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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)  Chapter2     TheTranscriptionFactorHacAMediatesthe UnfoldedProteinResponseinAspergillus niger,andUpRegulatesitsOwn Transcription        ƒ” Ǥ—Ž†‡”ǡƒ”—ƒŽ‘Š‡‹‘ǡ‡”Œƒ‡––‹Ž¡ǡ—•ƒǤƒ†”‹†          —„Ž‹•Š‡†‹‘Ž‡ —Žƒ” ‡‡–‹ •ƒ† ‡‘‹ •ǤʹͲͲͶǡʹ͹ͳȋʹȌǣͳ͵ͲǦͳͶͲ.

(3) Chapter2. Abstract  Š‡—ˆ‘Ž†‡†’”‘–‡‹”‡•’‘•‡ȋȌ‹˜‘Ž˜‡•ƒ ‘’Ž‡š•‹‰ƒŽŽ‹‰’ƒ–Š™ƒ›‹™Š‹ Š–Š‡ –”ƒ• ”‹’–‹‘ ˆƒ –‘” ƒ  ’Žƒ›• ƒ ‡–”ƒŽ ”‘Ž‡Ǥ ‡”‡ ™‡ ”‡’‘”– –Š‡ Ž‘‹‰ ƒ† Šƒ”ƒ –‡”‹•ƒ–‹‘ ‘ˆ –Š‡ hacA ‰‡‡ ƒ† ‹–• ’”‘†— – ˆ”‘ Aspergillus nigerǤ †‘’Žƒ•‹  ”‡–‹ —Ž—ȋȌ•–”‡••”‡•—Ž–•‹–Š‡•’Ž‹ ‹‰‘ˆƒ— ‘˜‡–‹‘ƒŽʹͲ–‹–”‘ˆ”‘–Š‡A. nigerhacAǡƒ†‹ƒ††‹–‹‘–‘–Š‡–”— ƒ–‹‘‘ˆ–Š‡ͷ̵Ǧ‡†‘ˆ–Š‡hacA„›ʹ͵Ͳ –Ǥ

(4) –Š‹••–—†›–Š‡™ƒ•–”‹‰‰‡”‡†„›‘˜‡”Ǧ‡š’”‡••‹‰–‹••—‡’Žƒ•‹‘‰‡ƒ –‹˜ƒ–‘”ȋ–Ǧ Ȍǡ ƒ† „› –”‡ƒ–‡– ‘ˆ › ‡Ž‹ƒ ™‹–Š †‹–Š‹‘–Š”‡‹–‘Ž ȋȌ ‘” –—‹ ƒ› ‹Ǥ ˜‡”Ǧ ‡š’”‡••‹‘‘ˆ–Š‡’”‘ ‡••‡†ˆ‘”‘ˆhacA‘–‘Ž›Ž‡†–‘–Š‡—’”‡‰—Žƒ–‹‘‘ˆbipAǡcypBƒ† pdiAǡ ‹‹ ‹‰ ƒ ǡ „—– ƒŽ•‘ Ž‡† –‘ –Š‡ —’”‡‰—Žƒ–‹‘ ‘ˆ –Š‡ hacA ‰‡‡ ‹–•‡ŽˆǤ In vitro „‹†‹‰ƒ••ƒ›• ‘ˆ‹”‡†–Šƒ––Š‡ ƒ ’”‘–‡‹„‹†•–‘–Š‡’”‘‘–‡”•‘ˆ‰‡‡•‡ ‘†‹‰ ǦŽ‘ ƒŽ‹•‡† Šƒ’‡”‘‡• ƒ† ˆ‘Ž†ƒ•‡•ǡ ƒ† –‘ –Š‡ ’”‘‘–‡” ‘ˆ hacA ‰‡‡ ‹–•‡ŽˆǤ ‹ƒŽŽ›ǡ ƒ. Ǧ ƒ ˆ—•‹‘™ƒ••Š‘™–‘Ž‘ ƒŽ‹•‡‹–Š‡— Ž‡—•Ǥ .  Introduction  Š‡ ‡†‘’Žƒ•‹  ”‡–‹ —Ž— ȋȌ •‡”˜‡• ƒ• ƒ ˆ‘Ž†‹‰ ‘’ƒ”–‡– ˆ‘” ‡„”ƒ‡ ƒ† •‡ ”‡–‘”› ’”‘–‡‹•Ǥ

(5)  –Š‡ ‘š‹†‹•‹‰ ‡˜‹”‘‡– ‘ˆ –Š‡ ǡ ’”‘–‡‹ ˆ‘Ž†‹‰ ‹• ƒ••‹•–‡† „› —‡”‘—•’”‘–‡‹ Šƒ’‡”‘‡•ƒ†ˆ‘Ž†ƒ•‡• ͵ͻǡƒ†ƒ˜ƒ”‹‡–›‘ˆ“—ƒŽ‹–› ‘–”‘Ž‡ Šƒ‹•• ‡•—”‡–Šƒ–‘Ž› ‘””‡ –Ž›ˆ‘Ž†‡†’”‘–‡‹•ƒ”‡”‡Ž‡ƒ•‡† ͵ͲǡͳͻͲǤ˜ƒ”‹‡–›‘ˆ’Š›•‹‘Ž‘‰‹ ƒŽ‘” ‡˜‹”‘‡–ƒŽ•–”‡•• ‘†‹–‹‘• ƒ†‹•–—”„’”‘–‡‹ˆ‘Ž†‹‰ƒ†Ž‡ƒ†–‘–Š‡ƒ ——Žƒ–‹‘ ‘ˆ —ˆ‘Ž†‡† ’”‘–‡‹• ‹ –Š‡ Ǥ ‡ŽŽ• ”‡•’‘† –‘ •— Š ƒ ƒ ——Žƒ–‹‘ „› ‹ ”‡ƒ•‹‰ –Š‡ –”ƒ• ”‹’–‹‘ ‘ˆ ‰‡‡• –Šƒ– ‘†‡ ˆ‘” Ǧ”‡•‹†‡– Šƒ’‡”‘‡• ƒ† ˆ‘Ž†ƒ•‡•ǡ –Š‡”‡„› ‹ ”‡ƒ•‹‰–Š‡ˆ‘Ž†‹‰ ƒ’ƒ ‹–›‘ˆ–Š‡ ‘’ƒ”–‡–ǤŠ‡•‹‰ƒŽŽ‹‰’ƒ–Š™ƒ›‹˜‘Ž˜‡†‹•’ƒ”– ‘ˆ –Š‡ —ˆ‘Ž†‡† ’”‘–‡‹ ”‡•’‘•‡ ȋȌ ͸Ͷǡͳʹ͵ǡͳͷͲǡͳ͹ͻǤ ‡‘‡Ǧ™‹†‡ ƒƒŽ›•‹• ‹ ›‡ƒ•–ǡ —•‹‰  ‹ ”‘ ƒ””ƒ›•ǡ Šƒ• Ž‹‡† ƒ Žƒ”‰‡ —„‡” ‘ˆ ‰‡‡• –‘ –Š‡ ǡ ™‹–Š ˆ— –‹‘• ˆƒ” „‡›‘† –Š‘•‡  Šƒ’‡”‘‡• ͵ͺǡͳ͸ͲǤ › ƒˆˆ‡ –‹‰ ˜‹”–—ƒŽŽ› ‡˜‡”› •–ƒ‰‡ ‘ˆ –Š‡ •‡ ”‡–‘”› ’ƒ–Š™ƒ›ǡ–Š‡ƒ’’‡ƒ”•–‘„‡ƒ˜‡”•ƒ–‹Ž‡”‡‰—Žƒ–‘”›•›•–‡–Šƒ–•‡”˜‡•–‘ƒ‹–ƒ‹–Š‡ Š‘‡‘•–ƒ•‹•‘ˆˆ— –‹‘•—†‡”•–”‡•• ‘†‹–‹‘•ǤŠ‡‡›•–‡’‹–Š‡ƒ –‹˜ƒ–‹‘‘ˆ–Š‡ ‹›‡ƒ•–‹•–Š‡•’Ž‹ ‹‰‘ˆƒ— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘HAC1ǡ™Š‹ Š•‡”˜‡•–‘ ”‡Ž‹‡˜‡ ƒ –”ƒ•Žƒ–‹‘ƒŽ „Ž‘ Ǥ ͸͸ǡͳ͵͹Ǥ HAC1  ‡ ‘†‡• –Š‡ „ƒ•‹  Ž‡— ‹‡ œ‹’’‡” ȋ„

(6) ȌǦ –›’‡ –”ƒ• ”‹’–‹‘ ˆƒ –‘” ƒ ͳ’ǡ ™Š‹ Š †‹”‡ –Ž› ƒ –‹˜ƒ–‡• –Š‡ –”ƒ• ”‹’–‹‘ ‘ˆ  ‰‡‡•Ǥ ’Ž‹ ‹‰‘ˆ–Š‡›‡ƒ•–HAC1‘ —”•‹ƒ‘Ǧ•’Ž‹ ‡‘•‘ƒŽƒ‡”Ǥ—”‹‰•–”‡••ǡ –Š‡ ‹–”‘ „‘”†‡”• ‘ˆ HAC1  ƒ”‡ Ž‡ƒ˜‡† „›

(7) ”‡ͳ’ Ͷ͵ǡͳͷʹǡ ƒˆ–‡” ™Š‹ Š –Š‡ ‡š‘• ƒ”‡ ”‡Œ‘‹‡† „› –Š‡ – Ž‹‰ƒ•‡ Ž‰ͳ’ ͳͷͳǤ

(8) ”‡ͳ’ǡ ƒ ‹ƒ•‡Ȁƒ•‡ ™Š‹ Š ”‡•‹†‡• ‹ –Š‡  40IsolationandCharacterisationoftheTranscriptionFactorHacA.

(9) Chapter2 ‡„”ƒ‡•‡•‡•–Š‡ƒ ——Žƒ–‹‘‘ˆ—ˆ‘Ž†‡†’”‘–‡‹˜‹ƒƒ†›ƒ‹ ‹–‡”ƒ –‹‘™‹–Š–Š‡ Šƒ’‡”‘‡ ‹ ͺǤ ‡Ž‡ƒ•‡ ‘ˆ ‹ –”‹‰‰‡”• –Š‡ †‹‡”‹•ƒ–‹‘ ƒ† •—„•‡“—‡– –”ƒ•Ǧ ’Š‘•’Š‘”›Žƒ–‹‘‘ˆ

(10) ”‡ͳ’ǡ–Š‡”‡„›ƒ –‹˜ƒ–‹‰–Š‡Ǥ

(11) ƒ‹ƒŽ ‡ŽŽ•ǡ‡ƒ”Ž›ƒŽŽ–Š‡ˆ‡ƒ–—”‡• ‘ˆ –Š‡ ›‡ƒ•–  •’Ž‹ ‹‰ •›•–‡ ƒ”‡ ‘•‡”˜‡†ǡ „—– ‹ ‘’ƒ”‹•‘ ™‹–Š ›‡ƒ•– –Š‡‹”  •–”‡••’ƒ–Š™ƒ›ƒ’’‡ƒ”•–‘„‡‘”‡‡š–‡•‹˜‡ͳͶͺǡͳͺ͸ǤŠ‡–”ƒ• ”‹’–‹‘ˆƒ –‘”ͳǡ™Š‹ Š‹• ‹˜‘Ž˜‡† ‹ –Š‡  •–”‡•• ”‡•’‘•‡ ‘ˆ Caenorhabditis elegans ƒ† ƒƒŽ‹ƒ ‡ŽŽ•ǡ ‹• ƒ –‹˜ƒ–‡† –Š”‘—‰Š ‹–”‘ •’Ž‹ ‹‰ ‡†‹ƒ–‡† „›

(12) ͳǡ ‹ ƒ ƒ‡” •‹‹Žƒ” –‘ –Šƒ– •‡‡ ‹ ›‡ƒ•–Ǥ

(13)  ƒ††‹–‹‘ǡ –”ƒ• ”‹’–‹‘ ‘ˆ –Š‡ XBP1 ‰‡‡ ‹• ‹†— ‡† „›  ͸ǡ ƒ  ‡„”ƒ‡ ƒ••‘ ‹ƒ–‡†–”ƒ• ”‹’–‹‘ˆƒ –‘”ǡ™Š‹ Š‹•’”‘–‡‘Ž›–‹ ƒŽŽ›”‡Ž‡ƒ•‡††—”‹‰•–”‡••ͺͳǤ

(14) –Š‹••–—†›™‡ˆ‘ —•‘–Š‡—ˆ‘Ž†‡†’”‘–‡‹”‡•’‘•‡‹–Š‡ˆ‹Žƒ‡–‘—•ˆ—‰—• Aspergillus nigerǡ ƒ ‹†—•–”‹ƒŽ ’”‘†— –‹‘ ‘”‰ƒ‹• ™Š‹ Š ‹• ƒ’ƒ„Ž‡ ‘ˆ •‡ ”‡–‹‰ Žƒ”‰‡ ƒ‘—–•‘ˆƒ–‹˜‡’”‘–‡‹•Ž‹‡‰Ž— ‘ƒ›Žƒ•‡Ǥ‹‡Ž†•‘ˆŠ‡–‡”‘Ž‘‰‘—•’”‘–‡‹•ǡŠ‘™‡˜‡”ǡƒ”‡ ‘ˆ–‡Ž‘™ʹǤ

(15) ‘”†‡”–‘‹’”‘˜‡A.nigerƒ•ƒŠ‘•–ˆ‘”Š‘‘Ž‘‰‘—•ƒ†Š‡–‡”‘Ž‘‰‘—•’”‘–‡‹ ’”‘†— –‹‘ǡ ƒ †‡–ƒ‹Ž‡† ‘™Ž‡†‰‡ ƒ„‘—– –Š‡ ˆ— –‹‘‹‰ ‘ˆ –Š‡ •‡ ”‡–‹‘ ’ƒ–Š™ƒ› ƒ† ‹–• „‘––Ž‡‡ • ‹• ‹’‘”–ƒ–Ǥ ‡”‡ ™‡ ”‡’‘”– –Š‡ ‹•‘Žƒ–‹‘ ƒ† Šƒ”ƒ –‡”‹•ƒ–‹‘ –Š‡ A. niger hacA‰‡‡ǡ–Š‡ˆ— –‹‘ƒŽŠ‘‘Ž‘‰—‡‘ˆ–Š‡›‡ƒ•–HAC1ǡ™Š‹ Š ‘†‡•ˆ‘”–Š‡–”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘”‘ˆ–Š‡Ǥ  . MaterialsandMethods  Strains,CultureConditionsandTransformations Escherichia coli •–”ƒ‹• ͳͲ ƒ†  ͷD ȋ

(16) ˜‹–”‘‰‡Ȍ ™‡”‡ —•‡† ƒ• Š‘•–• –‘ ’”‘’ƒ‰ƒ–‡ ’Žƒ•‹†•Ǥ Š‡  Ž‹„”ƒ”› ˆ”‘ Aspergillus niger ˜ƒ”Ǥ awamori •–”ƒ‹ ͳͶ͵ˆ ™ƒ• ‘•–”— –‡†‹–Š‡˜‡ –‘”’ʹȋ

(17) ˜‹–”‘‰‡ȌǤA.nigerͷͻʹȋcspA1,pyrA5Ȍ™ƒ•—•‡†ƒ•–Š‡ ”‡ ‹’‹‡– •–”ƒ‹ ˆ‘” –”ƒ•ˆ‘”ƒ–‹‘ǡ ƒ† A. niger ͶͲʹ ȋcspA1Ȍǡ ƒ† A. niger ͳͷǣǣ’glaAǦ–Ǧ ͓ͳͻǡ ™Š‹ Š ’”‘†— ‡• –Š‡ Š—ƒ –‹••—‡ ’Žƒ•‹‘‰‡ ƒ –‹˜ƒ–‘” ’”‘–‡‹ ȋ–ǦȌ ͳͺͳǡ ™‡”‡ —•‡†–‘‹˜‡•–‹‰ƒ–‡–Š‡—†‡”†‹ˆˆ‡”‡–•–”‡•• ‘†‹–‹‘•Ǥ ŽŽA.niger•–”ƒ‹•™‡”‡‰”‘™‘‹‹ƒŽ‡†‹ƒȋȌ’Žƒ–‡• ‘–ƒ‹‹‰’‡”Ž‹–”‡͸ ‰ ƒ͵ǡ ͳǤͷ ‰  ʹͶǡ ͲǤͷ ‰ ‰Ͷ ˜ ͹ ʹǡ ͲǤͷ ‰ Žǡ –”ƒ ‡ ‡Ž‡‡–• ƒ• †‡• ”‹„‡† „› ‹•Š‹ƒ ƒ†ƒ–‡” ͳ͹ʹƒ†ʹͲ‰ˆ”— –‘•‡ƒ•ƒ ƒ”„‘•‘—” ‡ȋ’ ͸ǤͲȌǤ‹“—‹† —Ž–—”‡•™‡”‡ ‰”‘™ ‹  •—’’Ž‡‡–‡† ™‹–Š ʹ ‰ ƒ•ƒ‹‘ ƒ ‹†• ƒ† ͷ ‰ ›‡ƒ•– ‡š–”ƒ – ȋȌǡ ƒ† ‹‘ —Žƒ–‡† ™‹–Š ͳͲ͸ •’‘”‡•ȀŽ ƒ† ‹ —„ƒ–‡† ƒ– ͵ͲqǤ ŽŽ ‰”‘™–Š ‡š’‡”‹‡–• ˆ‘” •—„•‡“—‡– ‘”–Š‡” ƒƒŽ›•‹• ™‡”‡ •–ƒ”–‡† ‹ ˆ”‡•Š  ‡†‹ƒ ™‹–Š ͳǤͷ ‰ › ‡Ž‹ƒ ‘ˆ ƒ ‘˜‡”‹‰Š– —Ž–—”‡Ǥ š ‡’– ˆ‘” A. niger ͳͷǣǣ’glaAǦ–Ǧ͓ͳͻǡ ™Š‹ Š ™ƒ• –”ƒ•ˆ‡””‡† –‘  ™‹–Š ʹͲ ‰ ƒŽ–‘†‡š–”‹ǡ ˆ‘” ‹†— –‹‘ ‘ˆ –Š‡ ‰Ž— ‘ƒ›Žƒ•‡ ’”‘‘–‡”Ǥ  •–”‡•• ™ƒ• ’”‘˜‘‡† „› ƒ††‹‰ ‡‹–Š‡” ͵ ‰ȀŽ †‹–Š‹‘–Š”‡‹–‘Ž ȋȌ ‘” ͳͲ ρ‰ȀŽ –—‹ ƒ› ‹Ǥ ŽŽ –Ͳ •ƒ’Ž‡•™‡”‡–ƒ‡Œ—•–„‡ˆ‘”‡–Š‡–”ƒ•ˆ‡”–‘–Š‡‡š’‡”‹‡–ƒŽ‡†‹—Ǥ IsolationandCharacterisationoftheTranscriptionFactorHacA41.

(18) Chapter2 A. niger ’”‘–‘’Žƒ•–• ™‡”‡ ‘Ǧ–”ƒ•ˆ‘”‡† ƒ ‘”†‹‰ –‘ –Š‡ ’”‘ ‡†—”‡ ‘ˆ —•–‡”•Ȃ˜ƒ ‘‡”‡ et al. ͹͸ ™‹–Š ȋͳȌ ’Žƒ•‹† ’ ͷͲǡ ™Š‹ Š ‡š’”‡••‡• –Š‡ ‘†‹‰ ”‡‰‹‘ ‘ˆ hacA ™‹–Š‘—––Š‡ʹͲ–— ‘˜‡–‹‘ƒŽ‹–”‘ȋhacAiȌ—†‡”–Š‡ ‘–”‘Ž‘ˆ–Š‡ ‘•–‹–—–‹˜‡gpdA ’”‘‘–‡” ƒ† –Š‡ trpC –‡”‹ƒ–‘”ǡ ƒ† ȋʹȌ ’ ͸͵ͷǡ ™Š‹ Š ‘–ƒ‹• –Š‡ pyrA ‰‡‡ ‘ˆ A. nigerͶͷǤ’ ͷʹ'Ͷǡ™Š‹ Š‡š’”‡••‡•ƒgfpǦhacAiˆ—•‹‘—†‡” ‘–”‘Ž‘ˆ–Š‡gpdA’”‘‘–‡”ǡ ƒŽ•‘ ‘–ƒ‹•–Š‡pyrA‰‡‡ǡ–Š—•ƒŽŽ‘™‹‰†‹”‡ ––”ƒ•ˆ‘”ƒ–‹‘‘ˆA.nigerͷͻʹǤ   GeneIsolation ™ƒ•‡š–”ƒ –‡†ˆ”‘A.niger˜ƒ”Ǥawamori•–”ƒ‹ͳͶ͵ˆƒˆ–‡”‰”‘™–Š‘ˆ› ‡Ž‹—‘ ‰Ž— ‘•‡ǡ •–ƒ” Š ‘” „‡‡– ’—Ž’ ƒ• ƒ •‘Ž‡ ƒ”„‘ •‘—” ‡ǡ ƒ† ˆ”‘ › ‡Ž‹— ‰”‘™ —†‡” ‹–”‘‰‡•–ƒ”˜ƒ–‹‘ǤŠ‡•™‡”‡‹š‡†ƒ†—•‡†ˆ‘”Ž‹„”ƒ”› ‘•–”— –‹‘ ͳ͹ͶǤ‘—–Š‡” Š›„”‹†‹•ƒ–‹‘ƒ†• ”‡‡‹‰‘ˆ–Š‡ Ž‹„”ƒ”›„› ‘Ž‘›Š›„”‹†‹•ƒ–‹‘™‡”‡’‡”ˆ‘”‡†ƒ• †‡• ”‹„‡†„›ƒ„”‘‘etal. ͳͶͳǤŠ‡Ž‹„”ƒ”› ‘Ž‘›Š›„”‹†‹•ƒ–‹‘™ƒ•†‘‡‘˜‡”‹‰Š–‹ƒ Š›„”‹†‹•ƒ–‹‘‹š™‹–Š‘—–ˆ‘”ƒ‹†‡ ͳͶͳƒ–ͷͺqǤ—„•‡“—‡–Ž›–Š‡ˆ‹Ž–‡”•™‡”‡™ƒ•Š‡†‹ ʹšǡͲǤͳΨˆ‘”ʹšͷ‹—–‡•ƒ–”‘‘–‡’‡”ƒ–—”‡ǡƒ†™‹–Š–Š‡•ƒ‡•‘Ž—–‹‘ƒ–ͷͺq ˆ‘”͵Ͳ‹—–‡•Ǥ„ƒ•‡†‰‡‘‹ ™ƒŽ‹‰™ƒ•’‡”ˆ‘”‡†ƒ•†‡• ”‹„‡†„›‹‡„‡”–etal. ͳͷ͵. –‘ ‹•‘Žƒ–‡ –Š‡ ˆŽƒ‹‰ ”‡‰‹‘• ‘ˆ –Š‡ hacA ‰‡‡Ǥ  •‡“—‡ ‹‰ ”‡ƒ –‹‘• ™‡”‡. ’‡”ˆ‘”‡†™‹–Š–Š‡‹‰†›‡•‡“—‡ ‹‰‹–ȋ’’Ž‹‡†‹‘•›•–‡•Ȍƒ†ƒƒŽ›•‡†™‹–Š–Š‡

(19)  ”‹•͵ͳͲͲ ‡‡–‹ ƒŽ›•‡”ȋ’’Ž‹‡†‹‘•›•–‡•ȌǤ   TabelI:Ž‹‰‘— Ž‡‘–‹†‡’”‹‡”•—•‡†‹–Š‹••–—†› Primer Sequence. Site. ͳ. ͷ̵Ǧ. .    . NcoI. ʹ. ͷ̵Ǧ  .    . PstI. ͵. ͷ̵Ǧ       . PstI. Ͷ. ͷ̵Ǧ.      . BamHI. ͷ. ͷ̵Ǧ.  . . . . .        . BamHI. ͸. ͷ̵Ǧ.   .   . NarI. ͹. ͷ̵Ǧ .   .  .  . NcoI. ͺ. ͷ̵Ǧ.  .     . NarI. ͻ. ͷ̵Ǧ    .   . 42IsolationandCharacterisationoftheTranscriptionFactorHacA. .

(20) Chapter2 PlasmidConstruction ‘”‘˜‡”Ǧ‡š’”‡••‹‘‘ˆhacA™‹–Š‘—––Š‡‡‡†ˆ‘”ƒ•–”‡••‹†— ‡†•’Ž‹ ‹‰‡˜‡–ǡƒ†ˆ‘” ‘•–”— –‹‘ ‘ˆ ƒ gfphacAi ˆ—•‹‘ǡ –Š‡ hacA   ™ƒ• ƒ’Ž‹ˆ‹‡† ‹ –™‘ ˆ”ƒ‰‡–• ˆ”‘ A. niger  „› Ǥ ‘‹‹‰ ‘ˆ –Š‡ –™‘ ‰‡‡ ˆ”ƒ‰‡–• ƒ– –Š‡ PstI •‹–‡ ”‡•—Ž–‡† ‹ ”‡ ‘•–‹–—–‹‘‘ˆ–Š‡hacA ™‹–Š‘—–‹–•— ‘˜‡–‹‘ƒŽ‹–”‘ǤŠ‡’”‹‡”•—•‡†‹–Š‹• •–—†› ƒ”‡ Ž‹•–‡† ‹ ƒ„Ž‡ ͳǡ ƒ† –Š‡ ’‘•‹–‹‘• ‘ˆ –Š‡ ’”‹‡”• —•‡† –‘ ƒ’Ž‹ˆ› hacAi ƒ”‡ ‹†‹ ƒ–‡†‹ ‹‰—”‡ͳǤ             Figure 1.  Š‡ƒ–‹  ”‡’”‡•‡–ƒ–‹‘ ‘ˆ hacAu  ™‹–Š ‹–• ƒ‹ ˆ‡ƒ–—”‡•Ǥ Š‡ Ž‘ ƒ–‹‘• ‘ˆ ‘Ž‹‰‘— Ž‡‘–‹†‡ ’”‹‡”• —•‡† ˆ‘” –Š‡ ‘•–”— –‹‘ ’ ͷͲǡ ’ ͷʹȟͶǡ ƒ† ’ ͵Ͳȟ͵ ƒ”‡ ‹†‹ ƒ–‡†„›–Š‡•ƒŽŽƒ””‘™•Ǥ.   ‘”‘˜‡”Ǧ‡š’”‡••‹‘‹A.niger–Š‡ͷǯƒ†͵ǯ’‘”–‹‘•‘ˆ–Š‡hacA ™‡”‡ƒ’Ž‹ˆ‹‡†—•‹‰ –Š‡’”‹‡”•‡–•ͳǦʹƒ†͵ǦͶǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡–™‘ˆ”ƒ‰‡–•™‡”‡–Š‡ˆ—•‡†ƒ––Š‡ PstI •‹–‡ ƒ† Ž‘‡† ‹–‘ ’ͷʹǦͳ‘–Ǥ A. nige” ͷͻʹ ™ƒ• –”ƒ•ˆ‘”‡† ™‹–Š –Š‡ ”‡•—Ž–‹‰ ’Žƒ•‹† ȋ’ ͷͲȌǤ ‘” ‡š’”‡••‹‘ ‹ E. coli ƒ ‘•–”— – ™ƒ• ‰‡‡”ƒ–‡† ‹ –Š‡ •ƒ‡ ™ƒ›ǡ ‡š ‡’– –Šƒ– ’”‹‡” Ͷ ™ƒ• ”‡’Žƒ ‡† „› ’”‹‡” ͷǡ ™Š‹ Š ‘†‡• ˆ‘” ƒ ȋ ‹•Ȍ͸ –ƒ‰ ƒ– –Š‡ Ǧ –‡”‹—•‘ˆ–Š‡ ƒ ’”‘–‡‹ǤŠ‹• ‘•–”— –™ƒ• Ž‘‡†‹–‘’͵†Ϊȋ‘˜ƒ‰‡Ȍǡƒ†–Š‡ ”‡•—Ž–‹‰ ’Žƒ•‹† ȋ’ ͵Ͳ'͵Ȍ ™ƒ• –”ƒ•ˆ‘”‡† ‹–‘ –Š‡ E. coli •–”ƒ‹ ʹͳ ȋ͵Ȍ’›• ȋ‘˜ƒ‰‡ȌǤ ƒ Ǧȋ ‹•Ȍ͸™ƒ•’—”‹ˆ‹‡†„› ȋ‡”•ŠƒŠƒ”ƒ ‹ƒ‹‘–‡ ŠȌǡ—•‹‰ƒ‹Ǧ  —’‡”ˆŽ‘™ ‘Ž— ȋ‹ƒ‰‡Ȍǡ ˆ‘ŽŽ‘™‡† „› ‰‡Ž ˆ‹Ž–”ƒ–‹‘ ‘ ƒ —’‡”†‡š ͹ͷ ‘Ž— ȋ‡”•ŠƒŠƒ”ƒ ‹ƒ‹‘–‡ ŠȌǤ ‘” ‘•–”— –‹‘‘ˆƒgfphacAiˆ—•‹‘ǡhacAi™ƒ•ƒ’Ž‹ˆ‹‡† ™‹–Š–Š‡’”‹‡”•Ͷƒ†͸—•‹‰’ ͷͲƒ•–‡’Žƒ–‡ǡƒ†gfp™ƒ•‰‡‡”ƒ–‡†™‹–Š’”‹‡”• ͹ƒ†ͺ—•‹‰’ ȋŽ‘–‡ ŠȌƒ•–‡’Žƒ–‡ǤŠ‡–™‘‰‡‡•™‡”‡Ž‹‰ƒ–‡†–‘‰‡–Š‡”ƒ––Š‡ NarI •‹–‡ǡ ƒ† Ž‘‡† ‹–‘ ’ͷʹǦͳ‘–Ǥ —„•‡“—‡– Ž‹‰ƒ–‹‘ ‘ˆ –Š‡ pyrA ‰‡‡ ‹–‘ –Š‹• ’Žƒ•‹† ”‡•—Ž–‡† ‹ ’ ͷʹ'Ͷǡ ™Š‹ Š ™ƒ• —•‡† –‘ –”ƒ•ˆ‘” A. niger ͷͻʹǤ ƒ’• ‘ˆ –Š‡ ’Žƒ•‹†•—•‡†‹–Š‹••–—†›ƒ”‡•Š‘™‹ ‹‰—”‡ʹǤ IsolationandCharacterisationoftheTranscriptionFactorHacA43.

(21) Chapter2                   . Figure2.ƒ’•‘ˆ–Š‡’Žƒ•‹†•—•‡†‹–Š‹••–—†›Ǥ.      NorthernBlotAnalysis ‘”‹•‘Žƒ–‹‘•ǡ› ‡Ž‹—™ƒ•‰”‘—†™‹–Šͳ‰Žƒ••„‡ƒ†•—•‹‰–Š‡ ƒ•–”‡’ ͳʹͲ ȋ

(22)  ͳͲͳ ƒ˜ƒ–Ȍǡ ƒ† –‘–ƒŽ  ™ƒ• ‹•‘Žƒ–‡† —•‹‰ –Š‡ ‡ƒ•› ’Žƒ– –‘–ƒŽ  ‹– ȋ‹ƒ‰‡ȌǤ ‘” ‘”–Š‡” ƒƒŽ›•‹• ͳͲ Ɋ‰ ƒŽ‹“—‘–• ‘ˆ –‘–ƒŽ  ™‡”‡ ˆ”ƒ –‹‘ƒ–‡† ‘ ƒ ͳǤͷΨ ȋ™Ȁ˜Ȍ ƒ‰ƒ”‘•‡Ǧˆ‘”ƒŽ†‡Š›†‡ ‰‡Ž ƒ† „Ž‘––‡† ‘–‘ ƒ ›„‘†  ‡„”ƒ‡ ȋ‡”•Šƒ Šƒ”ƒ ‹ƒ ‹‘–‡ ŠȌǤ ‘ ‰‡‡”ƒ–‡ ’”‘„‡•ǡ  ˆ”ƒ‰‡–• ™‡”‡ DǦ͵ʹǦŽƒ„‡ŽŽ‡† —•‹‰ –Š‡ ”‹‡Ǧ

(23) –

(24)

(25)  Žƒ„‡ŽŽ‹‰ ‹– ȋ–”ƒ–ƒ‰‡‡Ȍǡ ƒ† –Š‡ ’”‘„‡• ™‡”‡ ’—”‹ˆ‹‡† ‘ ‹‘‰‡Ž ͵Ͳ •’‹ ‘Ž—• ȋ‹‘”ƒ†ȌǤ ‘”–Š‡” Š›„”‹†‹•ƒ–‹‘• ™‡”‡ ’‡”ˆ‘”‡† ƒ– Ͷʹq ‹ ͷͲΨ ˆ‘”ƒ‹†‡ ‘–ƒ‹‹‰ ͳͲΨ ’‘Ž›‡–Š›Ž‡‡ ‰Ž› ‘Ž ͸ͲͲͲǡ ͷš ‡Šƒ”†–ǯ• •‘Ž—–‹‘ǡ ͷͲ  

(26) Ǧ Ž ȋ’  ͹ǤͷȌǡ ͳ  ƒŽǡ ͲǤͳΨ •‘†‹— ’›”‘’Š‘•’Šƒ–‡ǡ ͳΨ  ƒ† ͳͲͲ ρ‰ȀŽ •ƒŽ‘ •’‡”  ȋ‹‰ƒȌǤ ‹Ž–‡”•™‡”‡™ƒ•Š‡†ʹͲ‹—–‡•™‹–ŠͲǤʹšȀͲǤͳΨƒ–͸ͲqǤ. 44IsolationandCharacterisationoftheTranscriptionFactorHacA.

(27) Chapter2 WesternBlotAnalysis ”‹–‡”‹‘Ǧ’‘Ž›ƒ ”›Žƒ‹†‡ȋ Ȍ‰‡Ž•ȋ‹‘”ƒ†Ȍ™‡”‡—•‡†ˆ‘”’”‘–‡‹‡Ž‡ –”‘’Š‘”‡•‹•Ǥ Ǧ ‰‡Ž•™‡”‡•–ƒ‹‡†™‹–Š‘‘ƒ••‹‡”‹ŽŽ‹ƒ–Ž—‡ʹͷͲȋ‹‘”ƒ†Ȍ‘”™‡”‡„Ž‘––‡† ‘–‘ ’—”‡ ‹–”‘ ‡ŽŽ—Ž‘•‡ ‡„”ƒ‡• ȋ‹‘”ƒ†Ȍ ˆ‘” ‡•–‡” „Ž‘– ƒƒŽ›•‹•Ǥ ”‘–‡‹ ‘ ‡–”ƒ–‹‘•™‡”‡†‡–‡”‹‡†ƒ ‘”†‹‰–‘”ƒ†ˆ‘”†ͻ—•‹‰ƒ•ƒ•–ƒ†ƒ”†Ǥ‡•–‡” ƒƒŽ›•‹•™ƒ•’‡”ˆ‘”‡†—•‹‰•–ƒ†ƒ”†’”‘ ‡†—”‡• ͳͶͳǤ‡„”ƒ‡•™‡”‡‹ —„ƒ–‡†‹ͳͲͲ Ž  ȋͷͲ  

(28) Ǧ Žǡ ͳͷͲ  ƒŽǡ ’  ͺǤͲȌ ‘–ƒ‹‹‰ ͷ ‰ Ž‘™ ˆƒ– ‹Ž ’‘™†‡” ƒ† ’‘Ž› Ž‘ƒŽ ƒ–‹„‘†‹‡• •’‡ ‹ˆ‹  ˆ‘” 

(29) ǡ ǡ ƒ† 

(30) Ǥ

(31) —‘”‡ƒ –‹˜‡ ’”‘–‡‹• ™‡”‡ †‡–‡ –‡† —•‹‰ ƒ Ǧ ‘Œ—‰ƒ–‡† •‡ ‘†ƒ”› ƒ–‹„‘†› ƒ†  †‡–‡ –‹‘ ”‡ƒ‰‡–• ȋ‡”•ŠƒŠƒ”ƒ ‹ƒ‹‘–‡ ŠȌǤ  SouthernBlotAnalysis. ‡‘‹   ˆ‘” ‘—–Š‡” ƒƒŽ›•‹• ™ƒ• ‹•‘Žƒ–‡† „› ƒ††‹‰ ‡š–”ƒ –‹‘ „—ˆˆ‡” ȋͳͲͲ  

(32) Ǧ Ž’ ͺǤͲǡͷͲǡͷͲͲƒŽǡͳǡ͵ͷȌ–‘‰”‘—†› ‡Ž‹—Ǥ ˆ–‡”ͳͲǦ‹‹ —„ƒ–‹‘ƒ–͸ͷqǡ› ‡Ž‹—†‡„”‹•™ƒ••‡’ƒ”ƒ–‡†„› ‡–”‹ˆ—‰ƒ–‹‘ǡƒ†–Š‡  ‹ –Š‡ •—’‡”ƒ–ƒ– ™ƒ• ’”‡ ‹’‹–ƒ–‡† ™‹–Š ‹•‘’”‘’ƒ‘ŽǤ Š‡  ’‡ŽŽ‡– ™ƒ• ”‡•—•’‡†‡† ‹ ʹ ƒ† ‹ —„ƒ–‡† ™‹–Š ƒ•‡Ǧˆ”‡‡ ƒ•‡ ȋ‘ Š‡ ‘Ž‡ —Žƒ” ‹‘ Š‡‹ ƒŽ•ȌǤ ‘” ‡ƒ Š †‹‰‡•– ͳͲ ρ‰ ‘ˆ ‰‡‘‹   ™ƒ• ‹ —„ƒ–‡† ‘˜‡” ‹‰Š– ƒ– ͵͹ q ™‹–Š‡‹–Š‡”ʹͲ‘ˆ‡‹–Š‡”NotI‘”NcoI‡†‘— Ž‡ƒ•‡ǤŠ‡”‡ƒ –‹‘•™‡”‡ˆ”ƒ –‹‘ƒ–‡†‘ƒ ͲǤͺΨ  ƒ‰ƒ”‘•‡ ‰‡ŽǤ Ž‘––‹‰ ƒ† Š›„”‹†‹•ƒ–‹‘ ™‡”‡ †‘‡ ƒ ‘”†‹‰ –‘ •–ƒ†ƒ”† ’”‘ ‡†—”‡•ͳͶͳǤ  5'RACE ͷ̵ȋƒ’‹†’Ž‹ˆ‹ ƒ–‹‘‘ˆ †•Ȍ™ƒ•’‡”ˆ‘”‡†‘ͳρ‰‘ˆ–‘–ƒŽ—•‹‰–Š‡  ’Ž‹ˆ‹ ƒ–‹‘‹–ȋŽ‘–‡ ŠȌǤ”‹‡”ͻȋƒ„Ž‡ͳȌ™ƒ•—•‡†–‘ƒ’Ž‹ˆ› hacA Ǥ  ElectrophoreticMobilityShiftAssay(EMSA) Š‡’”‘‘–‡”ˆ”ƒ‰‡–•‘ˆbipAǡcypBǡpdiAǡprpAǡƒ†tigAȋƒ„Ž‡ʹȌ™‡”‡•›–Š‡•‹•‡†„›  ’‡”ˆ‘”‡† ‘ ‰ ‘ˆ A. nigerǤ Š‡ ”‡•—Ž–‹‰ ˆ”ƒ‰‡–• ™‡”‡ ‰‡Ž ’—”‹ˆ‹‡† ƒ† ‡†Ǧ Žƒ„‡ŽŽ‡†™‹–ŠɀǦ͵ʹ—•‹‰Ͷ’‘Ž›— Ž‡‘–‹†‡‹ƒ•‡ȋ‡™‰Žƒ†‹‘Žƒ„•Ȍƒ†’—”‹ˆ‹‡† ‘‹‘‰‡Ž͵Ͳ•’‹ ‘Ž—•ȋ‹‘ƒ†ȌǤ‹†‹‰”‡ƒ –‹‘•ȋͶͲɊŽȌ‹ Ž—†‡†ͲǤͷɊ‰‘ˆ’—”‹ˆ‹‡† ƒ ’”‘–‡‹ǡƒ†ͳš„‹†‹‰„—ˆˆ‡”ȋʹͲ ’ ͹ǤͷǡͷͲŽǡͳͲ‰ŽʹǡͲǤʹͷ ǡͲǤͷǡʹΨ ‹ ‘ŽŽʹͲͲǡͳͲͲρ‰ȀŽ’‘Ž›ȋ†

(33) Ǧ†Ȍǡƒ†ͷΨ‰Ž› ‡”‘ŽȌǤͳ‰‘ˆ ”ƒ†‹‘Žƒ„‡ŽŽ‡†’”‘„‡™ƒ•ƒ††‡†ƒ†„‹†‹‰™ƒ•ƒŽŽ‘™‡†–‘’”‘ ‡‡†‘‹ ‡ˆ‘”ͳͷ‹ǤŠ‡ ”‡ƒ –‹‘•™‡”‡Ž‘ƒ†‡†‘ƒͷΨ’‘Ž›ƒ ”›Žƒ‹†‡‰‡Žƒ†‡Ž‡ –”‘’Š‘”‡•‡†ƒ–ͳͲͲˆ‘”͵Š”‹ ͲǤʹͷšǤ . IsolationandCharacterisationoftheTranscriptionFactorHacA45.

(34) Chapter2 Table2.”‘‘–‡”ˆ”ƒ‰‡–•—•‡†‹•. . ‡‡ AccesionNo. PositionrelativetoATG. . bipA cypB pdiA prpA tigA hacA.     . Ͳͺͺ͸ͺ ͲͲͷͺ͸͹ ͻͺ͹ͻ͹  Ͳͻͷͺͻͻ ͻͺ͹Ͷͺ ͵Ͳ͵͸ͺͶ. ǦͶͳʹ–‘Ͳ Ǧ͵ͻ͸–‘Ͳ ǦͶ͵ʹ–‘Ͳ Ǧͷͺ͹–‘Ͳ Ǧ͵͵Ͳ–‘Ͳ Ǧͺ͸͵–‘Ǧʹ͸ͷ.   UVMicroscopy ”ƒ•ˆ‘”ƒ–•‡š’”‡••‹‰ Ǧ ƒ ™‡”‡‰”‘™‹‹‹ƒŽ‡†‹—ƒ•†‡• ”‹„‡†ƒ„‘˜‡Ǥ › ‡Ž‹— •ƒ’Ž‡• ™‡”‡ ‡šƒ‹‡† „› ˆŽ—‘”‡• ‡– ‹ ”‘• ‘’› ‘ ƒ ‘Ž›˜ƒ” ‹ ”‘• ‘’‡ ȋ‡‹ Š‡”–Ǧ —‰Ȍ ‘—’Ž‡† –‘ ƒ ‘‘Ž‡†  †‹‰‹–ƒŽ ƒ‡”ƒǡ —•‹‰

(35)  ˆ‹Ž–‡”•Ǥ ‹ –—”‡• ™‡”‡ ƒƒŽ›•‡† ™‹–Š –Š‡ ‡–ƒ‘”’Š •‘ˆ–™ƒ”‡ ’ƒ ƒ‰‡ ȋ˜‡”•‹‘ ͶǤͷȌǤ 

(36)  ȋ‘Ž‡ —Žƒ” ”‘„‡•Ȍ ™ƒ•—•‡†ƒ–ƒ ‘ ‡–”ƒ–‹‘‘ˆͳρˆ‘”˜‹•—ƒŽ‹•ƒ–‹‘‘ˆ— Ž‡‹Ǥ  . Results  CloningandSequenceAnalysis Š‡ ‰‡‡ ‡ ‘†‹‰ –Š‡ –”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘” ‘ˆ –Š‡ —ˆ‘Ž†‡† ’”‘–‡‹ ”‡•’‘•‡ ‹ A. nidulansǡhacAǡŠƒ•’”‡˜‹‘—•Ž›„‡‡ Ž‘‡†„ƒ•‡†‘‹–•Š‘‘Ž‘‰›–‘–Š‡HAC1‰‡‡‘ˆS. cerevisiae ͳͶͲǤŠ‡A.nidulanshacA•‡“—‡ ‡™ƒ•—•‡†‹–Š‹••–—†›ƒ•ƒ’”‘„‡–‘ˆƒ ‹Ž‹–ƒ–‡ Ž‘‹‰ ‘ˆ –Š‡ ‘””‡•’‘†‹‰ ‰‡‡ ˆ”‘ ƒ  Ž‹„”ƒ”› †‡”‹˜‡† ˆ”‘ –Š‡ A. niger ˜ƒ”Ǥ awamori•–”ƒ‹ͳͶ͵ˆǤ‡˜‡”ƒŽ’‘•‹–‹˜‡ Ž‘‡•™‡”‡‘„–ƒ‹‡†ǡƒ†•‡“—‡ ‹‰‘ˆ–Š‡‹”ͷǯ ‡†• ”‡˜‡ƒŽ‡† –Šƒ– –Š‡› ™‡”‡ ‹†‡‡† †‡”‹˜‡† ˆ”‘ ƒ Š‘‘Ž‘‰—‡ ‘ˆ –Š‡ A. nidulans hacA ‰‡‡Ǥƒ•‡†‘–Š‡•‡“—‡ ‡ǡ’”‹‡”•™‡”‡†‡•‹‰‡†ǡƒ†ƒ͸ǤʹǦ„‰‡‘‹ ˆ”ƒ‰‡– ‹ Ž—†‹‰ –Š‡ hacA ‘†‹‰ •‡“—‡ ‡ –‘‰‡–Š‡” ™‹–Š ‹–• ˆŽƒ‹‰ ”‡‰‹‘• ȋ ‡••‹‘ ‘Ǥ ͵Ͳ͵͸ͺͶȌ ™ƒ• Ž‘‡† ˆ”‘ A. niger ͶͲʹ „› ‰‡‘‹  ™ƒŽ‹‰Ǥ ‡“—‡ ‡ ‘’ƒ”‹•‘ ‘ˆ –Š‡hacA ˆ”‘A.niger˜ƒ”Ǥawamori™‹–Š–Š‡hacA‰‡‡ˆ”‘A.nigerͶͲʹ”‡˜‡ƒŽ‡† –Šƒ––Š‡–™‘‡ ‘†‡‹†‡–‹ ƒŽ’”‘–‡‹•Ǥ

(37)  S. cerevisiaeǡ ƒ — ‘˜‡–‹‘ƒŽ ʹͷʹǦ„’ ‹–”‘ ‹• •’Ž‹ ‡† ˆ”‘ –Š‡ HAC1  †—”‹‰ǡ™Š‡”‡ƒ•ƒ— Š•Š‘”–‡”ȋʹ͸Ǧ„’Ȍ‹–”‘‹•‡š ‹•‡†ˆ”‘–Š‡ƒƒŽ‹ƒXBP1  —†‡” •‹‹Žƒ” ‘†‹–‹‘• ͳʹǡʹʹǤ Š‡ hac1ȀhacA ‰‡‡• ˆ”‘ T. reesei ƒ† A. nidulans Šƒ˜‡ ƒŽ•‘ „‡‡ •Š‘™ –‘ ‘–ƒ‹ ‘Ǧ ‘˜‡–‹‘ƒŽ ‹–”‘• –Šƒ– ƒ”‡ •’Ž‹ ‡† †—”‹‰ –Š‡ ‹†— –‹‘‘ˆ ͳͶͲǤ‘’ƒ”‹•‘‘ˆ–Š‡ •‡“—‡ ‡‘ˆ‘‡‘ˆ–Š‡ Ž‘‡•‘„–ƒ‹‡†ˆ”‘ –Š‡ Ž‹„”ƒ”›‘ˆA.niger˜ƒ”Ǥawamori•–”ƒ‹ͳͶ͵ˆ™‹–Š–Š‡ ‘””‡•’‘†‹‰‰‡‘‹  46IsolationandCharacterisationoftheTranscriptionFactorHacA.

(38) Chapter2 •‡“—‡ ‡”‡˜‡ƒŽ‡†ƒ‘Ǧ ‘˜‡–‹‘ƒŽ‹–”‘‘ˆʹͲ–ǤŠ‡•’Ž‹ ‹‰•‹–‡‘ˆ–Š‡‹–”‘ ‘—Ž† ‘– „‡ †‡–‡”‹‡† —ƒ„‹‰—‘—•Ž› „› ‘’ƒ”‹‰ –Š‡ —•’Ž‹ ‡† ƒ† •’Ž‹ ‡† •‡“—‡ ‡•ǡ „‡ ƒ—•‡‘ˆ–Š‡’”‡•‡ ‡‘ˆƒ  •‡“—‡ ‡‘„‘–Š•‹†‡•‘ˆ–Š‡‹–”‘Ǥ ‘™‡˜‡”ǡ–Š‡ ‹–”‘„‘”†‡”•ƒ†•’Ž‹ ‡Ǧ•‹–‡•‘ˆ–Š‡›‡ƒ•–HAC1ƒ†ƒƒŽ‹ƒXBP1ƒ”‡™‡ŽŽ ‘•‡”˜‡† ͳʹǡƒ†–Š‡•‡ ‘•‡”˜‡†„ƒ•‡•™‡”‡ƒŽ•‘ˆ‘—†ƒ––Š‡„‘”†‡”•‘ˆ–Š‡A.nigerhacAȋ ‹‰Ǥ͵ȌǤ. ‘”‡‘˜‡”ǡ ƒ •–”‘‰ •‡ ‘†ƒ”›  •–”— –—”‡ ™ƒ• ’”‡†‹ –‡† ˆ‘” –Š‡ ‹–”‘ ”‡‰‹‘ ƒ† ‹–• ˆŽƒ‹‰•‡“—‡ ‡•ǤŠ‡•–”— –—”‡‘ˆ–Š‡ ‘•‡”˜‡†‹–”‘„‘”†‡”•Ȅ‘”‰ƒ‹•‡†‹–™‘Ž‘‘’• ‘ˆ ͹–‡ƒ ŠȄ™ƒ• •‹‹Žƒ” –‘ –Š‘•‡ ˆ‘”‡† „› „‘–Š –Š‡›‡ƒ•– HAC1 ƒ† ƒƒŽ‹ƒ XBP1 ‹–”‘•Ǥ‘•–’”‘„ƒ„Ž›ǡ–Š‡A.nigerhacA–”ƒ• ”‹’–‹•ƒŽ•‘•’Ž‹ ‡†™‹–Š‹–Š‡•‡Ž‘‘’•Ǥ                     . Figure 3. –”— –—”‡ ƒ† ‘–‡š– ‘ˆ –Š‡ hacA — ‘˜‡–‹‘ƒŽ ‹–”‘Ǥ (A) ”‡†‹ –‡† •‡ ‘†ƒ”› •–”— –—”‡‘ˆ–Š‡A.nigerhacA‹–”‘ǡ–Š‡ͷǯǦ‡†‘ˆ–Š‡hacAiǡ–Š‡ͷǯƒ†͵ǯ„‘”†‡”‘ˆ–Š‡S. cerevisiae HAC1 ‹–”‘ǡ ƒ† –Š‡ ƒƒŽ‹ƒ XBP1 ‹–”‘ǡ ”‡•’‡ –‹˜‡Ž› ƒ– –Š‡  Ž‡˜‡ŽǤ Š‡ ‹–”‘•‡“—‡ ‡•ƒ”‡•Š‘™‹Ž‘™‡” ƒ•‡Ž‡––‡”•ǡƒ†–Š‡ƒ””‘™•‹†‹ ƒ–‡–Š‡ Ž‡ƒ˜ƒ‰‡•‹–‡•Ǥ(B) Ž‹‰‡– ‘ˆ –Š‡  •‡“—‡ ‡ •—””‘—†‹‰ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ‹ A. niger hacAǡ S. cerevisiaeHAC1ǡƒ†–Š‡ƒƒŽ‹ƒXBP1ǤŠ‡ͷǯǦ‡†‘ˆA.nigerhacAi‹•ƒŽ•‘‹†‹ ƒ–‡†Ǥ ‘•‡”˜‡†”‡•‹†—‡•‹–Š‡Ž‘‘’•ƒ”‡‹†‹ ƒ–‡†‹–Š‡ ‘•‡•—••‡“—‡ ‡Ǥ. IsolationandCharacterisationoftheTranscriptionFactorHacA47.

(39) Chapter2                             . Figure4.Ž‹‰‡–‘ˆ–Š‡ƒ‹‘ƒ ‹†•‡“—‡ ‡• ‘ˆ–Š‡‹†— ‡†ƒ†—‹†— ‡†ˆ‘”•‘ˆ ƒ  ˆ”‘ A. nigerǡ A. nidulans ƒ† A. fumigatusǤ Ž› –Š‡  „‹†‹‰ †‘ƒ‹• ‘ˆ –Š‡ S. cerevisiae ƒ ͳ’ ƒ† –Š‡ T. reesei ƒ ͳ ’”‘–‡‹• ƒ”‡ •Š‘™Ǥ (A) Š‡ ’—–ƒ–‹˜‡  „‹†‹‰ †‘ƒ‹ ‹• —†‡”Ž‹‡† –Š‡ ”‡•‹†—‡• ‘ˆ –Š‡ ’—–ƒ–‹˜‡ Ž‡— ‹‡ œ‹’’‡” ƒ”‡ ‹†‹ ƒ–‡† „› –Š‡ asterisksǡ ƒ† –Š‡ ’‘•‹–‹‘‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘‹–Š‡–Š”‡‡Aspergillus•’‡ ‹‡•‹•‹†‹ ƒ–‡†„›–Š‡ƒ””‘™Ǥ (B)Ž‹‰‡–‘ˆ–Š‡—‹†— ‡†ˆ‘”•‘ˆ ƒ ˆ”‘A.nigerǡA.nidulansƒ†A.fumigatusˆ”‘ –Š‡•‹–‡‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘–‘–Š‡Ǧ–‡”‹—•Ǥ. 48IsolationandCharacterisationoftheTranscriptionFactorHacA.

(40) Chapter2 Š‡—•’Ž‹ ‡†hacAu ‘–ƒ‹•ƒ  ‘†‹‰ˆ‘”ƒͶ͵͸Ǧƒ‹‘ƒ ‹†’”‘–‡‹Ǥ’Ž‹ ‹‰ ”‡’Žƒ ‡•ƒǦ–‡”‹ƒŽ’‘”–‹‘ ‘†‹‰ˆ‘”ʹʹʹƒ‹‘ƒ ‹†•™‹–Šƒ†‹ˆˆ‡”‡–•‡“—‡ ‡ ‘†‹‰ ˆ‘”ͳʹͺƒ‹‘ƒ ‹†•ǤŠ‹•†‹ˆˆ‡”•ˆ”‘–Š‡•‹–—ƒ–‹‘‹›‡ƒ•–ǡ™Š‡”‡ƒͳͲǦ”‡•‹†—‡Ǧ–‡”‹ƒŽ –ƒ‹Ž‹•”‡’Žƒ ‡†„›ƒ•‡“—‡ ‡‘ˆͳͺƒ‹‘ƒ ‹†•ƒ•ƒ”‡•—Ž–‘ˆ•’Ž‹ ‹‰ʹʹǤ Š‡ ‘ˆ–Š‡A.nigerhacAi‡ ‘†‡•ƒ’”‘–‡‹‘ˆ͵Ͷʹƒ‹‘ƒ ‹†•ǡ™Š‹ Š‹• ͹͵Ψ ƒ† ͺͲΨ ‹†‡–‹ ƒŽ –‘ ‹–• ‘”–Š‘Ž‘‰—‡• ˆ”‘ A. nidulans ƒ† A. fumigatusǡ ”‡•’‡ –‹˜‡Ž› ȋ ‹‰ǤͶƒȌǤ ƒ „‡Ž‘‰•–‘–Š‡„

(41) –›’‡ˆƒ‹Ž›‘ˆ–”ƒ• ”‹’–‹‘ˆƒ –‘”•ǡ™Š‹ Š–›’‹ ƒŽŽ›Šƒ˜‡ ƒ Ž‡— ‹‡Ǧœ‹’’‡” †‹‡”‹•ƒ–‹‘ ‘–‹ˆ †‘™•–”‡ƒ ‘ˆ –Š‡ Ǧ„‹†‹‰ †‘ƒ‹Ǥ

(42)  A. niger ƒ –Š‡”‡ƒ”‡•‹šƒ‹‘ƒ ‹†•ȋǡǡǡǡƒ†Ȍ–Šƒ– ‘—Ž†’‘–‡–‹ƒŽŽ›ˆ‘”–Š‡Ž‡— ‹‡ œ‹’’‡”Ǥ

(43) –‡”‡•–‹‰Ž›ǡ ƒŽ‹‰‡– ‘ˆ –Š‡ ’”‘–‡‹ •‡“—‡ ‡• ‡ ‘†‡† „› –Š‡ —•’Ž‹ ‡† hacAu •ˆ”‘A.nigerǡA.nidulansƒ†A.fumigatus•Š‘™‡†ƒŠ‹‰Š†‡‰”‡‡‘ˆ‹†‡–‹–›ȋ͹ͳΨȂ ͹ͷΨȌƒ‘‰–Š‡•’‡ ‹‡•ǡ‡˜‡‹–Š‡’ƒ”–†‘™•–”‡ƒ‘ˆ–Š‡‘Ǧ ‘˜‡–‹‘ƒŽ‹–”‘ȋ ‹‰Ǥ Ͷ„ȌǤŠ‹• ‘—Ž†‹†‹ ƒ–‡–Šƒ––Š‡ ƒ ’”‘–‡‹‡ ‘†‡†„›hacAuƒŽ•‘Šƒ•ƒˆ— –‹‘ ˆ‘”™Š‹ Š–Š‡Ǧ–‡”‹ƒŽ•‡‰‡–‹•‹’‘”–ƒ–Ǥ  NorthernAnalysisofERStress ‡ ‘ˆ –Š‡ ‡› •–‡’• ‹ ƒ –‹˜ƒ–‹‘ ‘ˆ –Š‡  ’ƒ–Š™ƒ› ‹ ›‡ƒ•– ƒ† ƒƒŽ• ‹• –Š‡

(44) ”‡ͳ’Ȁ

(45) ͳ ‡†‹ƒ–‡† •’Ž‹ ‹‰ ‘ˆ ƒ — ‘˜‡–‹‘ƒŽ ‹–”‘ ˆ”‘ –Š‡  ‡ ‘†‹‰ –Š‡ –”ƒ• ”‹’–‹‘ƒŽƒ –‹˜ƒ–‘”‘ˆ–Š‡ ͳʹǡʹʹǤ•‹‹Žƒ”‡˜‡–Šƒ•„‡‡•Š‘™–‘‘ —”†—”‹‰ –Š‡‹†— –‹‘‘ˆ–Š‡‹T.reeseiƒ†A.nidulans ͳͶͲǤ‘•–—†›–Š‹•‡˜‡–‹A.nigerǡ •–”‡••™ƒ•–”‹‰‰‡”‡†„›–Š”‡‡‡ƒ•ǣȋͳȌ‘˜‡”Ǧ‡š’”‡••‹‘‘ˆƒŠ‡–‡”‘Ž‘‰‘—•’”‘–‡‹ȋ–ǦȌǡ ȋʹȌ –”‡ƒ–‡– ‘ˆ › ‡Ž‹— ™‹–Š ǡ ƒ† ȋ͵Ȍ –”‡ƒ–‡– ‘ˆ › ‡Ž‹— ™‹–Š –—‹ ƒ› ‹Ǥ  ƒ† –—‹ ƒ› ‹ ƒ”‡ „‘–Š ‘™ –‘ „‡ Š‡‹ ƒŽ ‹†— ‡”• ‘ˆ –Š‡ Ǥ —‹ ƒ› ‹ ’”‘˜‘‡•–Š‡„›‹Š‹„‹–‹‰–Š‡ ‘”‡‘Ž‹‰‘•ƒ Šƒ”‹†‡ƒ††‹–‹‘–‘ƒ• ‡–’‘Ž›’‡’–‹†‡• ‹ –Š‡ ǡ –Š‡”‡„› „Ž‘ ‹‰ ’”‘’‡” ˆ‘Ž†‹‰ ƒ† –”ƒ•‹– –Š”‘—‰Š –Š‡  ͳʹʹǤ • ƒ ”‡†— ‹‰ ƒ‰‡–ǡ  †‹•”—’–• –Š‡ ‘š‹†‹•‹‰ ‡˜‹”‘‡– ‘ˆ –Š‡ ǡ ’”‡˜‡–‹‰ –Š‡ ˆ‘”ƒ–‹‘ ‘ˆ †‹•—Ž’Š‹†‡ „‘†•Ǥ ‹••—‡ ’Žƒ•‹‘‰‡ ƒ –‹˜ƒ–‘” ȋ–ǦȌ ‹• ƒ •‡”‹‡ ’”‘–‡ƒ•‡ ™Š‹ Šǡ ™Š‡ ƒ –‹˜ƒ–‡†„›ˆ‹„”‹ǡ‹–•‡Žˆƒ –‹˜ƒ–‡•’Žƒ•‹‘‰‡ǡ‡ƒ„Ž‹‰–Š‡†‹••‘Ž—–‹‘‘ˆ„Ž‘‘† Ž‘–•Ǥ‘™ ‡š’”‡••‹‘Ž‡˜‡Ž•Šƒ˜‡Šƒ’‡”‡†‡ˆˆ‘”–•–‘’”‘†— ‡—•‡ˆ—Žƒ‘—–•‘ˆ–Ǧ‹˜ƒ”‹‘—•Š‘•– ‘”‰ƒ‹••ǡ ƒ† ‹ A. niger ‹– Šƒ• „‡‡ —•‡† ƒ• ƒ ‘†‡Ž ’”‘–‡‹ –‘ ƒ••‡•• –Š‡ ’”‘„Ž‡• ‘ˆ Š‡–‡”‘Ž‘‰‘—• ’”‘–‡‹ ‡š’”‡••‹‘ ͳͺͳǤ ‹ ‡ ‡š’”‡••‹‘ ‘ˆ –Ǧ ‹ A. niger Ž‡ƒ†• –‘ –Š‡ –”ƒ• ”‹’–‹‘ƒŽ —’”‡‰—Žƒ–‹‘ ‘ˆ ‰‡‡• ‡ ‘†‹‰ –Š‡  Šƒ’‡”‘‡• ƒ† ˆ‘Ž†ƒ•‡• 

(46) ǡ ǡ ƒ† 

(47) ǡ ‹– ™ƒ• Š‘•‡ ƒ• ƒ ‘†‡Ž •–”ƒ‹ –‘ ƒ••‡•• –Š‡  —†‡” ‘†‹–‹‘• ‘ˆ Š‡–‡”‘Ž‘‰‘—•’”‘–‡‹’”‘†— –‹‘Ǥ. IsolationandCharacterisationoftheTranscriptionFactorHacA49.

(48) Chapter2                              . Figure 5.  ‘”–Š‡” Š›„”‹†‹œƒ–‹‘ ƒƒŽ›•‡• •Š‘™‹‰ –Š‡ ‡ˆˆ‡ –• ‘ˆ  •–”‡••ǡ ‹’‘•‡† „› –”‡ƒ–‡–‘ˆ ‡ŽŽ•™‹–Š‘”–—‹ ƒ› ‹ȋȌƒ†„›–Š‡‡š’”‡••‹‘‘ˆƒŠ‡–‡”‘Ž‘‰‘—•‰‡‡ ȋͳͷǣǣpGLAǦ–ǦȌǡ ‘ –Š‡ ‡š’”‡••‹‘ ‘ˆ hacAǡ bipAǡ cypBǡ ƒ† pdiAǤ Š‡ –‹‡ ‹ ‹—–‡• ƒˆ–‡” ‹†— –‹‘ ‹• ‹†‹ ƒ–‡† ƒ„‘˜‡ –Š‡ Žƒ‡•Ǥ Š‡ –™‘ hacA  •’‡ ‹‡• –Šƒ– ™‡”‡ ’”‡•‡– ƒˆ–‡” ‹†— –‹‘ ‘ˆ  •–”‡•• ƒ”‡ ‹†‹ ƒ–‡† „› –Š‡ ƒ””‘™•ǡ ƒ† ƒ”‡ Žƒ„‡ŽŽ‡† hacAu ƒ† hacAiǡ ˆ‘” —‹†— ‡†ƒ†‹†— ‡†ǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡‰‡‡ ‘†‹‰ˆ‘”ͳͺ”™ƒ•—•‡†ƒ•ƒ ‘–”‘Ž’”‘„‡Ǥ. 50IsolationandCharacterisationoftheTranscriptionFactorHacA.

(49) Chapter2 ‘ ‡šƒ‹‡ –Š‡  ‹†— ‡† „› –Š‡ •›–Š‡•‹• ‘ˆ –Ǧǡ ‘” „› –”‡ƒ–‡– ™‹–Š  ‘” –—‹ ƒ› ‹ǡ‘”–Š‡”Š›„”‹†‹•ƒ–‹‘™ƒ•’‡”ˆ‘”‡†‘–‘–ƒŽ‹•‘Žƒ–‡†ˆ”‘› ‡Ž‹ƒ‘ˆ •— Š —Ž–—”‡•Ǥ   ˆ”ƒ‰‡– ‘’”‹•‹‰ –Š‡ ”‡‰‹‘ ˆ”‘ –Š‡ •–ƒ”– ‘†‘ —’ –‘ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ™ƒ• —•‡† ƒ• ƒ ’”‘„‡ –‘ †‡–‡ – hacA Ǥ ‘”–Š‡” ƒƒŽ›•‹• •Š‘™‡† –Šƒ– ƒ •‡ ‘†ǡ •ƒŽŽ‡” hacAi  –”ƒ• ”‹’– ‡‡”‰‡† ™‹–Š‹ ͵Ͳ ‹—–‡• ƒˆ–‡” ‡š’‘•—”‡‘ˆ–Š‡› ‡Ž‹—–‘ǡƒ†–Š‹• ‘‹ ‹†‡†™‹–Šƒ†‡ ”‡ƒ•‡‹–Š‡‹–‡•‹–›‘ˆ–Š‡ hacAu„ƒ†ȋ ‹‰ǤͷȌǤ‘‰‡–Š‡”™‹–Š–Š‡ƒ’’‡ƒ”ƒ ‡‘ˆ–Š‡•ƒŽŽ‡”hacAiǡ—’”‡‰—Žƒ–‹‘ ‘ˆ–Š‡ Šƒ’‡”‘‡Ǧ‡ ‘†‹‰‰‡‡ bipA ͳ͸ͺǡƒ†–Š‡ˆ‘Ž†ƒ•‡•Ǧ‡ ‘†‹‰‰‡‡ pdiA ͳͲͶ™ƒ• ‘„•‡”˜‡†ǤŽ–Š‘—‰Š•‘‡ˆŽ— –—ƒ–‹‘•‹–Š‡Ž‡˜‡Ž•ˆ‘”–Š‡ˆ‘Ž†ƒ•‡•›’ ʹ͹™‡”‡ ‘„•‡”˜‡†ǡ ‹–• ‡š’”‡••‹‘ ™ƒ• ‘– ‘„˜‹‘—•Ž› ƒˆˆ‡ –‡† „› –Š‡  –”‡ƒ–‡–Ǥ

(50)  ‘–”ƒ•–ǡ –—‹ ƒ› ‹–”‡ƒ–‡–Šƒ†ƒ’”‘ˆ‘—†‡ˆˆ‡ –‘–Š‡‡š’”‡••‹‘‘ˆcypBǡƒ†Ž‡†–‘ƒ•‡˜‡Ǧ ˆ‘Ž† —’”‡‰—Žƒ–‹‘ ™‹–Š‹ Ͷ Š ȋ’Š‘•’Š‘”Ǧ‹ƒ‰‡” †ƒ–ƒǡ ”‡•—Ž–• ‘– •Š‘™ȌǤ ‹‡  –”‡ƒ–‡–ǡ –—‹ ƒ› ‹ –”‡ƒ–‡– ƒŽ•‘ Ž‡† –‘ –Š‡ ƒ’’‡ƒ”ƒ ‡ ‘ˆ –Š‡ hacAi „ƒ†ǡ ƒ† –Š‡ •‹—Ž–ƒ‡‘—•—’”‡‰—Žƒ–‹‘‘ˆbipAƒ†pdiAǡƒŽ–Š‘—‰Š–Š‡‡ˆˆ‡ –ƒ’’‡ƒ”‡†Žƒ–‡”–Šƒ‹–Š‡  –”‡ƒ–‡† › ‡Ž‹ƒǤ ‘”–Š‡” ƒƒŽ›•‹• ‘ˆ A. niger ͳͷǣǣ‰ŽƒǦ–Ǧ͓ͳͻ •Š‘™‡† –Šƒ– –Š‡ ‡ˆˆ‡ – ‘ –Š‡ ™ƒ•ƒŽ”‡ƒ†› ƒ‹ˆ‡•–‡† „› ‹–Š‡ —’Ǧ”‡‰—Žƒ–‹‘ ‘ˆ bipAǡ cypB ƒ† pdiAǡ ƒ†–Š‡ˆ‘”ƒ–‹‘‘ˆƒ•ƒŽŽ‡”hacAi„ƒ†ǡ„‡ˆ‘”‡ Ž‡ƒ”‹†— –‹‘‘ˆ–Ǧ ‘—Ž†„‡†‡–‡ –‡† ȋƒˆ–‡”ʹŠ‘ˆ‹†— –‹‘Ȍǡ—†‡”• ‘”‹‰–Š‡•‡•‹–‹˜‹–›‘ˆ–Š‡Ǥ‘”–Š‡”„Ž‘–ƒƒŽ›•‹•ƒŽ•‘ ”‡˜‡ƒŽ‡† –Šƒ– –Š‡ –”ƒ• ”‹’– Ž‡˜‡Ž• ‘ˆ bipAǡ cypB ƒ† pdiA ‹ ”‡ƒ•‡† ‹ ’ƒ”ƒŽŽ‡Ž ™‹–Š –Š‡ ‹ ”‡ƒ•‡‹–Š‡–Ǧ–”ƒ• ”‹’–Ǥ ”‘–Š‡•‡”‡•—Ž–•‹–ƒ’’‡ƒ”•–Šƒ––Š‡‡ˆˆ‡ –•‘ˆ•–”‡••ǡ ‹’‘•‡†„›–Š‡‡š’”‡••‹‘‘ˆƒŠ‡–‡”‘Ž‘‰‘—•’”‘–‡‹‘”‡ Ž‘•‡Ž›”‡•‡„Ž‡•–Šƒ– ƒ—•‡† „›–—‹ ƒ› ‹–Šƒ–Šƒ–„›–”‡ƒ–‡–™‹–ŠǤŠ‹•‹•˜ƒŽ‹†ˆ‘”„‘–Š–Š‡”‡•’‘•‡–‹‡ǡ ƒ†–Š‡–”ƒ• ”‹’–‹‘Ž‡˜‡Ž•‘ˆbipAǡcypBƒ†pdiAǤŠ‡–‘–ƒŽƒ‘—–‘ˆ–Š‡hacA–”ƒ• ”‹’– ‹•Ž‘™‡”ƒ––͵Ͳ ‘’ƒ”‡†–‘–Ͳ‹ƒŽŽ–Š”‡‡•–”‡••–”‡ƒ–‡–•ǤŠ‹• ‘—Ž†„‡–Š‡”‡•—Ž–‘ˆ –”ƒ• ”‹’–‹‘ƒŽ †‘™Ǧ”‡‰—Žƒ–‹‘ †—‡ –‘ –Š‡ –”ƒ•ˆ‡” ‘ˆ › ‡Ž‹ƒ ƒ– –Š‡ •–ƒ”– ‘ˆ –Š‡ ‡š’‡”‹‡–•Ǥ‘–Š‡”’‘••‹„Ž‡‡š’Žƒƒ–‹‘‹•–Šƒ––Š‡’”‘ ‡••‡†hacAi‹•Ž‡•••–ƒ„Ž‡ –Šƒ–Š‡—‹†— ‡†hacAuǤŠ‹• ‘—Ž†‰‹˜‡–Š‡ˆ—‰—•–Š‡ƒ„‹Ž‹–›–‘•Š—–†‘™–Š‡‘”‡ ”ƒ’‹†Ž›™Š‡–Š‡•–”‡••Šƒ•„‡‡”‡Ž‹‡˜‡†Ǥ   5'TruncationofhacAmRNA ’Ž‹ ‹‰ ‘ˆ –Š‡ ʹͲǦ– ‘Ǧ ‘˜‡–‹‘ƒŽ ‹–”‘ —’‘  ‹†— –‹‘ ™ƒ• †‡‘•–”ƒ–‡† „› ‘’ƒ”ƒ–‹˜‡ ƒƒŽ›•‹• ‘ˆ –Š‡  ƒ† ‰‡‘‹  •‡“—‡ ‡•Ǥ ‘™‡˜‡”ǡ –Š‹• •’Ž‹ ‹‰ ‡˜‡– ƒŽ‘‡ ƒ‘– ƒ ‘—– ˆ‘” –Š‡ †‹ˆˆ‡”‡ ‡ ‹ •‹œ‡ „‡–™‡‡ –Š‡ hacAu ƒ† hacAi –”ƒ• ”‹’–• ‘„•‡”˜‡†„›‘”–Š‡”ƒƒŽ›•‹•Ǥ‡“—‡ ‡ƒƒŽ›•‹•‘ˆ–Š‡ Ž‘‡•‘„–ƒ‹‡†ˆ”‘–Š‡A.niger  Ž‹„”ƒ”› ‹†‹ ƒ–‡† –Šƒ– hacAi ‹‰Š– ƒŽ•‘ „‡ –”— ƒ–‡† ƒ– –Š‡ ͷǯǦ‡† ”‡Žƒ–‹˜‡ –‘ hacAuǤ —”–Š‡”‘”‡ǡ –Š‡ A. nidulans ƒ† T. reesei hacA/hac1 • ƒ”‡ Ž‡ƒ˜‡† ™‹–Š‹ –Š‡ ͷǯǦ ˆŽƒ‹‰”‡‰‹‘—’‘‹†— –‹‘Ǥ ‘™‡˜‡”ǡ‡‹–Š‡”–Š‡‡ Šƒ‹•‘”–Š‡‡šƒ –•‹–‡‘ˆ Ž‡ƒ˜ƒ‰‡Šƒ•„‡‡†‡–‡”‹‡†‹–Š‘•‡ ƒ•‡•ͳͶͲǤ‘ ‘ˆ‹”–Šƒ––”— ƒ–‹‘‘ˆ–Š‡ͷǯǦ‡†‘ˆ IsolationandCharacterisationoftheTranscriptionFactorHacA51.

(51) Chapter2 hacA–ƒ‡•’Žƒ ‡†—”‹‰‹A.nigerǡ‘”–Š‡”„Ž‘–•™‡”‡’”‘„‡†™‹–Šƒʹ͵ͲǦ„’ ˆ”ƒ‰‡– ‘’”‹•‹‰–Š‡‘•–—’•–”‡ƒ’ƒ”–‘ˆ–Š‡hacAͷǯȋ ‹‰ǤͷȌǤ•‡š’‡ –‡†ǡ–Š‹• ’”‘„‡ ‘Ž› Š›„”‹†‹•‡• –‘ –Š‡ ˆ—ŽŽǦŽ‡‰–Š hacAu ǡ ƒ† –Š‡ •‹‰ƒŽ †‡ ”‡ƒ•‡• —’‘ ‹†— –‹‘‘ˆ–Š‡Ǥ —”–Š‡”‘”‡ǡ–Š‹•’”‘„‡†‘‡•‘–Š›„”‹†‹•‡–‘–Š‡hacAiǤŠ‹• ‘ˆ‹”•–Šƒ––Š‡hacAi–”ƒ• ”‹’–ƒŽ•‘Žƒ •ƒͷǯǦ•‡‰‡–‘ˆ–Š‡hacAuǤ‘†‡–‡”‹‡ –Š‡•‹–‡‘ˆ–”— ƒ–‹‘ǡƒ†–‘ ‘ˆ‹”–Š‡•’Ž‹ ‹‰‘ˆ–Š‡ʹͲǦ–— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘ –Š‡A.nigerhacA—†‡” ‘†‹–‹‘•ǡƒͷǯ‡š’‡”‹‡–™ƒ•’‡”ˆ‘”‡†ǤŠ‡ ”‡ƒ –‹‘•™‡”‡’‡”ˆ‘”‡†‘‡š–”ƒ –‡†ˆ”‘A.nigerͳͷǣǣ’‰ŽƒǦ–Ǧ„‡ˆ‘”‡ǡƒ†ͶŠ ƒˆ–‡”  ‹†— –‹‘ ȋ ‹‰Ǥ ͷȌǤ  ‰‡‡Ǧ•’‡ ‹ˆ‹  ’”‹‡”ǡ Ž‘ ƒ–‡† †‘™•–”‡ƒ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ǡ ™ƒ• —•‡† –‘ ƒ’Ž‹ˆ› –Š‡ hacA Ǥ  •‹‰Ž‡ „ƒ† ™ƒ• ‘„–ƒ‹‡† ˆ”‘ ƒ › ‡Ž‹ƒŽ •ƒ’Ž‡ Šƒ”˜‡•–‡† „‡ˆ‘”‡ ‹†— –‹‘Ǥ ™‘ „ƒ†• ™‡”‡ ‘„–ƒ‹‡† ˆ”‘ –Š‡ •ƒ’Ž‡Šƒ”˜‡•–‡†ƒˆ–‡”‹†— –‹‘Ǣ–Š‡Žƒ”‰‡”‘ˆ–Š‡•‡–™‘„ƒ†• Šƒ†–Š‡•ƒ‡•‹œ‡ƒ•–Š‡ ˆ”ƒ‰‡– ‘„–ƒ‹‡† „‡ˆ‘”‡ ‹†— –‹‘Ǥ Š‹• ‹• ‹ ƒ‰”‡‡‡– ™‹–Š –Š‡ ‘”–Š‡” „Ž‘– †ƒ–ƒǡ ™Š‹ Š ‹†‹ ƒ–‡† –Šƒ– –™‘ hacA  •’‡ ‹‡• ȋhacAu ƒ† hacAiȌ ™‡”‡ ’”‡•‡– Ͷ Š ƒˆ–‡” ‹†— –‹‘ ȋ ‹‰Ǥ ͷȌǤ ŽŽ –Š‡ ˆ”ƒ‰‡–• ‘„–ƒ‹‡† ™‡”‡ Ž‘‡† ƒ† •‡“—‡ ‡†Ǥ Š‡ ˆ”ƒ‰‡– †‡”‹˜‡†ˆ”‘–Š‡•ƒ’Ž‡–ƒ‡„‡ˆ‘”‡‹†— –‹‘™ƒ•‹†‡–‹ ƒŽ–‘–Š‡Žƒ”‰‡”ˆ”ƒ‰‡–ˆ”‘ –Š‡•ƒ’Ž‡–ƒ‡ͶŠƒˆ–‡”–Ǧ‹†— –‹‘ȋhacAuȌǡƒ† ‘–ƒ‹‡†–Š‡— ‘˜‡–‹‘ƒŽʹͲǦ– ‹–”‘ǤŠ‡ͷǯǦ‡†•™‡”‡Ž‘ ƒ–‡†͵ͲͶ–—’•–”‡ƒ‘ˆ–Š‡hacA•–ƒ”– ‘†‘ȋ ‹‰Ǥ͸ȌǤ. Figure6.˜‡”˜‹‡™‘ˆ–Š‡•‡“—‡ ‡ƒ––Š‡ͷǯ‘ˆ–Š‡A.nigerhacA ‰‡‡ǤŠ‡ˆ‹”•–ˆ‹˜‡ƒ‹‘ ƒ ‹†•‘ˆ ƒ ƒ”‡‹†‹ ƒ–‡†‹—’’‡” ƒ•‡Ž‡––‡”•ǤŠ‡–”ƒ• ”‹’–‹‘•–ƒ”–•‹–‡‘ˆhacAu‹•‹†‹ ƒ–‡† „›–Š‡Š‘”‹œ‘–ƒŽƒ””‘™ǡƒ†–Š‡•–ƒ”–•‹–‡‘ˆhacAi„›–Š‡˜‡”–‹ ƒŽƒ””‘™ǤŠ‡ ‡ ‘†‹‰ƒͶͶǦ ƒ‹‘ƒ ‹†’‡’–‹†‡‹•ƒŽ•‘‹†‹ ƒ–‡†‹—’’‡” ƒ•‡Ǥ. 52IsolationandCharacterisationoftheTranscriptionFactorHacA.

(52) Chapter2 ‡“—‡ ‡ ƒƒŽ›•‹• ‘ˆ –Š‡ •ƒŽŽ‡” ˆ”ƒ‰‡–ǡ ‘„–ƒ‹‡† ˆ”‘ –Š‡ ‹†— ‡† •ƒ’Ž‡ ȋhacAiȌǡ ”‡˜‡ƒŽ‡† –Šƒ– ‹–• ͷǯǦ‡† Ž‹‡• ʹ͵Ͳ – ˆ—”–Š‡” †‘™•–”‡ƒǡ ‘Ž› ͹Ͷ– —’•–”‡ƒ ‘ˆ –Š‡ hacA •–ƒ”– ‘†‘ȋ ‹‰Ǥ͸ȌǤ —”–Š‡”‘”‡ǡ–Š‡— ‘˜‡–‹‘ƒŽʹͲǦ–‹–”‘™ƒ•‘–’”‡•‡–‹–Š‡•‡ ˆ”ƒ‰‡–•ǡ ‘ˆ‹”‹‰–Šƒ–•’Ž‹ ‹‰Šƒ†–ƒ‡’Žƒ ‡ǤŠ‡’ƒ”–”‡‘˜‡†ˆ”‘–Š‡ͷǯ‘ˆ hacA ‘–ƒ‹• ƒ •Š‘”–   ‡ ‘†‹‰ ƒ ͶͶǦƒ‹‘ ƒ ‹† ’‡’–‹†‡Ǥ Ž–Š‘—‰Š ‹– ‹• ‘– ‘™ ™Š‡–Š‡” –Š‹• ‡š–”ƒ ”‡‰‹‘ ‹• ˆ— –‹‘ƒŽǡ ‹– ‘—Ž† ‹’Ž› –Š‡ ‹˜‘Ž˜‡‡– ‘ˆ ƒ –”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž‡ Šƒ‹•Ǥ

(53) –‡”‡•–‹‰Ž›ǡˆ—”–Š‡”‹•’‡ –‹‘‘ˆ–Š‡•‡“—‡ ‡ƒ”‘—†–Š‡–”— ƒ–‹‘ •‹–‡”‡˜‡ƒŽ‡†ƒ•‡“—‡ ‡•–”‡– Š•‹‹Žƒ”–‘–Š‘•‡ˆ‘—†ƒ”‘—†–Š‡•‡“—‡ ‡„‘”†‡”•‘ˆ–Š‡ ʹͲǦ–‹–”‘ȋ ‹‰Ǥ͵ȌǤ   OverexpressionofhacA ‘†‡–‡”‹‡™Š‡–Š‡”‘˜‡”Ǧ‡š’”‡••‹‘‘ˆhacAi™‘—Ž†‹‹ –Š‡‡ˆˆ‡ –•‘ˆ–Ǧ‡š’”‡••‹‘ǡ  –”‡ƒ–‡– ‘” –—‹ ƒ› ‹ –”‡ƒ–‡–ǡ –Š‡ hacA ‘†‹‰ ”‡‰‹‘ ™ƒ• Ž‘‡† ‹ ƒ Aspergillus ‡š’”‡••‹‘ ˜‡ –‘”ǡ —†‡” –Š‡ ‘–”‘Ž ‘ˆ –Š‡ ‘•–‹–—–‹˜‡ A. nidulans gpdA ’”‘‘–‡”ǡƒ†–Š‡”‡•—Ž–‹‰’Žƒ•‹†ȋ’ ͷͲȌ™ƒ•‹–”‘†— ‡†‹–‘–Š‡‰‡‘‡‘ˆA.niger ͷͻʹǤ ‘—–Š‡”ƒƒŽ›•‹•‘ˆ–™‘–”ƒ•ˆ‘”ƒ–•†‡–‡ –‡†–™‘ƒ††‹–‹‘ƒŽ ‘’‹‡•‘ˆ–Š‡hacA ‰‡‡‹ͷͻʹǣǣ’ ͷͲ͓ͳǡƒ†‘‡ƒ††‹–‹‘ƒŽ ‘’›‹ͷͻʹǣǣ’ ͷͲ͓ʹǡ”‡Žƒ–‹˜‡–‘™‹Ž†–›’‡ ȋ ‹‰Ǥ͹ȌǤ‘”–Š‡”ƒƒŽ›•‹•‘ˆ–Š‡•‡•–”ƒ‹••Š‘™‡†•—„•–ƒ–‹ƒŽƒ ——Žƒ–‹‘‘ˆbipAǡcypBǡ ƒ†pdiA•‹ͷͻʹǣǣ’ ͷͲ͓ͳȋ ‹‰ǤͺƒȌǡƒ†ƒ‹‘”—’Ǧ”‡‰—Žƒ–‹‘‹–Š‡•‹‰Ž‡Ǧ ‘’› ‹–‡‰”ƒ– ͷͻʹǣǣ’ ͷͲ͓ʹǤ Š‡•‡ ”‡•—Ž–• ™‡”‡ ‘ˆ‹”‡† „› ‡•–‡” ƒƒŽ›•‹• ȋ ‹‰Ǥ ͺ„Ȍǡ ƒ†ƒ”‡ƒƒŽ‘‰‘—•–‘–Š‡”‡•—Ž–•‘„–ƒ‹‡†—’‘–”‡ƒ–‡–™‹–Š–—‹ ƒ› ‹ƒ†‹†— –‹‘ ‘ˆ –Ǧ ‡š’”‡••‹‘Ǥ —ƒ–‹ˆ‹ ƒ–‹‘ ‘ˆ –Š‡ hacAu  „› ’Š‘•’Š‘”Ǧ‹ƒ‰‡” ƒƒŽ›•‹• ȋ†ƒ–ƒ ‘– •Š‘™Ȍ ”‡˜‡ƒŽ‡† ˆ‘—”Ǧ ƒ† –™‘Ǧˆ‘Ž† Š‹‰Š‡” •‹‰ƒŽ• ‹ ͷͻʹǣǣ’ ͷͲ͓ͳ ƒ† ͷͻʹǣǣ’ ͷͲ͓ʹǡ ”‡•’‡ –‹˜‡Ž›ǡ ‘’ƒ”‡† –‘ ™‹Ž† –›’‡Ǥ Š‹• —’Ǧ”‡‰—Žƒ–‹‘ ‘ˆ hacAu —’‘ ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ˆ hacAi •—‰‰‡•–• –Šƒ–ǡ —†‡”  ‘†‹–‹‘•ǡ –”ƒ• ”‹’–‹‘ ‘ˆ –Š‡ hacA ‰‡‡‹•—’Ǧ”‡‰—Žƒ–‡†„›‹–•‘™‰‡‡’”‘†— –Ǥ. Figure7.‘—–Š‡” ƒƒŽ›•‹••Š‘™‹‰–™‘ƒ††‹–‹‘ƒŽ ‘’‹‡•‘ˆ–Š‡A. nigerhacA ‰‡‡‹•–”ƒ‹ ͷͻʹǣǣ’ ͷͲ͓ͳǡƒ†‘‡ƒ††‹–‹‘ƒŽ ‘’›‹ͷͻʹǣǣ’ ͷͲ͓ʹǡ ‘’ƒ”‡†–‘–Š‡™‹Ž†Ǧ–›’‡•–”ƒ‹ ͷͻʹǤ. IsolationandCharacterisationoftheTranscriptionFactorHacA53.

(54) Chapter2             Figure 8.  ˆˆ‡ –• ‘ˆ ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ˆ hacAi ‘ –Š‡ ‡š’”‡••‹‘ ‘ˆ bipAǡ cypBǡ ƒ† pdiAǤ (A) ‘”–Š‡” ƒƒŽ›•‹• •Š‘™‹‰ –Š‡ ‡ˆˆ‡ – ‘ˆ ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ˆ hacAi ‹ A. nigerǤ ”‘„‡• —•‡† ‹ Š›„”‹†‹•ƒ–‹‘•ƒ”‡‹†‹ ƒ–‡†‘–Š‡Ž‡ˆ–Ǥȋƒ‡•ǣ™–ǡ™‹Ž†Ǧ–›’‡A.niger•–”ƒ‹ͷͻʹǢ͓ͳǡA.niger •–”ƒ‹ͷͻʹǣǣ’ ͷͲ͓ͳǢ͓ʹǡA.niger•–”ƒ‹ͷͻʹǣǣ’ ͷͲ͓ʹȌǤŠ‡ƒ””‘™•‹†‹ ƒ–‡–Š‡–™‘hacA  •’‡ ‹‡• hacAu ƒ† hacAiǤ ‘–‡ –Šƒ– –Š‡ ‡š’‘•—”‡ –‹‡ ˆ‘” –Š‡ hacA „Ž‘– ™ƒ• ”‡†— ‡† ”‡Žƒ–‹˜‡–‘–Šƒ–—•‡†ˆ‘” ‹‰Ǥͷǡ†—‡–‘–Š‡Š‹‰Š‡š’”‡••‹‘Ž‡˜‡Ž‹•–”ƒ‹͓ʹǤ(B)‡•–‡”ƒƒŽ›•‹• •Š‘™‹‰ –Š‡ ‡ˆˆ‡ – ‘ˆ hacAi ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ –Š‡ Ž‡˜‡Ž• ‘ˆ ‹’ǡ ›’ ƒ† †‹ ’”‘–‡‹Ǥ –‹„‘†‹‡•ƒ”‡‹†‹ ƒ–‡†‹—’’‡” ƒ•‡Ž‡––‡”•‘–Š‡”‹‰Š–Ǥ.    ElectrophoreticMobilityShiftAnalysis(EMSA) Š‡‡š’‡”‹‡–•†‡• ”‹„‡†•‘ˆƒ”•Š‘™–Šƒ–—’Ǧ”‡‰—Žƒ–‹‘‘ˆ”‡•‹†‡– Šƒ’‡”‘‡•ƒ† ˆ‘Ž†ƒ•‡• ‘ —”• •› Š”‘‘—•Ž› ™‹–Š ’”‘ ‡••‹‰ ‘ˆ hacA  †—”‹‰  •–”‡••Ǥ ‹‹Žƒ” ”‡•—Ž–• ™‡”‡ ‘„–ƒ‹‡† ™‹–Š‘—– •‡ ”‡–‹‘ •–”‡•• —’‘ ‘˜‡”Ǧ‡š’”‡••‹‘ ‘ˆ hacAiǡ •—‰‰‡•–‹‰ –Šƒ– ƒ ‹•–Š‡ˆƒ –‘””‡•’‘•‹„Ž‡ǤŠ‡–Š‡” ƒ ‹†‡‡†„‹†•–‘–Š‡’”‘‘–‡”•‘ˆ–Š‡•‡ —’Ǧ”‡‰—Žƒ–‡† ‰‡‡• ™ƒ• •–—†‹‡† in vitro „› Ǥ —”‹ˆ‹‡† ƒ  ’”‘–‡‹ ™ƒ• —•‡† –‘ ‡˜ƒŽ—ƒ–‡„‹†‹‰–‘–Š‡’”‘‘–‡”•‘ˆbipAǡcypBǡpdiAƒ†hacA‹–•‡Žˆǡƒ†–‘–Š‡’”‘‘–‡”• ‘ˆ –™‘ ƒ††‹–‹‘ƒŽ  ˆ‘Ž†ƒ•‡Ǧ‡ ‘†‹‰ ‰‡‡•ǡ prpA ƒ† tigAǤ prpA ‹• Š‘‘Ž‘‰‘—• –‘ –Š‡ ’”‘–‡‹ †‹•—Žˆ‹†‡ ‹•‘‡”ƒ•‡ ‰‡‡ pdiAǡ ƒ† ‹– ™ƒ• •Š‘™ –‘ „‡ —’Ǧ”‡‰—Žƒ–‡† †—”‹‰ Š‡–‡”‘Ž‘‰‘—• ’”‘–‡‹„‹‘•›–Š‡•‹• ͳ͹ͶǤ Š‡ ’”‘†— – ‘ˆ tigA ƒŽ•‘ „‡Ž‘‰• –‘ –Š‡ 

(55)  ˆƒ‹Ž›ǡ ƒ†‹–•–”ƒ• ”‹’–‹‘‹•‹†— ‡†—’‘–”‡ƒ–‡–™‹–Š–—‹ ƒ› ‹͸ʹǤŠ‡’”‘‘–‡”‘ˆcypAǡ ™Š‹ Š ‡ ‘†‡• ƒ ›–‘’Žƒ•‹  › Ž‘’Š›Ž‹ ʹ͸ǡ ™ƒ• —•‡† ƒ• ƒ ‡‰ƒ–‹˜‡ ‘–”‘ŽǤ ƒ†Ǧ•Š‹ˆ–• ™‡”‡ ‘„•‡”˜‡† ˆ‘” ƒŽŽ –Š‡ Ǧ•’‡ ‹ˆ‹  ’”‘‘–‡”• ȋ ‹‰Ǥ ͻȌǤ

(56) –‡”‡•–‹‰Ž›ǡ „‹†‹‰ ™ƒ• ƒŽ•‘ ‘„•‡”˜‡†™‹–ŠƒhacA’”‘‘–‡”ˆ”ƒ‰‡–ǤŠ‹•‹•‹ƒ ‘”†ƒ ‡™‹–Š–Š‡ˆ‹†‹‰–Šƒ–‘˜‡”Ǧ ‡š’”‡••‹‘‘ˆhacAiŽ‡ƒ†•–‘—’Ǧ”‡‰—Žƒ–‹‘‘ˆhacAuǤ. 54IsolationandCharacterisationoftheTranscriptionFactorHacA.

(57) Chapter2           Figure 9.  Ž‡ –”‘’Š‘”‡–‹  ‘„‹Ž‹–› •Š‹ˆ– ƒƒŽ›•‹• ȋȌ •Š‘™‹‰ –Š‡ „‹†‹‰ ‘ˆ –Š‡ A. niger ƒ  ’”‘–‡‹ –‘ –Š‡ ’”‘‘–‡”• ‘ˆ ‰‡‡• ‡ ‘†‹‰  Šƒ’‡”‘‡• ƒ† ˆ‘Ž†ƒ•‡•ǡ ƒ† –‘ –Š‡ ’”‘‘–‡” ‘ˆ hacA ‹–•‡Žˆ ȋ ǡ ’”‘–‡‹Ǧ ‘’Ž‡šǢ ˆ’ǡ ˆ”‡‡ ’”‘„‡ȌǤ ƒ‡•ǣ ͳǡ bipAǢ ʹ cypBǢ ͵ǡ pdiAǢ Ͷǡ prpAǢ ͷǡ tigAǢ ͸ǡ cypA ȋ‡‰ƒ–‹˜‡ ‘–”‘ŽȌǢ ͹ǡ hacAǤ Š‡ •‹œ‡• ‘ˆ –Š‡ ’”‘‘–‡” ˆ”ƒ‰‡–•—•‡† ƒ„‡†‡†— ‡†ˆ”‘–Š‡†ƒ–ƒ‹ƒ„Ž‡ʹǤ.   CellularLocalisationofHacA ƒŽ›•‹• ‘ˆ ƒ  —•‹‰ –Š‡ 

(58)

(59)  ’”‘‰”ƒ ȋŠ––’ǣȀȀ ’•‘”–Ǥ‹„„Ǥƒ ǤŒ’ȀȌ ‹†‡–‹ˆ‹‡† –™‘ ’—–ƒ–‹˜‡ — Ž‡ƒ” ‘ ƒŽ‹•ƒ–‹‘ ‹‰ƒŽ• ȋ•Ȍǡ ƒ ’—–ƒ–‹˜‡ ‘‘’ƒ”–‹–‡  ȋ ‘’”‹•‹‰ ƒ‹‘ ƒ ‹† ”‡•‹†—‡• ͶͻȂ ͷͷȌǡ ƒ† ƒ ’—–ƒ–‹˜‡ „‹’ƒ”–‹–‡  ȋ

(60) ǡƒ‹‘ƒ ‹†”‡•‹†—‡•͹ͲȂͺ͸ȌǤŠ‡’—–ƒ–‹˜‡„‹’ƒ”–‹–‡‹•Ž‘ ƒ–‡† ‹–Š‡Ǧ„‹†‹‰†‘ƒ‹Ǥ‘‡šƒ‹‡–Š‡ ‡ŽŽ—Žƒ”Ž‘ ƒŽ‹•ƒ–‹‘‘ˆ ƒ ǡƒŠ›„”‹†gfphacA ‘•–”— – ™ƒ• ƒ†‡ǡ ‘•‹•–‹‰ ‘ˆ –Š‡ hacAi   ˆ—•‡† –‘ –Š‡ ͵ǯǦ‡† ‘ˆ –Š‡ gfp  Ǥ Ž—‘”‡• ‡ ‡‹ ”‘• ‘’›‘ˆͷͻʹǣǣ’ ͷʹͶǡ ™Š‹ Š‡š’”‡••‡• gfphacAi—†‡” ‘–”‘Ž‘ˆ –Š‡ gpdA ’”‘‘–‡”ǡ •Š‘™‡† ƒ •‹‹Žƒ” ’ƒ––‡” ‘ˆ ˆŽ—‘”‡• ‡ ‡ ƒ• Š›’Šƒ‡ •–ƒ‹‡† ™‹–Š –Š‡ — Ž‡ƒ”†›‡

(61) ǡ ‘ˆ‹”‹‰–Šƒ– ƒ ‹†‡‡†Ž‘ ƒŽ‹•‡•–‘–Š‡— Ž‡—•ȋ ‹‰ǤͳͲȌǤ   . Figure10. Ž—‘”‡• ‡ ‡‹ ”‘• ‘’›•Š‘™‹‰–Š‡Ž‘ ƒŽ‹•ƒ–‹‘‘ˆ Ǧ ƒ Ǥ(A)–ƒ‹‹‰‘ˆ–Š‡ Š›’Šƒ‡™‹–Š–Š‡— Ž‡ƒ”•–ƒ‹

(62) Ǥ(B)‘ ƒŽ‹•ƒ–‹‘‘ˆ ǤŠ‡„ƒ””‡’”‡•‡–•ͳͲɊǤ. IsolationandCharacterisationoftheTranscriptionFactorHacA55.

(63) Chapter2. Discussion  Š‡A.nigerhacA‰‡‡™ƒ•‹•‘Žƒ–‡†„ƒ•‡†‘‹–••‹‹Žƒ”‹–›–‘–Š‡A.nidulanshacA•‡“—‡ ‡Ǥ Š‡ ƒ  ’”‘–‡‹• ˆ”‘ A. niger ƒ† A. nidulans ƒ”‡ ˜‡”› ™‡ŽŽ ‘•‡”˜‡† ƒ† ƒ”‡ ͹͵Ψ ‹†‡–‹ ƒŽǤ Š‡ ’—–ƒ–‹˜‡ ƒ  ˆ”‘ A. fumigatus ˆ‘—† ‹ –Š‡ 

(64)  ‰‡‘‡ †ƒ–ƒ„ƒ•‡ ȋŠ––’ǣȀȀ™™™Ǥ–‹‰”Ǥ‘”‰Ȁ–†„Ȁ‡ʹͳȀƒˆ—ͳȀȌ ‹• ͺͲΨ ‹†‡–‹ ƒŽ –‘ –Š‡ A. niger ƒ Ǥ Š‡ Ǧ „‹†‹‰ †‘ƒ‹• ‘ˆ –Š‡ –Š”‡‡ ƒ  Š‘‘Ž‘‰—‡• ƒ”‡ ‹†‡–‹ ƒŽǡ ™Š‹Ž‡ –Š‡‹” Ž‡— ‹‡ œ‹’’‡” †‘ƒ‹•ƒ”‡˜‡”›•‹‹Žƒ”–‘‡ƒ Š‘–Š‡”Ǥ‡“—‡ ‡†‹ˆˆ‡”‡ ‡•ƒ”‡‘•–Ž›Ž‘ ƒ–‡†‹–Š‡Ǧ –‡”‹ƒŽƒ†Ǧ–‡”‹ƒŽ’ƒ”–•‘ˆ–Š‡’”‘–‡‹•Ǥ

(65) ›‡ƒ•–ǡHAC1‹•‡š’”‡••‡†”‡Žƒ–‹˜‡Ž›ƒ„—†ƒ–Ž›‹—•–”‡••‡† ‡ŽŽ•ǡ„—–‹–‹• ‘– –”ƒ•Žƒ–‡† †—‡ –‘ –Š‡ ’”‡•‡ ‡ ‘ˆ ƒ ʹͷʹǦ– ‹–”‘Ǥ Š‹• ‹–”‘ ’ƒ‹”• ™‹–Š –Š‡ ͷǯǡ –Š‡”‡„›’”‡˜‡–‹‰–Š‡”‡ƒ†Ǧ–Š”‘—‰Š„›–Š‡”‹„‘•‘‡•ͳ͵͹Ǥ’Ž‹ ‹‰‘ˆ–Š‹•‹–”‘„›

(66) ”‡ͳ’‹• ‡ ‡••ƒ”›ˆ‘”‹†— –‹‘ʹʹǤ Ž–Š‘—‰Š–Š‡ƒƒŽ‹ƒͳ‹••‹‹Žƒ”–‘–Š‡›‡ƒ•– ƒ ͳ’‘Ž›‹•‘ˆƒ”ƒ•„‘–Šƒ”‡ „

(67)  ’”‘–‡‹•ǡ ‹– Šƒ• „‡‡ •Š‘™–Šƒ– ‡ƒ”Ž›ƒŽŽ ˆ‡ƒ–—”‡• ‘ˆ –Š‡ ›‡ƒ•– •’Ž‹ ‹‰ ’ƒ––‡” ƒ”‡ ‘•‡”˜‡† ‹ ƒƒŽ•. ͳͺ͸Ǥ. ‘™‡˜‡”ǡ ‹ ‘–”ƒ•– –‘ HAC1ǡ ™Š‹ Š ‹• –”ƒ• ”‹„‡†. ‘•–‹–—–‹˜‡Ž›ǡ XBP1 ‹• ‡š’”‡••‡† ƒ– Ž‘™ Ž‡˜‡Ž• ƒ† –Š‡  ‹• –”ƒ•Žƒ–‡†ǡ •‹ ‡ ‘ ‹Š‹„‹–‘”› •‡ ‘†ƒ”›•–”— –—”‡ ƒ „‡ˆ‘”‡†Ǥ —”–Š‡”‘”‡ǡ ƒ –‹˜ƒ–‹‘‘ˆ–Š‡ƒƒŽ‹ƒ  ‹˜‘Ž˜‡• –™‘ –”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘”•ǡ  ͸ ƒ† ͳǤ  –‹˜ƒ–‹‘ •–ƒ”–• ™‹–Š –Š‡ ’”‘–‡‘Ž›–‹ ”‡Ž‡ƒ•‡‘ˆ ͸ˆ”‘–Š‡Ǥ ͸–Š‡—’Ǧ”‡‰—Žƒ–‡•–”ƒ• ”‹’–‹‘‘ˆ‰‡‡•ˆ‘”  Šƒ’‡”‘‡•ǡ ƒ• ™‡ŽŽ ƒ• –Š‡ ‡š’”‡••‹‘ ‘ˆ XBP1Ǥ XBP1 ‹• ƒ –‹˜ƒ–‡† „›

(68) ͳǦ‡†‹ƒ–‡† •’Ž‹ ‹‰‘ˆƒʹ͸Ǧ–‹–”‘ǡƒ†‹–•’”‘–‡‹’”‘†— –—’Ǧ”‡‰—Žƒ–‡•–Š‡•ƒ‡–ƒ”‰‡–•ƒ•  ͸ǡ ‹ Ž—†‹‰XBP1‹–•‡ŽˆǤ

(69) –Šƒ•„‡‡•—‰‰‡•–‡†–Šƒ––Š‹••›•–‡™‘—Ž†’”‘˜‹†‡–Š‡ƒƒŽ‹ƒ ‡ŽŽ™‹–Šƒ‘”‡ˆŽ‡š‹„Ž‡™Š‹ Š‹• ƒ’ƒ„Ž‡‘ˆ ‘’‹‰‘”‡‡ˆˆ‡ –‹˜‡Ž›™‹–Š•–”‡••ͳͺ͸Ǥ Š‡ •‹‹Žƒ”‹–› „‡–™‡‡ –Š‡ A. niger ƒ  ƒ† –Š‡ ›‡ƒ•– ƒ ͳ’ ‹• ‘ˆ‹‡† –‘ –Š‡  „‹†‹‰ †‘ƒ‹ǡ ƒ† ‘ •‹‰‹ˆ‹ ƒ– Š‘‘Ž‘‰› ‘—Ž† „‡ ˆ‘—† „‡–™‡‡ –Š‡ Aspergillus ’”‘–‡‹ƒ†‹–•ƒƒŽ‹ƒ‘”–Š‘Ž‘‰—‡ͳǤ‡•’‹–‡–Š‡•‡†‹ˆˆ‡”‡ ‡•ǡ‘—”ƒƒŽ›•‹•‘ˆ–Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ –Š‡ A. niger ƒ  ‹†‹ ƒ–‡• –Šƒ– ‹– •Šƒ”‡• ˆ‡ƒ–—”‡• ‘ˆ „‘–Š –Š‡ ›‡ƒ•– ƒ† –Š‡ ƒƒŽ‹ƒ‡“—‹˜ƒŽ‡–•ǡƒ†‹‰Š–‡˜‡’‘••‡••ˆ‡ƒ–—”‡•–Šƒ–ƒ”‡•’‡ ‹ˆ‹ ˆ‘”–Š‡‘ˆA. nigerƒ†‘–Š‡”ˆ‹Žƒ‡–‘—•ˆ—‰‹Ǥ Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘• ‘ˆ HAC1  ƒ† XBP1  ƒ”‡ •’Ž‹ ‡† „›

(70) ”‡ͳ’Ȁ

(71) ͳ—’‘‹†— –‹‘‘ˆ–Š‡ ͳͷʹǡͳͺ͸ǤŠ‡— ‘˜‡–‹‘ƒŽʹͲǦ–‹–”‘’”‡•‡–‹ –Š‡A.nigerhacAu•Š‘™•ƒ Ž‘•‡”‡•‡„Žƒ ‡–‘‹–• ‘—–‡”’ƒ”–•‹›‡ƒ•–ƒ†ƒƒŽ‹ƒ ‡ŽŽ•ȋ ‹‰Ǥ͵Ȍ ͳʹǡͳͷʹǤŠ‡•‡“—‡ ‡•ƒ––Š‡„‘—†ƒ”‹‡•‘ˆ–Š‡‹–”‘ƒ”‡™‡ŽŽ ‘•‡”˜‡†ǡƒ†ƒ •‹‹Žƒ”•‡ ‘†ƒ”›•–”— –—”‡‹•’”‡†‹ –‡†™Š‡”‡–Š‡‹–”‘„‘”†‡”•ƒ”‡‡ ‘’ƒ••‡†‹ –™‘Ž‘‘’•‘ˆ͹–ǤŠ‹••–”— –—”ƒŽƒ†•‡“—‡ ‡•‹‹Žƒ”‹–›•—‰‰‡•–•–Šƒ––Š‡A.nigerhacAu  ‹• •’Ž‹ ‡† ‹ ƒ •‹‹Žƒ” ƒ‡”ǡ ‘•– ’”‘„ƒ„Ž› „› –Š‡ ƒ –‹‘ ‘ˆ ƒ

(72) ”‡ͳ’Ȁ

(73) ͳ Ž‹‡. 56IsolationandCharacterisationoftheTranscriptionFactorHacA.

(74) Chapter2 ’”‘–‡‹Ǥ‡Šƒ˜‡”‡ ‡–Ž› Ž‘‡†ƒ‰‡‡ˆ”‘A.niger™‹–Š•‹‹Žƒ”‹–›–‘–Š‡›‡ƒ•–

(75) ”‡ͳ’ǡƒ† ’”‡Ž‹‹ƒ”›”‡•—Ž–••—‰‰‡•–‹–‹•‹˜‘Ž˜‡†‹–Š‡•’Ž‹ ‹‰‘ˆhacAǤ Š‡‘Ž›‘™‘†‹ˆ‹ ƒ–‹‘‘ˆ–Š‡›‡ƒ•–HAC1ƒ†ƒƒŽ‹ƒXBP1•–Šƒ– ‹•–”‹‰‰‡”‡†„›‹†— –‹‘‹•–Š‡•’Ž‹ ‹‰‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘Ǥ‡•—Ž–•ˆ”‘A. nidulans ƒ† T. reesei ͳͶͲǡ ƒ† †ƒ–ƒ ’”‡•‡–‡† ‹ –Š‹• ’ƒ’‡” ˆ‘” A. nigerǡ ‹†‹ ƒ–‡ –Šƒ– –Š‡ hacAu  —†‡”‰‘‡• ƒ ƒ††‹–‹‘ƒŽ ‘†‹ˆ‹ ƒ–‹‘ ‹ –Š‡•‡ ˆ—‰‹Ǥ

(76)  ƒ††‹–‹‘ –‘ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘Ǧ•’Ž‹ ‹‰ ‡˜‡– ‹†— ‡† „›  •–”‡••ǡ –Š‡ ͷǯ ‘ˆ hacA  ‹• –”— ƒ–‡†Ǥʹ͵ͲǦ–•‡‰‡–‹•”‡‘˜‡†ǡ™Š‹ Š ‘–ƒ‹•ƒ ™‹–Š–Š‡ ƒ’ƒ ‹–›–‘ ‘†‡ ˆ‘” ͶͶ ƒ‹‘ ƒ ‹†•Ǥ Š‡ ͷǯ ‘ˆ –Š‡ A. nidulans hacA ‰‡‡ ‘–ƒ‹• ƒ —  ˆ‘” •‡˜‡ ƒ‹‘ƒ ‹†•ǡƒ†–Š‡T.reeseihac1‰‡‡ ‘–ƒ‹•‘‡— ˆ‘”ͳͺƒ‹‘ƒ ‹†•ƒ†‘‡ˆ‘” –™‘ ƒ‹‘ ƒ ‹†•. ͳͶͲǤ. Š‡ ’”‡•‡ ‡ ‘ˆ –Š‡•‡ — • •—‰‰‡•–• –Š‡ ‹˜‘Ž˜‡‡– ‘ˆ ƒ. –”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž ‡ Šƒ‹•Ǥ ”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž ‹• ‘ˆ–‡ ƒ••‘ ‹ƒ–‡† ™‹–Š •Š‘”– —’•–”‡ƒ  • ‹ –Š‡ ͷǯ• ‘ˆ • ͻͲǤ

(77) †‡‡† –”ƒ•Žƒ–‹‘ ‘ˆ –Š‡ ›‡ƒ•– –”ƒ• ”‹’–‹‘ ˆƒ –‘” Ͷ’‹• ‘–”‘ŽŽ‡†„›ˆ‘—”— •ͷͻǡƒ†–”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž‘ˆ–Š‡•›–Š‡•‹•‘ˆ–Š‡ –”ƒ• ”‹’–‹‘ˆƒ –‘” Ͷǡ™Š‹ Š‹•‹˜‘Ž˜‡†‹–Š‡ƒƒŽ‹ƒ•–”‡••”‡•’‘•‹˜‡’ƒ–Š™ƒ›ǡ ‹• †‡’‡†‡– ‘ –™‘ — • ͷͳǤ Š‡ ‡ Šƒ‹• ”‡•’‘•‹„Ž‡ ˆ‘” –Š‡ –”— ƒ–‹‘ ‘ˆ A. niger hacA  —’‘  •–”‡•• ‹• •–‹ŽŽ —‘™Ǥ —” ”‡•—Ž–• ”‡˜‡ƒŽ‡† •‡“—‡ ‡ •‹‹Žƒ”‹–› „‡–™‡‡ –Š‡ –”— ƒ–‹‘ •‹–‡ ‹ –Š‡ ͷǯǦ’‘”–‹‘ ‘ˆ hacA  ƒ† –Š‡ „‘”†‡”• ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ȋ ‹‰Ǥ ͵ȌǤ ‘™‡˜‡”ǡ ‘  •‡ ‘†ƒ”› •–”— –—”‡ ‹ ‘”’‘”ƒ–‹‰ –Š‡ ’—–ƒ–‹˜‡–”— ƒ–‹‘•‹–‡‹ƒ•–ƒ„Ž‡•–‡ǦŽ‘‘’ ‘—Ž†„‡’”‡†‹ –‡†ˆ‘”–Š‡”‡‰‹‘ƒ”‘—†–Š‡ ͷǯǦ–”— ƒ–‹‘ •‹–‡Ǥ Š‹• ƒ‡• ƒ ‹˜‘Ž˜‡‡– ‘ˆ

(78) ”‡ ‹ –Š‡ –”— ƒ–‹‘ —Ž‹‡Ž›Ǥ —”–Š‡”‘”‡ǡ‹–Šƒ•„‡‡•’‡ —Žƒ–‡†–Šƒ–ǡ—’‘‹†— –‹‘‘ˆ–Š‡ǡ–”ƒ• ”‹’–‹‘‘ˆhac1 ‹T.reesei‹‰Š–‹‹–‹ƒ–‡ƒ–ƒ‡™•–ƒ”–•‹–‡ǡ–Š—•‰‹˜‹‰”‹•‡–‘–Š‡–”— ƒ–‡†hac1 ͳͶͲǤ—””‡ ‡–‹†‡–‹ˆ‹ ƒ–‹‘‘ˆƒcisǦƒ –‹‰‡Ž‡‡–‹–Š‡ͷǯ‘ˆ–Š‡A.nigerhacA. ‰‡‡•—’’‘”–•–Š‹•Š›’‘–Š‡•‹•ͳͲͲǤ A. niger ƒ  ‹• ‘•– ’”‘„ƒ„Ž› ‹˜‘Ž˜‡† ‹ –Š‡ —’”‡‰—Žƒ–‹‘ ‘ˆ ‹–• ‘™ ‰‡‡ǡ ƒ• ’—”‹ˆ‹‡† ƒ  ’”‘–‡‹ ™ƒ• ƒ„Ž‡ –‘ „‹† –‘ –Š‡ hacA ’”‘‘–‡” ȋ ‹‰Ǥ ͻȌ ‘”‡‘˜‡”ǡ ‘˜‡”Ǧ ‡š’”‡••‹‘‘ˆhacAi‘–‘Ž›Ž‡†–‘—’Ǧ”‡‰—Žƒ–‹‘‘ˆbipAǡcypBƒ†pdiAǡ„—–ƒŽ•‘Ž‡†–‘–Š‡ ƒ ——Žƒ–‹‘ ‘ˆ hacAu  ȋ ‹‰Ǥ ͺȌǤ Š‹• ‹• ‹ ƒ‰”‡‡‡– ™‹–Š –Š‡ ˆ‹†‹‰ –Šƒ– ‹ ƒƒŽ‹ƒ ‡ŽŽ•–Š‡–”ƒ• ”‹’–‹‘‘ˆXBP1‹•—’Ǧ”‡‰—Žƒ–‡†„›ͳ’”‘–‡‹—’‘–Š‡‘•‡– ‘ˆ–Š‡ͳͺ͸Ǥ

(79)  ‘ Ž—•‹‘ǡ–Š‡•‡˜‡”ƒŽŽ‹‡•‘ˆ‡˜‹†‡ ‡’”‡•‡–‡†Š‡”‡•–”‘‰Ž›‹†‹ ƒ–‡–Šƒ––Š‡ hacA‰‡‡‹A.niger‡ ‘†‡•ƒ–”ƒ• ”‹’–‹‘ˆƒ –‘”–Šƒ–”‡‰—Žƒ–‡•–Š‡Ǥ. IsolationandCharacterisationoftheTranscriptionFactorHacA57.

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