ’ǦŽ—–

(28)

(29) Ǧ ȋ

(30) ˜‹–”‘‰‡Ȍƒ†•‡“—‡ ‡†Ǥ  EstimationoftheRelativeAffinities Š‡ ƒ’’ƒ”‡– †‹••‘ ‹ƒ–‹‘ ‘•–ƒ– ȋKdȌ „‡–™‡‡ ƒ  ƒ† –Š‡ ˜ƒ”‹‘—• • ™ƒ• ƒŽ —Žƒ–‡† ˆ”‘  ‰‡Ž „ƒ† •Š‹ˆ– ”‡ƒ –‹‘•Ǥ – ‡“—‹Ž‹„”‹— –Š‡ ‘ ‡–”ƒ–‹‘ ‘ˆ ˆ”‡‡ ’”‘–‡‹ ™Š‡ ŠƒŽˆ ‘ˆ –Š‡ ’”‘„‡ ‹• ‘’Ž‡š‡†ǡ ‹• ‡“—‹˜ƒŽ‡– –‘ –Š‡ ƒ’’ƒ”‡– Kd ͳ͵͵Ǥ ”‘„‡ ‘ ‡–”ƒ–‹‘• ™‡”‡ ͳ  ‹ ƒŽŽ ƒ••ƒ›•ǡ ƒ† ’”‘–‡‹ ‘ ‡–”ƒ–‹‘• ˜ƒ”‹‡† ˆ”‘ ͷ  –‘ ͷɊǤ Š‡ ƒ’’ƒ”‡– Kd ‹• –Š‡”‡ˆ‘”‡ ‰‹˜‡ „› –Š‡ –‘–ƒŽ ‘ ‡–”ƒ–‹‘ ‘ˆ ’”‘–‡‹ ƒ– ŠƒŽˆ –Š‡ •ƒ–—”ƒ–‹‘‘ˆ –Š‡ ’”‘„‡ ‹—• ͲǤͷ Ǥ —ƒ–‹ˆ‹ ƒ–‹‘ ™ƒ• ’‡”ˆ‘”‡† „› ’Š‘•’Š‘”Ǧ‹ƒ‰‡” ƒƒŽ›•‹•ȋƒ ƒ”†Ǣ

(31) •–ƒ–

(32) ƒ‰‡”Ȍ  GUSReporterConstructs Š‡ ‡ ‘†‹‰‰‡‡ȋuidAȌ‘ˆE.coli™ƒ•’Žƒ ‡†—†‡” ‘–”‘Ž‘ˆƒʹͺͲ„’ˆ”ƒ‰‡–‘ˆ –Š‡ cypB ’”‘‘–‡” ‘–ƒ‹‹‰ –Š‡ ’—–ƒ–‹˜‡ Ǥ —–ƒ–‹‘• ™‡”‡ ‹–”‘†— ‡† ‹ –Š‡ cypB  „› •‹–‡ †‹”‡ –‡† —–ƒ‰‡‡•‹• —•‹‰ ’”‹‡”• ͳʹ ȋ—–ƒ–‹‘ ͸ÆȌ ƒ† ͳ͵ ȋ–Š‡ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ •‡“—‡ ‡ǡ ‘” ȌǤ  ͵Ǥͻ „ XbaI ˆ”ƒ‰‡– ˆ”‘ ’Žƒ•‹† ’ͶǤͳǡ „‡ƒ”‹‰ƒ‘Ǧˆ— –‹‘ƒŽpyrG*‰‡‡ǡ™ƒ•‹ Ž—†‡†‹–Š‡’’Žƒ•‹†ǡ™Š‹ ŠƒŽŽ‘™‡†ˆ‘” –ƒ”‰‡–‡†‹–‡‰”ƒ–‹‘‘ˆ–Š‡ ‘•–”— –ƒ––Š‡pyrGŽ‘ —•‘ˆA.nigerͶǤͳͳ͸ͻǤ  SouthernBlotAnalysis. ‡‘‹   ˆ‘” ‘—–Š‡” „Ž‘– ƒƒŽ›•‹• ™ƒ• ‹•‘Žƒ–‡† „› ƒ††‹‰ ͺͲͲ ɊŽ „—ˆˆ‡” ȋͳͲͲ  

(33) Ǧ Žȋ’ ͺǤͲȌǡͷͲǡͳǡ͵ͷȌ–‘‰”‘—†› ‡Ž‹—ǤŠ‡™ƒ• ’—”‹ˆ‹‡† „› ’Š‡‘ŽǦ ŠŽ‘”‘ˆ‘” ‡š–”ƒ –‹‘ ƒ ‘”†‹‰ –‘ •–ƒ†ƒ”† ’”‘ ‡†—”‡• ͳͶͳǤ Š‡  ’‡ŽŽ‡– ™ƒ• ”‡•—•’‡†‡† ‹ ʹ ƒ† ‹ —„ƒ–‡† ™‹–Š ƒ•‡ ˆ”‡‡ ƒ•‡ ȋ‘ Š‡ ‘Ž‡ —Žƒ” ‹‘ Š‡‹ ƒŽ•ȌǤ ‘”‡ƒ Š†‹‰‡•–ͳͲρ‰‘ˆ‰‡‘‹ ™ƒ•‹ —„ƒ–‡†͸Šƒ–͵͹q™‹–ŠʹͲ Eco

(34) ǡ ™Š‹ Š —–• ‘ ‡ ‹ ’Ǥ Š‡ ”‡ƒ –‹‘ ’”‘†— –• ™‡”‡ •‡’ƒ”ƒ–‡† ‘ ƒ ͲǤͺΨ  ƒ‰ƒ”‘•‡‰‡ŽǤŽ‘––‹‰ƒ†Š›„”‹†‹•ƒ–‹‘™ƒ•†‘‡ƒ ‘”†‹‰–‘•–ƒ†ƒ”†’”‘ ‡†—”‡•ͳͶͳǤ   GUSassay A. niger ͶǤͳ ”‡’‘”–‡” •–”ƒ‹• ™‡”‡ ‰”‘™ ‘˜‡”‹‰Š– ƒ– ͵Ͳι ‹ ‹‹ƒŽ ‡†‹— •—’’Ž‡‡–‡†™‹–ŠͲǤʹΨ™Ȁ˜›‡ƒ•–‡š–”ƒ –ǤŽ‹“—‘–•‘ˆͳ‰™‡–› ‡Ž‹—™‡”‡–”ƒ•ˆ‡””‡† –‘ ͳͲͲ Ž ˆ”‡•Š ‡†‹— ƒ† –Š‡ —Ž–—”‡• ™‡”‡ ‹ —„ƒ–‡† ˆ‘” ͵ Š „‡ˆ‘”‡ ’”‘˜‘‹‰ Ǧ •–”‡•• „› –Š‡ ƒ††‹–‹‘ ‘ˆ ‡‹–Š‡” ʹͲ Ɋ‰ȀŽ –—‹ ƒ› ‹ ‘” ʹͲ  Ǥ ƒ’Ž‡• ˆ”‘ •–”‡••‡† ƒ† —•–”‡••‡† —Ž–—”‡• ™‡”‡ –ƒ‡ ͳǡ ʹǡ Ͷǡ ͸ǡ ͺ ƒ† ͳͲ Š ƒˆ–‡” –Š‡ ƒ’’Ž›‹‰ –Š‡ •–”‡••ƒ‰‡–•ǡƒ†ˆŽƒ•Šˆ”‘œ‡‹Ž‹“—‹†‹–”‘‰‡Ǥ ‘”–‘–ƒŽ’”‘–‡‹’”‡’ƒ”ƒ–‹‘•ǡ› ‡Ž‹— 64CharacterisationoftheUnfoldedProteinResponseElement.

(35) Chapter3 ™ƒ•‰”‘—†‹”‡ƒ –‹‘„—ˆˆ‡”ȋͷͲƒͶ’ ͹ǤͲǡͳͲǡͳǡͲǤͳΨ–”‹–‘ ǦͳͲͲȌ™‹–Šͳ‰Žƒ••„‡ƒ†•—•‹‰ƒ ƒ•–”‡’ ͳʹͲȋ

(36) ͳͲͳƒ˜ƒ–ȌǤŠ‡–‘–ƒŽ’”‘–‡‹ ‘ ‡–”ƒ–‹‘‹–Š‡ ‡ŽŽ‡š–”ƒ –™ƒ•†‡–‡”‹‡†ƒ ‘”†‹‰–‘•–ƒ†ƒ”†’”‘ ‡†—”‡• ͻǤŠ‡.  ƒ –‹˜‹–› ™ƒ• †‡–‡”‹‡† ƒ– ͵͹ι ‹ ͳ Ž ”‡ƒ –‹‘ „—ˆˆ‡” ‘–ƒ‹‹‰ ͳ  pǦ ‹–”‘’Š‡›ŽǦȾǦǦ‰Ž— —”‘‹†‡Ǥ‡ƒ –‹‘•™‡”‡•–‘’’‡†„›–Š‡ƒ††‹–‹‘‘ˆͲǤͶŽͳƒʹ͵ǡ ƒ†–Š‡ƒ„•‘”„ƒ ‡ƒ–ͶͳͲ™ƒ•†‡–‡”‹‡†ǤŠ‡ƒ –‹˜‹–›‹—‹–•™ƒ•†‡–‡”‹‡†ƒ•–Š‡ ˆ‘”ƒ–‹‘‘ˆpǦ‹–”‘’Š‡‘Ž‹Ɋ‘Ž‡’‡”‹—–‡’‡”Ɋ‰‘ˆ–‘–ƒŽ’”‘–‡‹Ǥ‡ƒ†‹‰•ˆ”‘–Š‡ —–”ƒ•ˆ‘”‡†’ƒ”‡–ƒŽ•–”ƒ‹A.nigerͶǤͳ™‡”‡”‡‰ƒ”†‡†ƒ•„ƒ ‰”‘—†ƒ†•—„–”ƒ –‡† ˆ”‘–Š‡‡ƒ•—”‡‡–•ǤŠ‡‡š’‡”‹‡–ƒŽ‡””‘”‹–Š‡ ƒŽ —Žƒ–‡†ƒ –‹˜‹–›™ƒ•†‡”‹˜‡†„› ‹•‡”–‹‰ –Š‡ Š‹‰Š‡•– ƒ† Ž‘™‡•– ˜ƒŽ—‡• ‘ˆ –Š‡ ͸ͺΨ ‘ˆ‹†‡ ‡ ‹–‡”˜ƒŽ ‘ˆ „‘–Š –Š‡ pǦ ‹–”‘’Š‡‘Žƒ†–Š‡’”‘–‡‹†‡–‡”‹ƒ–‹‘‹–Š‡‡“—ƒ–‹‘ˆ‘”–Š‡—‹–†‡–‡”‹ƒ–‹‘Ǥ™‘ ƒ –‹˜‹–‹‡• ƒ”‡ •‹‰‹ˆ‹ ƒ–Ž› †‹ˆˆ‡”‡– ™Š‡ –Š‡ †‹ˆˆ‡”‡ ‡ „‡–™‡‡ –Š‡ ‹• Žƒ”‰‡” –Šƒ –Š‡ •—‘ˆ–Š‡‹”•–ƒ†ƒ”†‡””‘”•Ǥ  . Results  IdentificationoftheERStressResponseElement ‡ ‡–Ž›™‡•Š‘™‡†–Šƒ––Š‡ͷǯǦˆŽƒ‹‰”‡‰‹‘•‘ˆ–Š‡A.niger‰‡‡•bipA,cypB,pdiAprpA, tigAƒ†hacAƒŽŽ ‘–ƒ‹‡†•ǡ•‹ ‡–Š‡‹”’”‘‘–‡”ˆ”ƒ‰‡–•™‡”‡”‡–ƒ”†‡†‹ƒ‰‡Ž „ƒ† •Š‹ˆ– ‡š’‡”‹‡– ™‹–Š ƒ  ͳͲͳǤ ”‘‘–‡” †‡Ž‡–‹‘ ƒƒŽ›•‹• ƒ””‘™‡† †‘™ –Š‡ Ž‘ ƒ–‹‘‘ˆ–Š‡cypB–‘ƒͳʹͲ„’”‡‰‹‘ˆŽƒ‹‰–Š‡ͷǯǦ‡†‘ˆ–Š‡cypB‰‡‡ȋ†ƒ–ƒ‘– •Š‘™ȌǤ ‹ ‡ –Š‡ ƒ  Š‘‘Ž‘‰—‡• ˆ”‘ A. nigerǡ A. nidulans ƒ† Aspergillus fumigatus Šƒ˜‡ ƒ ‹†‡–‹ ƒŽ  „‹†‹‰ †‘ƒ‹ǡ ƒ† –Š‡”‡ˆ‘”‡ ‹‰Š– ”‡ ‘‰‹•‡ •‹‹Žƒ” –ƒ”‰‡–•ǡ ƒ •‡“—‡ ‡ ‘’ƒ”‹•‘„‡–™‡‡–Š‡ͷǯǦˆŽƒ‹‰”‡‰‹‘•‘ˆ–Š‡cypBŠ‘‘Ž‘‰—‡•ˆ”‘–Š‡•‡ –Š”‡‡Aspergilli™ƒ•ƒ†‡ǤŠ‹•”‡˜‡ƒŽ‡†ƒŠ‹‰ŠŽ› ‘•‡”˜‡†„‘šǡ’”‡•‡–™‹–Š‹–Š‡ͳʹͲ„’ ͷǯǦˆŽƒ‹‰”‡‰‹‘‘ˆ–Š‡cypB‰‡‡ȋ ‹‰ǤͳȌǤ—”‹ˆ‹‡† ƒ ’”‘–‡‹™ƒ•ƒ„Ž‡–‘„‹†invitro –‘ƒ͵Ͷ„’‘Ž‹‰‘— Ž‡‘–‹†‡ ‘–ƒ‹‹‰–Š‡’—–ƒ–‹˜‡cypBǡ†‡†— ‡†ˆ”‘•‡“—‡ ‡ ƒŽ‹‰‡–•ȋ ‹‰ǤʹȌǤŠ‹•„‹†‹‰’”‘˜‡†–‘„‡•’‡ ‹ˆ‹ „‡ ƒ—•‡‡˜‡ƒͷͲͲǦˆ‘Ž†‡š ‡••‘ˆ ƒ —Žƒ„‡ŽŽ‡† ’”‘„‡ ‘–ƒ‹‹‰ ƒ ’‘‹– —–ƒ– ‘ˆ –Š‡ cypB  †‹† ‘– ‘’‡–‡ •‹‰‹ˆ‹ ƒ–Ž›‹–Š‡„‹†‹‰ƒ••ƒ›Ǥ. CharacterisationoftheUnfoldedProteinResponseElement65.

(37) Chapter3 . Figure 1.

(38) †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ ’—–ƒ–‹˜‡ ˆ‘Ž†‡† ”‘–‡‹ ‡•’‘•‡ Ž‡‡–• ȋ•Ȍ ˆ”‘ AspergilliǤ (A) Ž‹‰‡– ‘ˆ ‘•‡”˜‡† ’”‘‘–‡” ”‡‰‹‘• –Šƒ– •Šƒ”‡† •‡“—‡ ‡ •‹‹Žƒ”‹–› ™‹–Š –Š‡A.nigercypBǤŽ‹‰‡–•ƒ”‡–”‹’Ž‡™‹•‡ƒ‘‰–Š‡Š‘‘Ž‘‰—‡•‘ˆ–Š”‡‡AspergilliǢA. niger (A.nig)ǡ A. nidulans (A.nid)ǡ ƒ† A. fumigatus (A.fum). —„‡”• „‡•‹†‡ –Š‡ •‡“—‡ ‡• ‹†‹ ƒ–‡ –Š‡ ’‘•‹–‹‘ ”‡Žƒ–‹˜‡ –‘ –Š‡   ‘ˆ –Š‡ †‘™•–”‡ƒ Ž‘ ƒ–‡† ‰‡‡Ǥ Š‡ ͳͳ „’ ‘”‡ •‡“—‡ ‡•‘ˆ–Š‡•ƒ”‡—†‡”Ž‹‡†Ǥ(B)Ž‹‰‡–‘ˆ–Š‡•‹†‡–‹ˆ‹‡†‹–Š‡’”‘‘–‡”• ‘ˆ–Š‡A.niger‰‡‡•bipA,cypB,pdiA,prpA,tigAǡƒ†hacAǤŠ‡„ƒ•‡•‘ˆ–Š‡ ‘•‡”˜‡† ‘”‡ƒ”‡ —„‡”‡†ƒ„‘˜‡–Š‡ƒŽ‹‰‡–ǤŠ‡ͳͳ„’ ‘•‡•—••‡“—‡ ‡‹•‹†‹ ƒ–‡†—†‡”–Š‡ƒŽ‹‰‡–. 66CharacterisationoftheUnfoldedProteinResponseElement.

(39) Chapter3 ‘ ‹†‡–‹ˆ› ’—–ƒ–‹˜‡ • ‹ –Š‡ ’”‘‘–‡”• ‘ˆ bipA, pdiA, prpA, tigA ƒ† hacA, ƒ •‹‹Žƒ” ƒ’’”‘ƒ Š ™ƒ• ˆ‘ŽŽ‘™‡†Ǥ Žƒ•– •‡ƒ” Š‡• ™‡”‡ ’‡”ˆ‘”‡† –‘ ”‡–”‹‡˜‡ ‘Ǧƒ‘–ƒ–‡† Š‘‘Ž‘‰—‡•ˆ”‘–Š‡A.fumigatusƒ†A.nidulans‰‡‘‡†ƒ–ƒ„ƒ•‡• ȋŠ––’ǣȀȀ™™™Ǥ–‹‰”Ǥ‘”‰Ȁ–†„Ȁ‡ʹͳȀƒˆ—ͳȀƒ† Š––’ǣȀȀ™™™Ǥ„”‘ƒ†Ǥ‹–Ǥ‡†—Ȁƒ‘–ƒ–‹‘Ȁˆ—‰‹Ȁƒ•’‡”‰‹ŽŽ—•Ȁ‹†‡šǤŠ–ŽȌǤ ƒ Š •‡– ‘ˆ Š‘‘Ž‘‰‘—• ’”‘‘–‡”• ˆ”‘ –Š‡ –Š”‡‡ Aspergilli ™ƒ• ƒƒŽ›•‡† ˆ‘” –Š‡ ’”‡•‡ ‡ ‘ˆ ‘•‡”˜‡† „Ž‘ • —•‹‰ –Š‡ ’”‘‰”ƒ . ͳͶͷǤ. ‘•‡”˜‡† „‘š‡• ™‹–Š. •‹‹Žƒ”‹–‹‡•–‘–Š‡cypB™‡”‡ˆ‘—†‹ƒŽŽ’”‘‘–‡”•ȋ ‹‰ǤͳȌǤ—–•‹†‡–Š‡•‡„‘š‡•ǡ –Š‡ •‡“—‡ ‡ ‹†‡–‹–› ‹• — Š Ž‘™‡”ǡ ™Š‹ Š ‹• ‘•– ‘„˜‹‘—• ˆ”‘ –Š‡ ƒŽ‹‰‡–• ‘ˆ –Š‡ cypB ƒ† tigA ’”‘‘–‡” ”‡‰‹‘•Ǥ  •Š‘™‡† –Šƒ– ƒŽŽ •‡“—‡ ‡• ‹†‡–‹ˆ‹‡† ƒ• ’‘–‡–‹ƒŽ • ™‡”‡ „‘—† „› ƒ  ȋ ‹‰Ǥ ʹȌǤ

(40) –‡”‡•–‹‰Ž›ǡ –™‘ ’—–ƒ–‹˜‡ • ȋ‹†‹ ƒ–‡† ƒ• hacA1ƒ†hacA2Ȍ™‡”‡‹†‡–‹ˆ‹‡†‹–Š‡’”‘‘–‡”•‘ˆ–Š‡hacAŠ‘‘Ž‘‰—‡•ȋ ‹‰ǤͳȌǤŠ‡ ˆƒ – –Šƒ– „‘–Š • ƒ”‡ ™‡ŽŽ ‘•‡”˜‡† ƒ‘‰ –Š‡ –Š”‡‡ Aspergilliǡ ƒ† –Š‡ ‘„•‡”˜ƒ–‹‘ –Šƒ–„‘–ŠA.nigerhacA•ƒ”‡„‘—†„› ƒ invitroȋ ‹‰ǤʹȌǡ ‘—Ž†‹†‹ ƒ–‡ƒinvivo ˆ— –‹‘ ˆ‘” „‘–Š „‘š‡•Ǥ ȋŠ‡ –Š‹”†  ‹ –Š‡ hacA ’”‘‘–‡”ǡ hacA3, ™ƒ• †‹• ‘˜‡”‡† Žƒ–‡”ǡ ƒ† ™‹ŽŽ „‡ †‹• —••‡† •‡’ƒ”ƒ–‡ǡ Žƒ–‡” ‘ ‹ –Š‡ ”‡•—Ž–• •‡ –‹‘ȌǤ ‹‰—”‡ͳ •Š‘™• –Š‡ ƒŽ‹‰‡– ‘ˆ –Š‡ •‡˜‡ A. niger • ‹†‡–‹ˆ‹‡† ‹ –Š‹• •–—†›Ǥ Š‡ ‘•‡•—• •‡“—‡ ‡ǡ ȋ Ȁ) T/GCCTǡ — ‘˜‡”‡† „› –Š‹• ƒŽ‹‰‡– •Š‘™• •‹‹Žƒ”‹–‹‡• ™‹–Š –Š‡ S. cerevisiaeǤŠ‡S.cerevisiaeKAR2ǡ ȋȌ  ǡ‹•ƒ’ƒ”–Ž›’ƒŽ‹†”‘‹ •‡“—‡ ‡ •‡’ƒ”ƒ–‡† „› ƒ •’ƒ ‡” ‘ˆ ‘‡ — Ž‡‘–‹†‡. ͻ͹Ǥ. Š‡ ’ƒŽ‹†”‘‹  ”‡•‹†—‡•  ƒ†  . ȋ—†‡”Ž‹‡†Ȍƒ”‡ ‘‘ˆ‘”ƒ—„‡”‘ˆS.cerevisiae•ǡƒ†–Š‡•’ƒ ‡”— Ž‡‘–‹†‡ ȋ„‡–™‡‡ „”ƒ ‡–•Ȍ ‹• ‘•‡”˜‡† ‹ ƒŽ‘•– ƒŽŽ S. cerevisiae •Ǥ ”‘ –Š‡ ƒŽ‹‰‡– ‘ˆ –Š‡•ˆ‘—†‹–Š‡’”‘‘–‡”•‘ˆ•‡˜‡”ƒŽA.niger‰‡‡•‹–‹• Ž‡ƒ”–Šƒ––Š‡A.niger ƒŽ•‘Šƒ•’ƒ”–‹ƒŽ•›‡–”›ƒ”‘—†ƒ•’ƒ ‡”— Ž‡‘–‹†‡Ǥ ‘™‡˜‡”ǡ‹ ‘–”ƒ•––‘S.cerevisiaeǡ ‹–Š‡A.niger•–Š‡•’ƒ ‡”‹•‡‹–Š‡” ‘”Ǥ —”–Š‡”‘”‡ǡ–Š‡’ƒ”–Ž›’ƒŽ‹†”‘‹ „‘š‹ A.niger‹•ˆ‘ŽŽ‘™‡†„›–Š‡Š‹‰ŠŽ› ‘•‡”˜‡†•‡“—‡ ‡T/GCCTȋ‹„‘Ž†Ȍǡ™Š‹ Š‹•‘–ˆ‘—† ‹S.cerevisiaeǤ Š‡ ƒŽ‹‰‡–• •Š‘™ ‹ ‹‰—”‡ ͳ ”‡˜‡ƒŽ‡† –Šƒ– –Š‡ • ƒ”‡ ”‡ƒ”ƒ„Ž› ™‡ŽŽ ‘•‡”˜‡†ƒ‘‰Š‘‘Ž‘‰‘—•‰‡‡•ƒ ”‘••–Š‡•’‡ ‹‡•„‘—†ƒ”›ǡ™Š‡”‡ƒ•‘”‡˜ƒ”‹ƒ–‹‘ ‘ —”• ƒ‘‰ –Š‡ • ‘ˆ –Š‡ †‹ˆˆ‡”‡–  ”‡‰—Žƒ–‡† ‰‡‡• ™‹–Š‹ ƒ •‹‰Ž‡ Aspergillus •’‡ ‹‡•Ǥ ‘” ‡šƒ’Ž‡ǡ –Š‡ ͵Ͷ „’ ”‡‰‹‘• ‘–ƒ‹‹‰ –Š‡ • ‘ˆ –Š‡ pdiAǡ prpAǡ ƒ† tigA Š‘‘Ž‘‰—‡•ȋ ‹‰ǤͳȌ•Šƒ”‡͸ͺΨǡͷͲΨǡƒ†͵ͺΨ‹†‡–‹ ƒŽ”‡•‹†—‡•ǡ”‡•’‡ –‹˜‡Ž›Ǥ ‘™‡˜‡”ǡ ƒ‘‰ –Š‡• ‘ˆ –Š‡•‡ –Š”‡‡’”‘–‡‹ †‹•—Ž’Š‹†‡ ‹•‘‡”ƒ•‡• ‘ˆ A. niger ȋ ‹‰ǤͳȌǡ –Š‡”‡ ƒ”‡‘Ž›ʹͻΨ‹†‡–‹ ƒŽ”‡•‹†—‡•Ǥ. CharacterisationoftheUnfoldedProteinResponseElement67.

(41) Chapter3                     Figure2.Ž‡ –”‘’Š‘”‡–‹ ‘„‹Ž‹–›•Š‹ˆ–ƒ••ƒ›ȋȌ•Š‘™‹‰–Š‡„‹†‹‰‘ˆ–Š‡A.niger ƒ   –‘ ͵Ͷ „’ ‘Ž‹‰‘— Ž‡‘–‹†‡• ‡ ‘’ƒ••‹‰ –Š‡ ‹†‡–‹ˆ‹‡† •Ǥ Š‡ „‹†‹‰ ”‡ƒ –‹‘• ™‡”‡  ’‡”ˆ‘”‡†™‹–ŠʹͲͲ ƒ ’”‘–‡‹ƒ†ͳ ͵ʹǦŽƒ„‡ŽŽ‡†’”‘„‡Ǥ(A)’‡ ‹ˆ‹ „‹†‹‰‘ˆ ƒ   –‘ –Š‡ cypB Ǥ ȋǦȌ ™‹–Š‘—– Ǥ ȋΪȌ ™‹–Š Ǥ ȋͳȌ ‘’‡–‹–‹‘ ™‹–Š ͷǡ ͷͲ ƒ† ͷͲͲ . —Žƒ„‡ŽŽ‡†cypBǡ”‡•’‡ –‹˜‡Ž›ǤȋʹȌ‘’‡–‹–‹‘™‹–ŠͷǡͷͲƒ†ͷͲͲ—Žƒ„‡ŽŽ‡†—–ƒ– cypB Ǥ Š‡ —–ƒ– cypB  ‘–ƒ‹‡† –Š‡ —–ƒ–‹‘ ͸Æ ȋ ˆ ƒ„Ž‡ ʹȌǤ (B) ‹†‹‰ ‘ˆ  ƒ –‘–Š‡bipA,cypB,pdiA,prpA,tigA,hacA1,andhacA2•ǤȋͳȌ‹–Š‘—– ‘’‡–‹–‹‘ǤȋʹȌ  ‘’‡–‹–‹‘™‹–ŠͷͲͲ—Žƒ„‡ŽŽ‡†—–ƒ–cypBǤǦ ƒ Ǧ ‘’Ž‡šǡ Ǧ ”‡‡’”‘„‡Ǥ. . .  MutationAnalysisofthecypBUPRE ‘‡˜ƒŽ—ƒ–‡–Š‡ ‘–”‹„—–‹‘‘ˆ‡ƒ Š— Ž‡‘–‹†‡‘ˆ–Š‡ ‘•‡•—••‡“—‡ ‡–‘„‹†‹‰ǡ ™‡ ƒ••‡••‡† –Š‡ ‡ˆˆ‡ – ‘ˆ ͳͶ —–ƒ–‹‘• ƒ– †‹ˆˆ‡”‡– ’‘•‹–‹‘• ‹ –Š‡ cypB  ȋƒ„Ž‡ ʹȌǤ Š‡ —–ƒ–‹‘• ͳÆ ƒ† ʹÆǡ „‘–Š Ž‘ ƒ–‡† ‹ –Š‡ Ž‡ˆ– ŠƒŽˆǦ•‹–‡ ‘ˆ –Š‡ ‹’‡”ˆ‡ – ’ƒŽ‹†”‘‡ǡ”‡•—Ž–‡†‹ƒʹǤͷǦˆ‘Ž††‡ ”‡ƒ•‡ƒ†ƒʹǦˆ‘Ž†‹ ”‡ƒ•‡‹„‹†‹‰ǡ”‡•’‡ –‹˜‡Ž›Ǥ

(42)  ‘–”ƒ•–ǡ —–ƒ–‹‘ ‘ˆ –Š‡ ’ƒŽ‹†”‘‹  ”‡•‹†—‡• ‹ –Š‡ ”‹‰Š– ŠƒŽˆǦ•‹–‡ ‘ˆ –Š‡ ‹’‡”ˆ‡ – ’ƒŽ‹†”‘‡ ‘’Ž‡–‡Ž›ƒ„‘Ž‹•Š‡†„‹†‹‰ȋ—–ƒ–‹‘•͸Æǡ͸Æ ƒ†͹ ÆȌǤ••–ƒ–‡† „‡ˆ‘”‡ǡ ‘•– S. cerevisiae • Šƒ˜‡ ƒ •’ƒ ‡” — Ž‡‘–‹†‡ǡ —•—ƒŽŽ› ƒ ǡ ‹–‡””—’–‹‰ –Š‡ ’ƒŽ‹†”‘‡Ǥ––Š‡ ‘””‡•’‘†‹‰’‘•‹–‹‘‹–Š‡‘ˆAspergillus,—•—ƒŽŽ›ƒ ‘”ƒ‹• ’”‡•‡–Ǥ Ž–Š‘—‰Š ‘  ™ƒ• ˆ‘—† ƒ– –Š‡ ’‘•‹–‹‘ ‘ˆ –Š‡ •’ƒ ‡” — Ž‡‘–‹†‡ǡ –Š‡ —–ƒ–‹‘. 68CharacterisationoftheUnfoldedProteinResponseElement.

(43) Chapter3 Ͷ Æ ”‡•—Ž–‡† ‹ ƒ •—„•–ƒ–‹ƒŽ ‹ ”‡ƒ•‡ ‹ „‹†‹‰ ‘ˆ –Š‡ cypB Ǥ ‘•‹–‹‘ ͺ ‹ –Š‡ Aspergillus‹•‘ —’‹‡†„›‡‹–Š‡”ƒ‘”ƒ ȋ ‹‰ǤͳȌǡƒ†—–ƒ–‹‘ͺ Æ‹–Š‡cypB  ‘Ž› Šƒ† ƒ ‹‘” ‡ˆˆ‡ – ‘ „‹†‹‰Ǥ ‘™‡˜‡”ǡ „‹†‹‰ ™ƒ• ‘’Ž‡–‡Ž› ƒ„‘Ž‹•Š‡† ™Š‡–Š‹•”‡•‹†—‡™ƒ•—–ƒ–‡†–‘ƒǤŠƒ‰‡•‹–Š‡Žƒ•––Š”‡‡ ‘•‡”˜‡†”‡•‹†—‡•ȋȌ Šƒ††”ƒƒ–‹ ‡ˆˆ‡ –•Ǥ‹†‹‰‹•Ž‘•–„›–Š‡—–ƒ–‹‘ͻÆǡƒ†™‡ƒ‡‡†•—„•–ƒ–‹ƒŽŽ›„› –Š‡—–ƒ–‹‘•ͳͲÆǡͳͳÆǡƒ†ͳͳÆ Ǥ  Table2.—ƒ”›‘ˆ–Š‡‰‡Ž•Š‹ˆ–ƒƒŽ›•‹•‘ˆ†‘—„Ž‡Ǧ•–”ƒ†‡†‘Ž‹‰‘— Ž‡‘–‹†‡•”‡’”‡•‡–‹‰ –Š‡ ›’ƒ† ‘–ƒ‹‹‰—–ƒ–‹‘•‹–Š‡ ‘”‡„‹†‹‰•‡“—‡ ‡Ǥ Probe. Sequence. %ofbindingrelative towildtypeprobe ™– ͷǯǦ   .  ͳͲͲ ͳ ͷ̵Ǧ  A .  ͶͲ ʹ ͷ̵Ǧ  C .  ʹͲͻ ͵ ͷ̵Ǧ  C .  ͸ Ͷ ͷ̵Ǧ  T.  ͳ͸ͻ ͷ  ͷ̵Ǧ   G.  ͳͷͷ ͸ ͷ̵Ǧ  . C.  Ͳ ͸  ͷ̵Ǧ  . G.  Ͳȗ ͹ ͷ̵Ǧ   T  Ͳ ͺ ͷ̵Ǧ    T ͺͳ ͺ ͷ̵Ǧ    C Ͳȗ ͻ ͷ̵Ǧ   . A Ͳ ͳͲ ͷ̵Ǧ   . A ͵ͻ ͳͳ ͷ̵Ǧ   . C ʹͳ ͳͳ  ͷ̵Ǧ   . G ͳͷȗ —–ƒ–‹‘• ƒ”‡ ‹†‹ ƒ–‡† ‹ boldǤ Š‡ „‹†‹‰ ”‡Žƒ–‹˜‡ –‘ –Š‡ ™‹Ž†Ǧ–›’‡ ™ƒ• †‡–‡”‹‡† „ƒ•‡† ‘ ƒ’’ƒ”‡–Kd˜ƒŽ—‡•ǡ‡š ‡’–ˆ‘”–Š‘•‡ƒ”‡†™‹–Šƒ†asteriskǡ™Š‹ Š™‡”‡†‡–‡”‹‡†ˆ”‘•‹‰Ž‡ „ƒ†•Š‹ˆ–‡š’‡”‹‡–•Ǥ.   SelectionforHighAffinityBindingSites ‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ ‹• ƒ ”ƒ’‹† ‡–Š‘† –‘ ‹•‘Žƒ–‡ ‘Ž‹‰‘— Ž‡‘–‹†‡• ™‹–Š Š‹‰Š ƒˆˆ‹‹–› ˆ‘”  „‹†‹‰ ’”‘–‡‹• ͳʹͺǤ ‘ †‡–‡”‹‡ –Š‡ ‘’–‹ƒŽ „‹†‹‰ •‹–‡ ˆ‘” ƒ ǡ ƒ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘•–”ƒ–‡‰›™ƒ•‡’Ž‘›‡†Ǥ‹†‹‰”‡ƒ –‹‘•™‡”‡’‡”ˆ‘”‡†™‹–Šƒ͹Ͷ„’†‘—„Ž‡Ǧ •–”ƒ†‡† ‘Ž‹‰‘— Ž‡‘–‹†‡ ‹ Ž—†‹‰ ʹͷ „’ ˆŽƒ• ˆ‘”  ƒ’Ž‹ˆ‹ ƒ–‹‘Ǥ Š‡ ‡–”ƒŽ ʹͶ „’ ™‡”‡’ƒ”–Ž›”ƒ†‘‹•‡†Ǥƒ•‡•™Š‹ Š™‡”‡ ‘•‡”˜‡†‹ƒŽŽA.niger•ȋͳǡ͸ǡ͹ ǡͻǡ ͳͲ ƒ† ͳͳȌ ™‡”‡ ‡’– ƒ– –Š‡‹” ‘”‹‰‹ƒŽ ’‘•‹–‹‘• ȋ ˆ ‹‰Ǥ ͳ ƒ† ͻ ‹ ƒ„Ž‡ ʹȌǤ Š‡ ‡š’‡”‹‡–™ƒ•’‡”ˆ‘”‡†‹’ƒ”ƒŽŽ‡Ž™‹–Šƒ†‘—„Ž‡•–”ƒ†‡†‘Ž‹‰‘— Ž‡‘–‹†‡ˆ‘”™Š‹ Š–Š‡ ‡–”ƒŽ ’ƒ”– ™ƒ• ‘’Ž‡–‡Ž› ”ƒ†‘‹•‡†Ǥ ‘ ‡•—”‡ •–”‹‰‡– •‡Ž‡ –‹‘ ‘†‹–‹‘•ǡ –Š‡ „‹†‹‰ ”‡ƒ –‹‘ ‹ Ž—†‡† ͳͲ  ‘ˆ –Š‡ ͵Ͷ „’ cypB  ƒ• ‘’‡–‹–‘” Ǥ  •‡“—‡ ‡ ƒŽ‹‰‡–‘ˆ–Š‡‘Ž‹‰‘— Ž‡‘–‹†‡••‡Ž‡ –‡†ƒˆ–‡”‡‹‰Š–”‘—†•‘ˆ–Š‡•‡Ž‡ –‹‘’”‘ ‡†—”‡‹• •Š‘™‹ ‹‰—”‡͵Ǥ CharacterisationoftheUnfoldedProteinResponseElement69.

(44) Chapter3. Figure3.‡Ž‡ –‹‘‘ˆ„‹†‹‰•‹–‡•ˆ”‘”ƒ†‘‘Ž‹‰‘— Ž‡‘–‹†‡•™‹–Š ǦȋŠ‹•Ȍ͸Ǥ(A)’’‡” ’ƒ‡ŽǢ ‡“—‡ ‡• •‡Ž‡ –‡† ™‹–Š –Š‡ ’ƒ”–Ž› ”ƒ†‘ ‘Ž‹‰‘— Ž‡‘–‹†‡ ͻǤ Š‡ ˆ‹š‡† — Ž‡‘–‹†‡• ‹ –Š‹•’”‘„‡ǡ”‡’”‡•‡–‹‰–Š‡ ‘•‡”˜‡†„ƒ•‡•‹–Š‡A.niger•ȋ ˆ ‹‰ǤͳȌǡƒ”‡†‡’‹ –‡†‹ ‹–ƒŽ‹ •ƒ†ƒ”‡‘–ƒ ‘—–‡†ˆ‘”‹–Š‡ƒŽ‹‰‡–Ǥ‘™‡”’ƒ‡ŽǢ‡“—‡ ‡••‡Ž‡ –‡†™‹–Š”ƒ†‘ ‘Ž‹‰‘ ͺǤ ‡Ž‘™ –Š‡ ƒŽ‹‰‡– ƒ”‡ ‹†‹ ƒ–‡† –Š‡ — Ž‡‘–‹†‡• ‘•– ˆ”‡“—‡–Ž› ’”‡•‡– ƒ– ‡ƒ Š ’‘•‹–‹‘ȋ‘”‡–Šƒ͸ͲΨȌǤŠ‡„ƒ•‡•‹ Ž—†‡†‹–Š‡ ‘•‡”˜‡† ‘”‡ƒ”‡—„‡”‡†ƒƒŽ‘‰‘—•–‘ ‹‰—”‡ͳǤ‘–‡–Šƒ–”‡•‹†—‡”ǤͶǡ–Š‡•’ƒ ‡”— Ž‡‘–‹†‡‹•ƒ„•‡–Ǥ(B)•Š‘™‹‰–Š‡„‹†‹‰ ’”‘’‡”–‹‡• ‘ˆ –Š‡ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ „‘š ȋȌ ‘’ƒ”‡† –‘ –Š‡ cypB Ǥ  –Š‡ Ž‡ˆ–ǡ „‹†‹‰‘ˆ ƒ –‘ ͵ʹǦŽƒ„‡ŽŽ‡†cypBˆ‘ŽŽ‘™‡†„› ‘’‡–‹–‹‘™‹–ŠȋͳȌͷǡͷͲƒ†ͷͲͲ —Žƒ„‡ŽŽ‡†—–ƒ–cypBǡƒ†ȋʹȌͷǡͷͲƒ†ͷͲͲ—Žƒ„‡ŽŽ‡†Ǥ–Š‡”‹‰Š–ǡ„‹†‹‰‘ˆ ƒ –‘ ͵ʹǦŽƒ„‡ŽŽ‡†ˆ‘ŽŽ‘™‡†„› ‘’‡–‹–‹‘™‹–Š—Žƒ„‡ŽŽ‡†cypBǡƒ†—Žƒ„‡ŽŽ‡†ǤŠ‡ cypBƒ†‘Ž‹‰‘— Ž‡‘–‹†‡•. 70CharacterisationoftheUnfoldedProteinResponseElement.

(45) Chapter3 

(46) –‡”‡•–‹‰Ž›ǡ ƒ ’‡”ˆ‡ – ’ƒŽ‹†”‘‹  •‡“—‡ ‡ǡ   ǡ ™ƒ• ’”‡•‡– ‹ ‡ƒ”Ž› ƒŽŽ ƒ•‡•Ǥ –”‹‹‰Ž›ǡƒ•’ƒ ‡”— Ž‡‘–‹†‡„‡–™‡‡–Š‡–™‘’ƒŽ‹†”‘‹ ŠƒŽˆǦ•‹–‡•™ƒ•ƒ„•‡–‹ƒŽŽ‘ˆ –Š‡ •‡Ž‡ –‡† ‘Ž‹‰‘•Ǥ ‡•‹†‡• –Š‡ Žƒ  ‘ˆ ƒ •’ƒ ‡” — Ž‡‘–‹†‡ ƒ† ‹–• ’‡”ˆ‡ – ’ƒŽ‹†”‘‹  ƒ–—”‡ǡ –Š‡ •‡Ž‡ –‡† „‘š ȋˆ—”–Š‡” ”‡ˆ‡””‡† –‘ ƒ•  ˆ‘” „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ •‡“—‡ ‡Ȍ ”‡•‡„Ž‡•–Š‡•ˆ‘—†‹A.nigerǤŠ‡’ƒŽ‹†”‘‹ — Ž‡‘–‹†‡•ƒ† ƒ–’‘•‹–‹‘• ͳǦʹƒ†͸Ǧ͹”‡•’‡ –‹˜‡Ž›ǡ™‡”‡ˆ‘—†„‘–Š‹–Š‡•ƒ†‹–Š‡•Ǥ‘•‹–‹‘ͺ‹–Š‡  ƒ”‡ ‹ ƒŽŽ ƒ•‡• ‘ —’‹‡† „› ‡‹–Š‡” ƒ  ‘”  — Ž‡‘–‹†‡ǡ •‹‹Žƒ” –‘ ’‘•‹–‹‘ ͺ ‹ –Š‡ •Ǥ —”–Š‡”‘”‡ǡ–Š‡ ‘•‡”˜‡†•–”‡– Šƒ–’‘•‹–‹‘ͻǦͳͳ‹–Š‡A.niger•ǡ™ƒ• ƒŽ•‘ˆ”‡“—‡–Ž›ˆ‘—†‹–Š‡•Ǥ–’‘•‹–‹‘Ȃͳǡ†‹”‡ –Ž›’”‡ ‡†‹‰–Š‡’ƒŽ‹†”‘‹  ‘”‡ǡ ƒ  — Ž‡‘–‹†‡ ™ƒ• ’”‡•‡– ‘•– ˆ”‡“—‡–Ž›Ǥ Š‹• ‘—Ž† „‡ ’ƒŽ‹†”‘‹  ™‹–Š –Š‡ — Ž‡‘–‹†‡ƒ–’‘•‹–‹‘ͺǡ™Š‹ Š‹•‘•–ˆ”‡“—‡–Ž›ƒǡ–Š‡”‡„›‡š–‡†‹‰–Š‡’ƒŽ‹†”‘‹  ‘”‡–‘‡‹‰Š–„ƒ•‡•Ǥ ”‘–Š‡•‡†ƒ–ƒǡ‹–ƒŽ•‘ƒ’’‡ƒ”•–Šƒ– ƒ ’”‡ˆ‡”•’›”‹‹†‹‡•ƒ–’‘•‹–‹‘ͳʹƒ†ͳ͵ ƒ†’—”‹‡•ƒ–’‘•‹–‹‘Ȃʹƒ†Ȃ͵ǤŠ‡’—”‹‡• ƒ†™‡”‡’”‡•‡–ƒ–’‘•‹–‹‘•Ȃʹƒ†Ȃ͵ǡ ”‡•’‡ –‹˜‡Ž›‹‹‡ƒ†–‡‘ˆ–Š‡–Š‹”–‡‡•ǤŠ‡’›”‹‹†‹‡•ƒ†™‡”‡’”‡•‡–ƒ– ’‘•‹–‹‘•ͳʹƒ†ͳ͵ǡ‹‡Ž‡˜‡ƒ†‹‡ ƒ•‡•”‡•’‡ –‹˜‡Ž›Ǥ‹‹Žƒ”Ž›ǡ ƒ†— Ž‡‘–‹†‡• ™‡”‡‘”‡ˆ”‡“—‡–Ž›’”‡•‡–ƒ–’‘•‹–‹‘•Ȃ͵ƒ†Ȃʹǡƒ†ƒ†— Ž‡‘–‹†‡•ƒ–’‘•‹–‹‘• ͳʹƒ†ͳ͵‘ˆ–Š‡•ȋ ‹‰ǤͳȌǤ ƒ•‡† ‘ –Š‡ ƒ„‘˜‡ ˆ‹†‹‰•ǡ ˆ‹˜‡ —–ƒ–‹‘• ™‡”‡ ‹–”‘†— ‡† ‹ –Š‡ cypB  –‘ Šƒ‰‡ ‹–• ’ƒ”–Ž› ’ƒŽ‹†”‘‹  ‘”‡ –‘™ƒ”†• –Š‡  ‘•‡•—• •‡“—‡ ‡ ȋ ‹‰Ǥ ͵ǡ „’ —„‡” ͳǦͷȌǤ Š‡ „‹†‹‰ ’”‘’‡”–‹‡• ‘ˆ –Š‹•  ™‡”‡ ‘’ƒ”‡† ™‹–Š –Š‡ ™‹Ž† –›’‡ cypB  „›  ƒƒŽ›•‹•Ǥ ‘ǦŽƒ„‡ŽŽ‡†  ‘—–Ǧ ‘’‡–‡† –Š‡ cypB  ˆ‘” „‹†‹‰ „› ƒ Ǥ‘˜‡”•‡Ž›ǡƒ‡š ‡••‘ˆ–Š‡ ‘Ž†cypB™ƒ•—ƒ„Ž‡–‘•‹‰‹ˆ‹ ƒ–Ž›†‡ ”‡ƒ•‡–Š‡ ‹–‡•‹–›‘ˆ–Š‡ ƒ Ǧ ‘’Ž‡šǤŠ‹•‹†‹ ƒ–‡•ƒŠ‹‰Š‡”„‹†‹‰ƒˆˆ‹‹–›ˆ‘”–Š‡•‹–‡ ‘’ƒ”‡†–‘–Š‡cypBǤ   ThehacAUPRE3 Š‡  ‹•‘Žƒ–‡† „› „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ Žƒ ‡† ƒ •’ƒ ‡” — Ž‡‘–‹†‡ǡ ƒ† ™ƒ• „‘—† •–”‘‰‡”„› ƒ –Šƒ–Š‡cypBȋ ‹‰Ǥ͵ȌǤ”‘‘–‡”•‡“—‡ ‡•‘ˆA.nigerǡA.nidulans ƒ†A.fumigatus™‡”‡–Š‡”‡ˆ‘”‡ƒƒŽ›•‡†ˆ‘”–Š‡’”‡•‡ ‡‘ˆǦŽ‹‡•Ǥ—”’”‹•‹‰Ž›ǡ •— Š ƒ  ™ƒ• ˆ‘—† ‹ –Š‡ hacA ’”‘‘–‡” ‹–•‡Žˆ ȋ ‹‰Ǥ ͳȌǤ ‹‡ –Š‡ ‘–Š‡” •ǡ ‹–• •‡“—‡ ‡ ƒ† ’‘•‹–‹‘ ‹ –Š‡ ’”‘‘–‡” ‹• Š‹‰ŠŽ› ‘•‡”˜‡† ƒ‘‰ –Š‡ ͵ AspergilliǤ

(47) –• ‘•‡•—••‡“—‡ ‡‹• TCCT,™Š‹ Š‹•‡ƒ”Ž›‹†‡–‹ ƒŽ–‘–Š‡ǡƒ†Ž‹‡–Š‡ –Š‡ ‘”‡ •‡“—‡ ‡ ‹• ’”‡ ‡†‡† „› •‡˜‡”ƒŽ ’—”‹‡ ”‡•‹†—‡•ǡ ƒ† ˆ‘ŽŽ‘™‡† „› •‡˜‡”ƒŽ ’›”‹‹†‹‡•. CharacterisationoftheUnfoldedProteinResponseElement71.

(48) Chapter3 BindingAffinitiesoftheUPREs ‹‰—”‡ Ͷ •Š‘™• ƒ –›’‹ ƒŽ ’ƒ––‡” ‘ˆ ƒ  ‘„–ƒ‹‡† ™‹–Š ‹ ”‡ƒ•‹‰ ‘ ‡–”ƒ–‹‘• ‘ˆ ƒ ’”‘–‡‹ƒ†ͳcypBǤ‡Ž‘™–Š‡‹•ƒ‰”ƒ’Š™Š‡”‡–Š‡•ƒ–—”ƒ–‹‘ȋȌ‹• ’Ž‘––‡† ƒ‰ƒ‹•– –Š‡ ‘ ‡–”ƒ–‹‘ ‘ˆ ƒ Ǥ ‹ ‡ ƒ  ™ƒ• ˆ‘—† –‘ „‹†  ƒ• ƒ Š‘‘†‹‡”ȋ•‡‡Žƒ–‡”–Š‹•’ƒ’‡”Ȍǡ–Š‡•ƒ–—”ƒ–‹‘ȋȌ‘ˆ–Š‡™ƒ•†‡ˆ‹‡†ƒ•ǣ  αȏ ʹ˜ȐȀȋȏ ʹ˜ȐΪȏȐȌ  ‘”†‡–‡”‹ƒ–‹‘‘ˆKdǡ–Š‡†ƒ–ƒ™ƒ•ˆ‹––‡†–‘–Š‡ˆ‘ŽŽ‘™‹‰‡“—ƒ–‹‘ǣ  αͲǤͷȏ ȐƒšȀȋȋKdΪͲǤͷȌͲǤͷȏ ȐȌǤ . Figure4.ƒ–—”ƒ–‹‘‘ˆ–Š‡͵Ͷ„’cypB ȋȌ˜‡”•—•–Š‡ ‘ ‡–”ƒ–‹‘‘ˆ ƒ ǤŠ‡Kd ™ƒ• ƒŽ —Žƒ–‡†„›ˆ‹––‹‰–Š‡†ƒ–ƒ–‘‡“—ƒ–‹‘ƒ„‘˜‡—•‹‰Ž‹†‡”‹–‡Ž—•˜‡”•‹‘͵ǤͲͲǤKd˜ƒŽ—‡•ˆ‘” –Š‡†‹ˆˆ‡”‡–A.niger•ƒ”‡Ž‹•–‡†‹ƒ„Ž‡͵Ǥ. 72CharacterisationoftheUnfoldedProteinResponseElement.

(49) Chapter3 Š‡†‹••‘ ‹ƒ–‹‘ ‘•–ƒ–•ˆ‘”–Š‡ȏ ȐʹǦ ‘’Ž‡š‡•ˆ‘”–Š‡†‹ˆˆ‡”‡–•ƒ†–Š‡ ƒ”‡Ž‹•–‡†‹ƒ„Ž‡͵ǤŠ‡Kd˜ƒŽ—‡•ˆ‘”–Š‡•‘ˆbipA,pdiA,prpAƒ†tigAƒ”‡”ƒ–Š‡” •‹‹Žƒ”ƒ–ƒ’’”‘š‹ƒ–‡Ž›͵ͲǤŠ‹•‹• ‘’ƒ”ƒ„Ž‡–‘–Š‡ƒ’’ƒ”‡–Kd‘ˆ͵ͷ”‡’‘”–‡†ˆ‘” S.cerevisiae–”ƒ• ”‹’–‹‘ˆƒ –‘” Ͷ ͵ͷǤŠ‡Kd˜ƒŽ—‡ˆ‘”–Š‡cypB™ƒ•ˆ‘—†–‘„‡ — ŠŠ‹‰Š‡”ƒ–ͻ͸ǡ™Š‹ Š™ƒ•ƒŽ•‘‡˜‹†‡–ˆ”‘ ‹‰—”‡ʹǤ

(50)  ‘–”ƒ•–ǡ–Š‡–Š”‡‡hacA •Šƒ˜‡Kd˜ƒŽ—‡•— ŠŽ‘™‡”–Šƒ–Š‘•‡‘ˆ–Š‡•’”‡•‡–‹–Š‡’”‘‘–‡”•‘ˆ–Š‡ ˆ‘Ž†ƒ•‡•Ǥ Š‡–Š‡” –Š‹• ”‡ˆŽ‡ –• ƒ ’Š›•‹‘Ž‘‰‹ ƒŽ ”‘Ž‡ ”‡ƒ‹• –‘ „‡ ‹˜‡•–‹‰ƒ–‡†Ǥ

(51) –‡”‡•–‹‰Ž›ǡ–Š‡hacA3ƒ†–Š‡ǡ™Š‹ Š„‘–ŠŽƒ –Š‡•’ƒ ‡”— Ž‡‘–‹†‡ǡŠƒ˜‡–Š‡ Ž‘™‡•– Kd ˜ƒŽ—‡• ‘ˆ –Š‡ • –Šƒ– ™‡”‡ ƒƒŽ›•‡† Š‡”‡Ǥ ‘’ƒ”‡† –‘ –Š‡ cypB  –Š‡ ƒ”–‹ˆ‹ ‹ƒŽ   Šƒ• ƒ †”ƒ•–‹ ƒŽŽ› ‹ ”‡ƒ•‡† „‹†‹‰ ƒˆˆ‹‹–›Ǥ ‹˜‡ —–ƒ–‹‘• ™‡”‡ ‹–”‘†— ‡†‹ –Š‡ cypB  –‘ ‘„–ƒ‹ –Š‡ ǡ „—– ™‹–Š ƒ Kd ˜ƒŽ—‡ ‘ˆ ͳǤͻ ǡ ‹–• „‹†‹‰ ƒˆˆ‹‹–›‹•ƒ’’”‘š‹ƒ–‡Ž›ͷͲ–‹‡•Š‹‰Š‡”Ǥ  Table3.‹••‘ ‹ƒ–‹‘ ‘•–ƒ–•‘ˆ–Š‡A.nigerǦ ƒ  ‘’Ž‡š‡••–—†‹‡†Š‡”‡ UPRE. KdinnM. bipA cypB pdiA prpA tigA hacA1 hacA2 hacA3. ʹͺǤͻrͳǤ͵ ͻ͸ǤͲrͷǤͲ ͵ͳǤ͸rʹǤͶ ͵ʹǤͷrͳǤͷ ͵ͶǤͺrͳǤͷ ͳͲǤͻrͲǤͷ ͳͶǤʹrͲǤ͹ ͷǤ͹rͲǤͷ. . ͳǤͻrͲǤͳ.   InvivoActivityofthecypBandtheBSSUPRElement Š‡invivoƒ –‹˜‹–›‘ˆ–Š‡cypB™ƒ•ƒ••‡••‡†™‹–Š”‡’‘”–‡” ‘•–”— –•Šƒ˜‹‰–Š‡uidA ‰‡‡—†‡” ‘–”‘Ž‘ˆƒʹͺͲ„’’”‘‘–‡”ˆ”ƒ‰‡–‘ˆ–Š‡A.nigercypB‰‡‡ȋ’Ǧ™–ȌǤ Šƒ‰‡•™‡”‡ƒ†‡‹–Š‡‘ˆ–Š‡cypB’”‘‘–‡”ǡ™Š‹ Š”‡•—Ž–‡†‹–Š”‡‡ƒ††‹–‹‘ƒŽ ‘•–”— –•ǣ̵’Ǧ—–̵ǡ ‘–ƒ‹‹‰—–ƒ–‹‘͸Æ™Š‹ ŠŽ‡†–‘ƒ ‘’Ž‡–‡Ž‘••‘ˆ ƒ  „‹†‹‰ ‹  ‰‡Ž •Š‹ˆ–• ȋ ˆ ƒ„Ž‡ ʹȌǡ ̵’Ǧ„••̵ǡ ‘–ƒ‹‹‰ –Š‡ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ „‘šǡƒ†̵’Ǧ„••ͷš̵ǡ ‘–ƒ‹‹‰–Š‡„‹†‹‰•‹–‡•‡Ž‡ –‹‘„‘š”‡’‡ƒ–‡†ˆ‹˜‡–‹‡•ȋŠ‡ƒ† –‘–ƒ‹ŽȌ‹–Š‡cypB’”‘‘–‡”ǤA.nigerͶǤͳ–”ƒ•ˆ‘”ƒ–•Šƒ˜‹‰–Š‡”‡’‘”–‡” ‘•–”— –ƒ• ƒ •‹‰Ž‡ ‘’› ‹–‡‰”ƒ–‡† ƒ– –Š‡ pyrG Ž‘ —• ™‡”‡ •‡Ž‡ –‡† „› ‘—–Š‡” ƒƒŽ›•‹• ȋ†ƒ–ƒ ‘– •Š‘™ȌǤŠ‡”‡’‘”–‡”•–”ƒ‹•™‡”‡‰”‘™‹Ž‹“—‹†‡†‹ƒǡ•–”‡••™ƒ•’”‘˜‘‡†„›–Š‡ ƒ††‹–‹‘ ‘ˆ –—‹ ƒ› ‹ ƒ† ǡ ƒ† –Š‡ †‡˜‡Ž‘’‡– ‘ˆ ‹–”ƒ ‡ŽŽ—Žƒ” ȾǦ‰Ž— —”‘‹†ƒ•‡ ƒ –‹˜‹–›™ƒ•ˆ‘ŽŽ‘™‡†ˆ‘”ͳͲŠȋ ‹‰ǤͷȌǤ. CharacterisationoftheUnfoldedProteinResponseElement73.

(52) Chapter3. Figure5.invivoƒƒŽ›•‹•‘ˆ–Š‡‹A.nigerǤ‡’‘”–‡”•–”ƒ‹• ‘–ƒ‹‡†ƒ•‹‰Ž‡ ‘’›‘ˆ–Š‡ ‘•–”— – ’ ‹–‡‰”ƒ–‡† ƒ– –Š‡ pyrG Ž‘ —•Ǥ

(53) –”ƒ ‡ŽŽ—Žƒ” ȾǦ‰Ž— —”‘‹†ƒ•‡ ƒ –‹˜‹–› ƒ† •–ƒ†ƒ”† ‡””‘”• ™‡”‡ †‡–‡”‹‡† ƒ• ‡–‹‘‡† ‹ –Š‡ š’‡”‹‡–ƒŽ ’”‘ ‡†—”‡•Ǥ cǦA. niger ͶǤͳǣǣ’Ǧ—–ǡ ‘Ǧ A. niger ͶǤͳǣǣ’Ǧ™–ǡ UǦA. niger ͶǤͳǣǣ’Ǧ„••ǡ ǦA. niger ͶǤͳǣǣ’Ǧ„••ͷšǤ(A)‡˜‡Ž‘’‡–‘ˆȾǦ‰Ž— —”‘‹†ƒ•‡ƒ –‹˜‹–›‹—•–”‡••‡† —Ž–—”‡•ǡ(B)‹ —Ž–—”‡•–”‡ƒ–‡†™‹–ŠʹͲɊ‰ȀŽ–—‹ ƒ› ‹ǡƒ†(C)‹ —Ž–—”‡•–”‡ƒ–‡†™‹–ŠʹͲǤ. 74CharacterisationoftheUnfoldedProteinResponseElement.

(54) Chapter3 ‘’ƒ”‡† –‘ A. niger ͶǤͳǣǣ’Ǧ™–ǡ –Š‡ „ƒ•ƒŽ ‡š’”‡••‹‘ Ž‡˜‡Ž ™ƒ• ‡Ž‡˜ƒ–‡† ‘•‹†‡”ƒ„Ž›‹–Š‡”‡’‘”–‡”•–”ƒ‹• ƒ””›‹‰‡‹–Š‡”’Ǧ„••‘”’Ǧ„••ͷšȋ ‹‰ǤͷȌǤA. niger ͶǤͳǣǣ’Ǧ—– ‘ –Š‡ ‘–Š‡” Šƒ†ǡ ‡šŠ‹„‹–‡† ƒ •Ž‹‰Š–Ž› Ž‘™‡” „ƒ•ƒŽ ƒ –‹˜‹–› ‘’ƒ”‡† –‘ –Š‡ ’Ǧ™–Ǥ ’‘  •–”‡••ǡ –”‹‰‰‡”‡† „› –—‹ ƒ› ‹ ‘” ǡ –Š‡ ȾǦ ‰Ž— —”‘‹†ƒ•‡ ƒ –‹˜‹–› ‹ ”‡ƒ•‡† •‹‰‹ˆ‹ ƒ–Ž› ‹ •–”ƒ‹• ƒ””›‹‰ ’Ǧ™–ǡ ’Ǧ„••ǡ ƒ†’Ǧ„••ͷšǤŠ‡‡ˆˆ‡ –™ƒ•‘•–’”‘‘— ‡†ˆ‘”’Ǧ„••ͷšǡˆ‘ŽŽ‘™‡†„›’Ǧ „••ƒ†’Ǧ™–Ǥ‘”‡•’‘•‡™ƒ•‘„•‡”˜‡†ˆ‘”’Ǧ—–ǡ‡‹–Š‡”‘–—‹ ƒ› ‹‘” ‘ Ǥ ’Ǧ„••ͷš •Š‘™‡† –Š‡ Žƒ”‰‡•– ‹ ”‡ƒ•‡ ‹ ƒ –‹˜‹–›ǡ „‘–Š ™‹–Š –—‹ ƒ› ‹ ƒ† ǤŠ‹•‹†‹ ƒ–‡•–Šƒ––Š‡‡ˆˆ‡ –‘ˆƒ–Ž‡ƒ•–•‘‡‘ˆ–Š‡ƒ††‹–‹‘ƒŽ ‘’‹‡•‘ˆ–Š‡‹ –Š‹•’”‘‘–‡”‹• ——Žƒ–‹˜‡Ǥ

(55) ƒŽŽ ƒ•‡•ǡ‰ƒ˜‡ƒ•–”‘‰‡””‡•’‘•‡–Šƒ–—‹ ƒ› ‹Ǥ —‹ ƒ› ‹ •’‡ ‹ˆ‹ ƒŽŽ› ’”‘˜‘‡• Ǧ•–”‡•• „› ‹Š‹„‹–‹‰ Ǧ‰Ž› ‘•›Žƒ–‹‘ ‘ˆ ‡™Ž› •›–Š‡•‹•‡† ’”‘–‡‹•ǡ –Š‡”‡„› „Ž‘ ‹‰ –Š‡‹” ˆ‘Ž†‹‰ ƒ† –”ƒ•‹– –Š”‘—‰Š –Š‡  ͳʹʹǤ  ƒ –• ‘”‡ ‰‡‡”ƒŽŽ› „› †‹•”—’–‹‰ –Š‡ ‘š‹†‹•‹‰ ‡˜‹”‘‡– ‘ˆ –Š‡  ƒ† –Š‡”‡„› ’”‡˜‡–‹‰ –Š‡ ˆ‘”ƒ–‹‘ ‘ˆ †‹•—Ž’Š‹†‡ „‘†•Ǥ ƒ‡ –‘‰‡–Š‡”ǡ –Š‡•‡ ”‡•—Ž–• †‡‘•–”ƒ–‡ –Šƒ––Š‡ ƒ Ǧ„‹†‹‰•‹–‡•‡•–ƒ„Ž‹•Š‡†invitro,ƒ”‡‘ˆ’Š›•‹‘Ž‘‰‹ ƒŽ”‡Ž‡˜ƒ ‡Ǥ   HacABindsDNAasaDimer Š‡ ‘•‡•—•  ‘ˆ A. niger ™ƒ• ˆ‘—† –‘ „‡ ƒ ’ƒ”–Ž› ’ƒŽ‹†”‘‹  •‡“—‡ ‡ǡ ƒ† ƒ ’‡”ˆ‡ – ’ƒŽ‹†”‘‹  „‘š ™ƒ• ‹•‘Žƒ–‡† „› ƒ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ ƒ’’”‘ƒ ŠǤ —”–Š‡”‘”‡ǡ ƒ  ‡šŠ‹„‹–• ˆ‡ƒ–—”‡• –›’‹ ƒŽ ˆ‘” –Š‡ ˆƒ‹Ž› ‘ˆ „ƒ•‹  Ž‡— ‹‡Ǧœ‹’’‡” –›’‡ –”ƒ• ”‹’–‹‘ ˆƒ –‘”• ȋ„

(56) ȌǤ ƒ‡ –‘‰‡–Š‡” –Š‹• •—‰‰‡•–• –Šƒ– ƒ  „‹†• –‘  ƒ• ƒ Š‘‘†‹‡”Ǥ ‘ ƒ••‡••™Š‡–Š‡” ƒ ‹†‡‡†„‹†•–‘–Š‡ƒ•ƒ†‹‡”ǡƒ–”— ƒ–‡†ˆ‘”‘ˆ ƒ ™ƒ• ‡š’”‡••‡†‹E.coli.•–‘’ ‘†‘™ƒ•‹–”‘†— ‡†ƒ––Š‡•–ƒ”–‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘ ‘ˆhacAǡ™Š‹ Š”‡•—Ž–‡†‹ƒ ƒ ˜ƒ”‹ƒ–Žƒ ‹‰ͳʹͺƒ‹‘ƒ ‹†•ƒ––Š‡Ǧ–‡”‹—•ǤŠ‹• –”— ƒ–‡† ƒ ™ƒ•—•‡†‹ƒ„‹†‹‰‡š’‡”‹‡––‘‰‡–Š‡”™‹–Š–Š‡ˆ—ŽŽŽ‡‰–Š ǡ ƒ†–Š‡͵ͶǦ„’͵ʹǦŽƒ„‡ŽŽ‡†cypBȋ ‹‰Ǥ͸ȌǤ. Figure 6.  •Š‘™‹‰ –Šƒ– ƒ  „‹†• in vitro –‘  ƒ• ƒ Š‘‘†‹‡”Ǥ ȋ™–Ȍǡ –Š‡ ˆ—ŽŽ Ž‡‰–Š Ǧ ȋ ‹•Ȍ͸Ǥ ȋ–”Ȍǡ –Š‡ –”— ƒ–‡† ͳǦʹͳͶǦȋ ‹•Ȍ͸Ǥ Š‡ ’‘•‹–‹‘‘ˆ–Š‡†‹‡”ǡˆ‘”‡†„‡–™‡‡–Š‡ˆ—ŽŽŽ‡‰–Š ƒ†–Š‡–”— ƒ–‡† ƒ ‹•‹†‹ ƒ–‡†„›ƒƒ””‘™Ǥͳ ͵ʹǦŽƒ„‡ŽŽ‡†cypB™ƒ•—•‡†ƒ•ƒ’”‘„‡Ǥ. CharacterisationoftheUnfoldedProteinResponseElement75.

(57) Chapter3 Š‡ ‘’Ž‡š‡•ˆ‘”‡†„‡–™‡‡–Š‡cypBƒ†‡‹–Š‡”–Š‡ˆ—ŽŽŽ‡‰–Š‘”–”— ƒ–‡† ƒ  ’”‘–‡‹•ǡ Šƒ† †‹ˆˆ‡”‡– ‹‰”ƒ–‹‘ ”ƒ–‡• „—– ’‘••‡••‡† ƒ’’”‘š‹ƒ–‡Ž› –Š‡ •ƒ‡ ‹–‡•‹–›ǡ ™Š‹ Š ‹†‹ ƒ–‡• •‹‹Žƒ” „‹†‹‰ ’”‘’‡”–‹‡•Ǥ Š‡ „‘–Š ƒ  ’”‘–‡‹• ™‡”‡ ƒ††‡† –‘ –Š‡ •ƒ‡„‹†‹‰”‡ƒ –‹‘ƒƒ††‹–‹‘ƒŽ ‘’Ž‡š‘ˆƒ‹–‡”‡†‹ƒ–‡•‹œ‡™ƒ•ˆ‘”‡†ǡƒ•Œ—†‰‡† „›–Š‡ƒƒŽ›•‹•ȋ ‹‰Ǥ͸ȌǤŠ‹• ‘’Ž‡š ‘””‡•’‘†•–‘ƒ†‹‡”ˆ‘”‡†„‡–™‡‡‘‡ ˆ—ŽŽ Ž‡‰–Š ƒ† ‘‡ –”— ƒ–‡† ‘Ž‡ —Ž‡ ‘ˆ ƒ ǡ ‘ˆ‹”‹‰ –Šƒ– ‹– ˆ— –‹‘• ƒ• ƒ †‹‡”Ǥ ‹ ‡ –Š‡ †‹•–”‹„—–‹‘‘ˆ –Š‡ –Š”‡‡ ‘’Ž‡š‡• ‹• ƒŽ•‘ ƒ•‡š’‡ –‡†ǡ ƒ’’”‘š‹ƒ–‡Ž› ʹͷΨ ˆ—ŽŽ Ž‡‰–Š ƒ †‹‡”ǡͷͲΨˆ—ŽŽŽ‡‰–Š Ȁ–”— ƒ–‡† ƒ †‹‡”ǡƒ†ʹͷΨ–”— ƒ–‡† ƒ  †‹‡”ǡ –Š‹• ‹†‹ ƒ–‡• –Šƒ– –Š‡ Ǧ–‡”‹—• ‘ˆ ƒ  †‘‡• ‘– •‹‰‹ˆ‹ ƒ–Ž› ƒˆˆ‡ – –Š‡  „‹†‹‰’”‘’‡”–‹‡•‘ˆ–Š‡’”‘–‡‹invitro.  . Figure 7.  Š‡ƒ–‹  ”‡’”‡•‡–ƒ–‹‘ ‘ˆ –Š‡ ’‘•‹–‹‘ ‘ˆ –Š‡ A. niger • ‹ ”‡Žƒ–‹‘ –‘ –Š‡ ’—–ƒ–‹˜‡ –”ƒ• ”‹’–‹‘ •–ƒ”– •‹–‡ǡ ƒ† –Š‡   ‘†‘ ‘ˆ –Š‡ ‘””‡•’‘†‹‰ ‰‡‡Ǥ —„‡”• ‹†‹ ƒ–‡–Š‡’‘•‹–‹‘‘ˆ–Š‡–”ƒ• ”‹’–‹‘•–ƒ”–ǡƒ†–Š‡’‘•‹–‹‘‘ˆ–Š‡ ‘•‡”˜‡†ƒ–’‘•‹–‹‘ͳ ‘ˆ–Š‡ȋ ˆ ‹‰ǤͳȌ”‡Žƒ–‹˜‡–‘–Š‡  ‘†‘ǤŠ‡–”ƒ• ”‹’–‹‘•–ƒ”–•ƒ”‡‹†‹ ƒ–‡†„›„Žƒ  „‘š‡•ǡ ƒ† –Š‡ • „› ‘’‡ „‘š‡•. hacAi ”‡ˆ‡”• –‘ –Š‡ ‹†— ‡† ˆ‘” ‘ˆ –Š‡ hacA –”ƒ• ”‹’–ǡ hacAu–‘–Š‡—‹†— ‡†ˆ‘”ǤŠ‡‘’‡ƒ””‘™‹†‹ ƒ–‡•–Š‡•ƒŽŽ‘’‡”‡ƒ†‹‰ˆ”ƒ‡ǡ’”‡•‡–‹ –Š‡ͷ̵‘ˆhacAuǤ. 76CharacterisationoftheUnfoldedProteinResponseElement.

(58) Chapter3 DeterminationoftheDistanceBetweenUPRelementsandTranscriptionStartSites Š‡ †‹•–ƒ ‡ „‡–™‡‡ –Š‡ • ‹ –Š‡ ͷ̵ǦˆŽƒ‹‰ ”‡‰‹‘• ƒ† –Š‡ ‘””‡•’‘†‹‰   ‘†‘•ǡ‹†‹ˆˆ‡”‡–Ǧ ‘–”‘ŽŽ‡†‰‡‡•ǡ˜ƒ”‹‡•„‡–™‡‡͵͵Ͷ–ȋbipAȌƒ†͹ͺ–ȋcypBȌǤ ‘†‡–‡”‹‡™Š‡–Š‡”–Š‡”‡‹•Ž‡••˜ƒ”‹ƒ–‹‘„‡–™‡‡–Š‡’‘•‹–‹‘•‘ˆ–Š‡•‡ ƒ „‹†‹‰ •‹–‡•‹”‡Žƒ–‹‘–‘–Š‡–”ƒ• ”‹’–‹‘•–ƒ”–•‹–‡ǡͷǯ‡š’‡”‹‡–•™‡”‡’‡”ˆ‘”‡†ˆ‘”ˆ‹˜‡ –ƒ”‰‡–‰‡‡•Ǥ ‹‰—”‡͹•Š‘™•• Š‡ƒ–‹ ƒŽŽ›–Š‡’‘•‹–‹‘‘ˆ–Š‡‹”‡Žƒ–‹‘–‘„‘–Š–Š‡ –”ƒ• ”‹’–‹‘•–ƒ”–•‹–‡ƒ†–Š‡ Ǧ ‘†‘‹ƒ—„‡”‘ˆ ƒ ”‡‰—Žƒ–‡†’”‘‘–‡”•Ǥ

(59)  ƒŽŽ ƒ•‡•ǡ –Š‡  ‹• ‹ Ž‘•‡ ˜‹ ‹‹–› –‘ –Š‡ –”ƒ• ”‹’–‹‘ •–ƒ”– •‹–‡Ǥ Š‡ †‹•–ƒ ‡ „‡–™‡‡–Š‡ ‘•‡”˜‡†ƒ–’‘•‹–‹‘ͳ‘ˆ–Š‡•ȋ ˆ ‹‰ǤͳȌƒ†–Š‡–”ƒ• ”‹’–‹‘•–ƒ”– •‹–‡˜ƒ”‹‡•„‡–™‡‡͹ͻ„’ˆ‘”bipAƒ†ʹͷ„’ˆ‘”cypBǤ”‡˜‹‘—•Ž›™‡†‡–‡”‹‡†–Š‡•–ƒ”– •‹–‡•‘ˆ–Š‡hacAuƒ†hacAi–”ƒ• ”‹’–• ͳͲͳǤ

(60) ––—”‡†‘—––Šƒ––Š‡•‡•–ƒ”–•‹–‡•ƒ”‡’”‡ ‡†‡† „› ͳ ƒ† ʹ ”‡•’‡ –‹˜‡Ž›ǡ ™Š‡”‡ƒ• ͵ ‹• •‹–—ƒ–‡† ‹ „‡–™‡‡ –Š‡ –™‘ ‘–Š‡” •ǡƒ†‘˜‡”Žƒ’•™‹–Š–Š‡•–ƒ”–‘ˆ–Š‡— ȋ ‹‰Ǥ͹ȌǤ  . Discussion  ‡ ‡–Ž› ™‡ Ž‘‡† ƒ† Šƒ”ƒ –‡”‹•‡† –Š‡ A. niger –”ƒ• ”‹’–‹‘ ˆƒ –‘” ƒ ǡ ‹˜‘Ž˜‡† ‹ •‹‰ƒŽŽ‹‰–Š‡’”‡•‡ ‡‘ˆ•–”‡••–‘–Š‡— Ž‡—•Ǥ˜‡”Ǧ‡š’”‡••‹‘‘ˆhacAiŽ‡†–‘–Š‡—’Ǧ ”‡‰—Žƒ–‹‘‘ˆ–ƒ”‰‡–‰‡‡•ǡƒ†„ƒ†•Š‹ˆ–ƒƒŽ›•‹•™‹–Š–Š‡’”‘‘–‡”ˆ”ƒ‰‡–• ‘ˆ–Š‘•‡‰‡‡• ‘ˆ‹”‡†–Š‡†‹”‡ –‹–‡”ƒ –‹‘‘ˆ ƒ ™‹–Š–Š‘•‡’”‘‘–‡”• ͳͲͳǤ ‡”‡™‡ ‹†‡–‹ˆ‹‡†–Š‡cisǦƒ –‹‰‡Ž‡‡–•ǡ’”‡•‡–‹–Š‡’”‘‘–‡”•‘ˆ–Š‡•‡”‡•’‘•‹˜‡‰‡‡• ‘ˆA.nigerǤŠ‡A.nigerƒ’• Ž‘•‡Ž›–‘–Š‡ ‘””‡•’‘†‹‰–”ƒ• ”‹’–‹‘•–ƒ”–’‘‹– ȋ ‹‰Ǥ͹Ȍǡƒ†ƒ– Š‡•–Š‡ˆ‘ŽŽ‘™‹‰ ‘•‡•—••‡“—‡ ‡•–”‡– Šǣȋ ȀȌ T/GCCT. ’ƒ”–Ž› ’ƒŽ‹†”‘‹  •‡“—‡ ‡ ȋ—†‡”Ž‹‡†Ȍ ƒ”‘—† ƒ •‹‰Ž‡ •’ƒ ‡” — Ž‡‘–‹†‡ ȋ„‡–™‡‡ „”ƒ ‡–•Ȍǡˆ‘ŽŽ‘™‡†„›ƒŠ‹‰ŠŽ› ‘•‡”˜‡†•–”‡– Š‘ˆˆ‘—”„ƒ•‡•ȋ‹„‘Ž†ȌǤŠ‡’ƒŽ‹†”‘‹  ‘”‡‘ˆ–Š‡ ‘•‡•—•A.niger•Š‘™••‹‹Žƒ”‹–‹‡•–‘–Š‡KAR2‘ˆS.cerevisiaeǡ ™Š‹ Š‹•ǣ ȋȌ  ͻ͹ǡͻͻǤ‘–Š• ‘–ƒ‹ƒǦ„‘šŽ‹‡’ƒŽ‹†”‘‡ȋ Ȍƒ”‘—† ƒ•‹‰Ž‡•’ƒ ‡”— Ž‡‘–‹†‡ ͶͶǤŠ‡•‹‹Žƒ”‹–›„‡–™‡‡–Š‡A.nigerƒ†S.cerevisiae•‹• ƒŽ•‘”‡ˆŽ‡ –‡†„›•‡˜‡”ƒŽ‘–Š‡”ˆ‹†‹‰•Ǥ ‹”•–Ž›ǡƒŽ–Š‘—‰Š–Š‡‘˜‡”ƒŽŽ‹†‡–‹–›„‡–™‡‡–Š‡A. niger ƒ ƒ†S.cerevisiae ƒ ͳ’‹•‘Ž›ʹͷΨǡ–Š‡›•Šƒ”‡͹͹Ψ‹†‡–‹ ƒŽ”‡•‹†—‡•‹–Š‡‹” „‹†‹‰†‘ƒ‹•ͳͲͳǤ‡ ‘†Ž›ǡ–Š‡A.niger ƒ ™ƒ•ˆ‘—†–‘„‹†ƒ‘Ž‹‰‘— Ž‡‘–‹†‡ ”‡’”‡•‡–‹‰ –Š‡ S. cerevisiae KAR2  ‹ ƒ  ‰‡Ž •Š‹ˆ– ƒ••ƒ› ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ —”–Š‡”‘”‡ǡ–Š‡A.niger ƒ •Šƒ”‡•ʹ͹Ψ‘˜‡”ƒŽŽ‹†‡–‹–›ƒ†ͻʹΨ‹†‡–‹ ƒŽ”‡•‹†—‡•‹ –Š‡„‹†‹‰†‘ƒ‹™‹–Š–Š‡Trichodermareesei ƒ ͳ’”‘–‡‹ǤŠ‡T.reeseihac1‰‡‡ ‘—Ž†’ƒ”–Ž› ‘’Ž‡‡––Š‡S.cerevisiaeHAC1†‹•”—’–‹‘ͳͶͲǡƒ†‘˜‡”Ǧ‡š’”‡••‹‘‘ˆ–Š‡T. reeseihac1‹S.cerevisiae”‡•—Ž–‡†‹ƒ ——Žƒ–‹‘‘ˆ–Š‡KAR2–”ƒ• ”‹’–ǡ™Š‹ Š‹†‹ ƒ–‡• ”‡ ‘‰‹–‹‘‘ˆ–Š‡S.cerevisiaeKAR2„›–Š‡T.reesei ƒ ͳ’”‘–‡‹ͳ͸ͶǤ CharacterisationoftheUnfoldedProteinResponseElement77.

(61) Chapter3 Ž–Š‘—‰Š–Š‡A.nigerƒ†–Š‡S.cerevisiae•Šƒ”‡•‘‡ ‘‘ˆ‡ƒ–—”‡•ǡ–Š‡”‡ ƒ”‡ –Š”‡‡ ƒŒ‘” †‹ˆˆ‡”‡ ‡•Ǣ –Š‡ ƒ–—”‡ ‘ˆ –Š‡ •’ƒ ‡” — Ž‡‘–‹†‡ǡ –Š‡ Š‹‰ŠŽ› ‘•‡”˜‡† •–”‡– Šˆ‘ŽŽ‘™‹‰–Š‡’ƒŽ‹†”‘‹  ‘”‡‘ˆ–Š‡Aspergillusǡƒ†–Š‡†‹•’‡•ƒ„‹Ž‹–›‘ˆ–Š‡ •’ƒ ‡”— Ž‡‘–‹†‡Ǥ Š‡ˆ‹”•–‘–ƒ„Ž‡†‹ˆˆ‡”‡ ‡„‡–™‡‡–Š‡A.nigerƒ†S.cerevisiae‹•–Š‡ƒ–—”‡ ‘ˆ –Š‡ •’ƒ ‡” — Ž‡‘–‹†‡ǡ ™Š‹ Š ‹ –Š‡  ‘ˆ S. cerevisiae ‘•– ˆ”‡“—‡–Ž› ‹• ƒ  — Ž‡‘–‹†‡ǡƒ† ƒ ͳ’’”‡ˆ‡”•ƒ•’‡ ‹ˆ‹ •’ƒ ‡”— Ž‡‘–‹†‡‹–Š‡‘”†‡”ε εε ͻͷǤ

(62) –Š‡ A.niger•ǡŠ‘™‡˜‡”ǡƒ ‘”ƒ™ƒ•’”‡•‡–ƒ••’ƒ ‡”— Ž‡‘–‹†‡ǡ‰‹˜‡–Š‡•‹–—ƒ–‹‘ ‹ ‹‰—”‡ ͳ ‹ ™Š‹ Š –Š‡ ƒŒ‘”‹–› ‘ˆ –Š‡ ƒŽ‹‰‡† • ƒ”‡ •Š‘™ ‹ –Š‡‹” ƒ–—”ƒŽ ‘”‹‡–ƒ–‹‘Ǥ‘”‡‘˜‡”ǡ–Š‡—–ƒ–‹‘Ͷ Æ‘ˆ–Š‡•’ƒ ‡”— Ž‡‘–‹†‡‹–Š‡cypBŽ‡†–‘ •‹‰‹ˆ‹ ƒ–Ž› ‹ ”‡ƒ•‡† „‹†‹‰ ȋƒ„Ž‡ ʹȌǡ –Š‹• ‹ ‘–”ƒ•– –‘ –Š‡ ‘”†‡” ‘ˆ ’”‡ˆ‡”‡ ‡ ”‡’‘”–‡†ˆ‘”–Š‡S.cerevisiae ƒ ͳ’Ǥ Š‡ •‡ ‘† ‹’‘”–ƒ– †‹ˆˆ‡”‡ ‡ ‹• –Š‡ ’”‡•‡ ‡ ‘ˆ ƒ Š‹‰ŠŽ› ‘•‡”˜‡† •–”‡– Š ȋȀ Ȍƒ†Œƒ ‡––‘–Š‡’ƒŽ‹†”‘‹  ‘”‡‘ˆ–Š‡Aspergillus•ǡ™Š‹ Šƒ’’‡ƒ”•–‘„‡ ƒ„•‡–‹S.cerevisiaeǤŠ‹••–”‡– Š™ƒ•ˆ‘—†‹ƒŽŽA.niger•ƒƒŽ›•‡†ǡƒ†™‹–Š‘‡ ‡š ‡’–‹‘ȋ ‹–Š‡A.fumigatustigAȌ‹–Š‡Š‘‘Ž‘‰—‡•ˆ”‘A.nidulansƒ†A. fumigatus ƒ•™‡ŽŽȋ ‹‰ͳȌǤŠ‡‹’‘”–ƒ ‡‘ˆ–Š‡•‡— Ž‡‘–‹†‡•ˆ‘”„‹†‹‰‘ˆ ƒ –‘–Š‡ ǡ™ƒ••Š‘™„›—–ƒ–‹‘ƒŽƒƒŽ›•‹•‘ˆ–Š‡cypBǡ†‡‘•–”ƒ–‹‰–Šƒ–—–ƒ–‹‘• ‹ ƒ› ‘ˆ –Š‡ Žƒ•– –Š”‡‡ ”‡•‹†—‡• ‘ˆ –Š‡ •–”‡– Š ȋȌ ƒˆˆ‡ –‡† „‹†‹‰ •‹‰‹ˆ‹ ƒ–Ž› ȋƒ„Ž‡ ʹǤȌǤ —”–Š‡”‘”‡ǡ„‹†‹‰•‹–‡•‡Ž‡ –‹‘ƒŽ•‘Ž‡†–‘ƒ„‘šŠƒ˜‹‰–Š‹••–”‡– Šǡˆ‘ŽŽ‘™‹‰–Š‡ ’ƒŽ‹†”‘‹  ‘”‡ȋ ‹‰Ǥ͵ȌǤ Š‡–Š‹”††‹ˆˆ‡”‡ ‡‹•–Š‡†‹•’‡•ƒ„‹Ž‹–›‘ˆ–Š‡•’ƒ ‡”— Ž‡‘–‹†‡‹–Š‡Aspergillus Ǥ‡Ž‡–‹‘‘ˆ–Š‡•’ƒ ‡”‹–Š‡S.cerevisiaeKAR2”‡†— ‡†–Š‡”‡•’‘•‡–‘ ͳ͵Ψ ‘ˆ –Š‡ ™‹Ž† –›’‡ Ž‡˜‡Žǡ ƒ† ‹–”‘†— –‹‘ ‘ˆ –™‘ ‘” ‘”‡  „ƒ•‡• „‡–™‡‡ –Š‡ ’ƒŽ‹†”‘‹  ŠƒŽˆǦ•‹–‡• ƒ„‘Ž‹•Š‡† ƒ ͳ’Ǧ‡†‹ƒ–‡† –”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‹‘ ƒŽ‘•– ‘’Ž‡–‡Ž› ͻͷǤ

(63)  ‘–”ƒ•––‘–Š‡•‹–—ƒ–‹‘”‡’‘”–‡†ˆ‘”–Š‡S.cerevisiae ƒ ͳ’ǡ–Š‡Aniger ƒ ’”‡ˆ‡”•ƒ™‹–Š‘—–ƒ•’ƒ ‡”— Ž‡‘–‹†‡ǤŽ–Š‘—‰Š‘•–‘ˆ–Š‡•‹†‡–‹ˆ‹‡† ‹–Š‡’”‘‘–‡””‡‰‹‘•‘ˆ–Š‡ ƒ Ǧ ‘–”‘ŽŽ‡†‰‡‡•‹A.nigerƒ –—ƒŽŽ› ‘–ƒ‹ƒ•’ƒ ‡” — Ž‡‘–‹†‡ǡ ™‡ †‡‘•–”ƒ–‡† –Šƒ– †‡•’‹–‡ –Š‡ Žƒ  ‘ˆ ‹–ǡ –Š‡ „‹†‹‰ •‹–‡ •‡Ž‡ –‹‘ „‘š ‘’‡”ƒ–‡•ƒ•ƒ’Š›•‹‘Ž‘‰‹ ƒŽŽ›”‡Ž‡˜ƒ–cisǦƒ –‹‰‡Ž‡‡–™Š‡‹–”‘†— ‡†‹A.nigerȋ ‹‰Ǥ ͷȌǤŠ‹••—‰‰‡•–•–Šƒ–ƒ™‹–Š‘—–ƒ•’ƒ ‡”— Ž‡‘–‹†‡ ‘—Ž†„‡‘ˆ„‹‘Ž‘‰‹ ƒŽ”‡Ž‡˜ƒ ‡ǡ ƒ† •‡˜‡”ƒŽ ‘–Š‡” ˆ‹†‹‰• •–”‡‰–Š‡ –Š‹• Š›’‘–Š‡•‹•Ǥ ‹”•–Ž›ǡ –Š‡ hacA ’”‘‘–‡” ‹–•‡Žˆ ‘–ƒ‹• •— Š ƒ  ȋ ‹‰Ǥ ͳȌǤ Š‡ –”ƒ• ”‹’–‹‘ ‘ˆ hacA ‹• ƒ—–‘Ǧ”‡‰—Žƒ–‡†. ͳͲͳǡ. ƒ†. –”ƒ• ”‹’–‘‡ƒƒŽ›•‹•‘ˆ–Š‡‹A.nidulans’—–•hacAƒ‘‰–Š‡Š‹‰Š‡•–—’Ǧ”‡‰—Žƒ–‡† ‰‡‡• ͳͷͶǡ™Š‹ Š‹•‹ƒ ‘”†ƒ ‡™‹–Š‘—”ˆ‹†‹‰‘ˆ–Š‡hacA͵Šƒ˜‹‰–Š‡Ž‘™‡•–Kd ˜ƒŽ—‡ȋƒ„Ž‡͵ȌǤ

(64) –•Š‘—Ž†„‡‘–‡†ǡŠ‘™‡˜‡”ǡ–Šƒ––Š‡hacA’”‘‘–‡” ‘–ƒ‹•͵•ǡƒŽŽ Šƒ˜‹‰ Ž‘™‡” Kd ˜ƒŽ—‡• –Šƒ –Š‡ ‘–Š‡” • •–—†‹‡† Š‡”‡Ǥ Š‡”‡ˆ‘”‡ǡ —–ƒ–‹‘ ƒƒŽ›•‹• •Š‘—Ž†„‡ ƒ””‹‡†‘—–ǡ–‘†‡–‡”‹‡–Š‡ ‘–”‹„—–‹‘‘ˆ–Š‡†‹ˆˆ‡”‡–•ǡƒ†‡•’‡ ‹ƒŽŽ› –Š‡͵ǡ–‘–Š‡–”ƒ• ”‹’–‹‘ƒŽ”‡‰—Žƒ–‹‘‘ˆ hacAǤ‡ ‘†Ž›ǡ–Š‡A.nidulans bipA 78CharacterisationoftheUnfoldedProteinResponseElement.

(65) Chapter3 Žƒ • –Š‡ •’ƒ ‡” — Ž‡‘–‹†‡ ȋ ˆ ‹‰Ǥ ͳȌǤ š ‡’– ˆ‘” –Š‡ ‹••‹‰ •’ƒ ‡” — Ž‡‘–‹†‡ǡ ‹–• •‡“—‡ ‡ȋˆ”‘Ȃ͹–‘Ϊͳ͹Ȍ‹•‹†‡–‹ ƒŽ–‘‹–•Š‘‘Ž‘‰—‡•ˆ”‘A.nigerƒ†A.fumigatusǤŠ‡ „‹†‹‰ ƒˆˆ‹‹–› ‘ˆ –Š‡ A. nidulans bipA  ȋKd α͵ͻ r ͻ Ȍ ‹• ‹ –Š‡ •ƒ‡ ”ƒ‰‡ ‘ˆ –Š‡ „‹†‹‰ƒˆˆ‹‹–‹‡•‘ˆ–Š‡A.niger•ȋƒ„Ž‡͵Ȍǡƒ†‘Ž›•Ž‹‰Š–Ž›Ž‘™‡” ‘’ƒ”‡†–‘–Š‡A. nigerbipAǤŠ‹•‹†‹ ƒ–‡•–Šƒ––Š‡•’ƒ ‡”— Ž‡‘–‹†‡‹•†‹•’‡•ƒ„Ž‡ǡƒ–Ž‡ƒ•–‹•‘‡ Aspergillus•Ǥ ‹ƒŽŽ›ǡƒ Ž‘•‡”‹•’‡ –‹‘‘ˆ–Š‡A.nigertigA”‡˜‡ƒŽ‡†–Šƒ–‹–ƒŽ•‘ ‹‰Š–Žƒ ƒ•’ƒ ‡”— Ž‡‘–‹†‡Ǥ

(66) –• ‘”‡•‡“—‡ ‡ ƒ„‡’”‡•‡–‡†™‹–Šƒ•’ƒ ‡”— Ž‡‘–‹†‡ ƒ• ȋȌ  TCCTǡ „—– ‘”‡ Ž‹‡Ž› ™‹–Š‘—– ƒ •’ƒ ‡” — Ž‡‘–‹†‡ ƒ•   TCCTǤ —”–Š‡”‘”‡ǡ –Š‡ ˆƒ – –Šƒ– –Š‡ A. nidulans tigA  ‘Ž› ‹ –Š‡ Žƒ––‡” ƒ•‡ ’‘••‡••‡• ’ƒŽ‹†”‘‹  ”‡•‹†—‡• ȋ ‹‰Ǥ ͳȌ •—‰‰‡•–• –Šƒ– –Š‡ •‹–—ƒ–‹‘ ™‹–Š‘—– ƒ •’ƒ ‡” — Ž‡‘–‹†‡ ‹• ‘”‡ Ž‹‡Ž›Ǥ ‘™‡˜‡”ǡ—–ƒ–‹‘ƒŽƒƒŽ›•‹• •Š‘—Ž† „‡ ƒ””‹‡† ‘—– –‘ —ƒ„‹‰—‘—•Ž› ’”‘˜‡ –Š‡Žƒ ‘ˆƒ•’ƒ ‡”— Ž‡‘–‹†‡‹–Š‡tigAǤ‘„‹‡†ǡ–Š‡•‡ˆ‹†‹‰••–”‘‰Ž›ƒ”‰—‡ ˆ‘” –Š‡ †‹•’‡•ƒ„‹Ž‹–› ‘ˆ –Š‡ •’ƒ ‡” — Ž‡‘–‹†‡ǡ ƒ† –Š‡ „‹‘Ž‘‰‹ ƒŽ ”‡Ž‡˜ƒ ‡ ‘ˆ •— Š ƒ Ǥ Š‡ • ˆ”‘ A. niger, A. nidulans ƒ† A. fumigatusǡ ƒ”‡ ”‡ƒ”ƒ„Ž› ™‡ŽŽ ‘•‡”˜‡†ƒ‘‰Š‘‘Ž‘‰‘—•‰‡‡•ƒ ”‘••–Š‡•’‡ ‹‡•ǡ™Š‹Ž‡‘”‡•‡“—‡ ‡˜ƒ”‹ƒ–‹‘‹• ’”‡•‡– ƒ‘‰ –Š‡ • ‘ˆ †‹ˆˆ‡”‡– ‰‡‡• ˆ”‘ A. niger ȋ ‹‰Ǥ ͳȌǤ Š‡ †‹••‘ ‹ƒ–‹‘ ‘•–ƒ–• ȋKdȌ „‡–™‡‡ •‡˜‡”ƒŽ ‘ˆ –Š‡ A. niger • ƒŽ•‘ ˜ƒ”› •‹‰‹ˆ‹ ƒ–Ž› ȋƒ„Ž‡ ͵ȌǤ ƒ‡ –‘‰‡–Š‡” –Š‹• ‹‰Š– ‹†‹ ƒ–‡ –Šƒ– †—”‹‰ –Š‡ ‘—”•‡ ‘ˆ ‡˜‘Ž—–‹‘ǡ –Š‡ ‡‡† ˆ‘” †‹ˆˆ‡”‡–‹ƒŽ—’Ǧ”‡‰—Žƒ–‹‘‘ˆ–Š‡†‹ˆˆ‡”‡–Ǧ–ƒ”‰‡–’”‘–‡‹•—’‘•–”‡••ǡŠƒ•‡’––Š‡ •‘ˆŠ‘‘Ž‘‰‘—•‰‡‡•”ƒ–Š‡”•‹‹Žƒ”ǤInvivoƒƒŽ›•‡••Š‘™‡†–Šƒ–„‹†‹‰Ǧ‡Šƒ ‹‰ Šƒ‰‡•‹–Š‡cypB‡Ž‡˜ƒ–‡†–Š‡‡š’”‡••‹‘‘ˆ ǡƒŽ•‘—†‡”‘Ǧ•–”‡•• ‘†‹–‹‘•Ǥ

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