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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)  Chapter4     AHacADependentTranscriptional SwitchReleaseshacAmRNA fromaTranslationalBlock uponEndoplasmic Reticulum Stress    ƒ” Ǥ—Ž†‡”ǡ

(3) ‰‘”‹‘Žƒ‡˜          —„Ž‹•Š‡†‹—ƒ”›‘–‹ ‡ŽŽǤʹͲͲͻǡͺȋͶȌǣ͸͸ͷǦ͸͹ͷ .

(4) Chapter4. Abstract   –‹˜ƒ–‹‘ ‘ˆ –Š‡ —ˆ‘Ž†‡† ’”‘–‡‹ ”‡•’‘•‡ ȋȌ ‹ ‡—ƒ”›‘–‡• ‹˜‘Ž˜‡• •’Ž‹ ‹‰ ‘ˆ ƒ — ‘˜‡–‹‘ƒŽ ‹–”‘ ˆ”‘ –Š‡  ‡ ‘†‹‰ –Š‡ –”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘” ‘ˆ –Š‡ ’ƒ–Š™ƒ›Ǥ

(5) Saccharomycescerevisiaeƒʹͷʹ–— ‘˜‡–‹‘ƒŽ‹–”‘‹••’Ž‹ ‡†‘—–‘ˆ–Š‡ –”ƒ• ”‹’–‘ˆHAC1, Šƒ‰‹‰–Š‡͵ǯ‡†‘ˆ–Š‡HAC1 ƒ†”‡Ž‹‡˜‹‰–Š‡–”ƒ• ”‹’–ˆ”‘ƒ –”ƒ•Žƒ–‹‘ƒŽ„Ž‘ ‹ƒ•‹‰Ž‡•–‡’ǤŠ‡–”ƒ•Žƒ–‹‘ƒŽ„Ž‘ ‹• ƒ—•‡†„›„ƒ•‡Ǧ’ƒ‹”‹‰‘ˆ’ƒ”– ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ™‹–Š –Š‡ ͷǯǤ

(6)  Aspergillus niger ƒ† ‘–Š‡” Aspergilli –Š‡ — ‘˜‡–‹‘ƒŽ‹–”‘‹hacA‹•‘Ž›ʹͲ–Ž‘‰Ǥ‹ ‡–Š‹•‹–”‘‹•’ƒ”–‘ˆƒ•–ƒ„Ž‡ •–‡ǦŽ‘‘’•–”— –—”‡ǡ„ƒ•‡Ǧ’ƒ‹”‹‰™‹–Š–Š‡ͷ̵ǡ‹ ‘–”ƒ•––‘–Š‡›‡ƒ•–HAC1 ƒ•‡‹•‘– ’‘••‹„Ž‡Ǥ ‘™‡˜‡”ǡƒƒŽ›•‹•‘ˆ–Š‡hacA”‡˜‡ƒŽ‡†ƒ ”‹ Š‹˜‡”–‡†”‡’‡ƒ–ȋͳͺ„ƒ•‡ ’ƒ‹”‹‰•ȌǤ’‘ƒ –‹˜ƒ–‹‘‘ˆ–Š‡ǡ–Š‡ͷ̵‘ˆ–Š‡hacA‹•–”— ƒ–‡†„›ʹ͵Ͳ–ǡ ”‡‘˜‹‰ –Š‡ Ž‡ˆ– ’ƒ”– ‘ˆ –Š‹• ‹˜‡”–‡† ”‡’‡ƒ–Ǥ Š‹• ‹’Ž‹‡• ƒ •‹‹Žƒ” ”‡Ž‡ƒ•‡ ‘ˆ ƒ –”ƒ•Žƒ–‹‘ƒŽ„Ž‘ ƒ•‹–Š‡ ƒ•‡‘ˆ–Š‡S.cerevisiaeHAC1,„—–‹–™‘•–‡’•ǤŠ‡‡ Šƒ‹• „‡Š‹† –Š‡ ͷ̵Ǧ–”— ƒ–‹‘ǡ ™Š‹ Š †‘‡• ‘– –ƒ‡ ’Žƒ ‡ ‹ ‡‹–Š‡” ›‡ƒ•– HAC1 ‘” ƒƒŽ‹ƒ XBP1ǡŠƒ•„‡‡—‘™Š‹–Š‡”–‘Ǥ ‡”‡™‡•Š‘™–Šƒ–†—”‹‰•‡ ”‡–‹‘•–”‡••‹A. nigerǡ hacA –”ƒ• ”‹’–‹‘ •–ƒ”–• ˆ”‘ ƒ ‡™ •–ƒ”– •‹–‡ Ž‘•‡” –‘ –Š‡  ǡ ”‡Ž‹‡˜‹‰ –Š‡ –”ƒ• ”‹’–ˆ”‘–”ƒ•Žƒ–‹‘ƒŽƒ––‡—ƒ–‹‘ǤŠ‹•–”ƒ• ”‹’–‹‘ƒŽ•™‹– Š‹•‡†‹ƒ–‡†„› ƒ  ‹–•‡Žˆƒ†–Š‡ ƒ „‹†‹‰•‹–‡ʹ‹–Š‡hacA’”‘‘–‡”Ǥ  . Introduction 

(7)  ‡—ƒ”›‘–‹  ‡ŽŽ• –Š‡ ƒŒ‘”‹–› ‘ˆ –Š‡ •‡ ”‡–‡† ƒ† ‡„”ƒ‡ ’”‘–‡‹• ƒ”‡ ˆ‘Ž†‡†ǡ ƒ••‡„Ž‡†ƒ†‘†‹ˆ‹‡†‹–Š‡‡†‘’Žƒ•‹ ”‡–‹ —Ž—ȋȌǤŠƒ’‡”‘‡•ƒ†ˆ‘Ž†ƒ•‡•ƒ••‹•– ‹ˆ‘Ž†‹‰–Š‡‡™Ž›•›–Š‡•‹•‡†’”‘–‡‹• ͵ͻǡƒ† ‘””‡ –Ž›ˆ‘Ž†‡†’”‘–‡‹•ƒ”‡–”ƒ•’‘”–‡† –‘ –Š‡ ‘Ž‰‹ ‘’ƒ”–‡– ™Š‡”‡ ˆ—”–Š‡” ‘†‹ˆ‹ ƒ–‹‘ –ƒ‡• ’Žƒ ‡Ǥ Š‡ •‡ ”‡–‘”› ’”‘–‡‹• ƒ”‡ –”ƒ•’‘”–‡† ‹ ˜‡•‹ Ž‡• ˆ”‘ –Š‡ ‘Ž‰‹ ƒ’’ƒ”ƒ–—• –‘ –Š‡ ‡ŽŽ ‡„”ƒ‡ ™Š‡”‡ –Š‡ ‘–‡–‘ˆ–Š‡˜‡•‹ Ž‡‹•”‡Ž‡ƒ•‡†‹–‘–Š‡‡†‹—ǤŠ‡–Š‡ƒ‘—–‘ˆ‡™Ž›•›–Š‡•‹•‡† ’”‘–‡‹•‡š ‡‡†•–Š‡ˆ‘Ž†‹‰ ƒ’ƒ ‹–›‘ˆ–Š‡ǡ‘”‹ˆˆ‘Ž†‹‰‹–Š‡‹•‹’ƒ‹”‡†„›‘–Š‡” ‡ƒ•ǡ –Š‡ ‡—ƒ”›‘–‹  ‡ŽŽ –”‹‰‰‡”• ƒ ‹–”ƒ ‡ŽŽ—Žƒ” •‹‰ƒŽŽ‹‰ ’ƒ–Š™ƒ› ‹ ‘”†‡” –‘ ‘—–‡”ƒ ––Š‡”‡•—Ž–‹‰•‡ ”‡–‹‘•–”‡••ǤŠ‹•”‡•’‘•‡‹•‘™ ƒ• –Š‡ —ˆ‘Ž†‡† ’”‘–‡‹ ”‡•’‘•‡ȋȌ͸Ͷǡͳʹ͵ǡͳͷͲǡͳ͹ͻǡƒ†Ž‡ƒ†•–‘–Š‡‹ ”‡ƒ•‡†•›–Š‡•‹•‘ˆ”‡•‹†‡–ˆ‘Ž†ƒ•‡•ƒ† Šƒ’‡”‘‡• ƒ• ™‡ŽŽ ƒ• ƒ› ‘–Š‡” ‘’‘‡–• ‘ˆ –Š‡ •‡ ”‡–‘”› ’ƒ–Š™ƒ› ͳ͸ͲǤ ‡‘‹  ƒƒŽ›•‹•‘ˆ–Š‡‹S.cerevisiaeǡArabidopsisthalianaǡA.nidulansǡT.reeseiǡƒ†A.niger ƒŽŽ Ž‹‡† Žƒ”‰‡ •‡–• ‘ˆ ‰‡‡• ȋ•‡˜‡”ƒŽ Š—†”‡†•Ȍ –‘ –Š‡ ǡ ƒˆˆ‡ –‹‰ ƒŽŽ •–ƒ‰‡• ‘ˆ –Š‡ •‡ ”‡–‘”›’ƒ–Š™ƒ› ͶǡͶͺǡͺͻǡͳͷͶǡͳ͸ͲǤŠ‡ ‡–”ƒŽƒ –‹˜ƒ–‹‘•–‡’‘ˆ–Š‡‹˜‘Ž˜‡•–Š‡•’Ž‹ ‹‰. 84ThehacATranscriptionalSwitch.

(8) Chapter4 ‘ˆƒ— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘–Š‡ ‡ ‘†‹‰–Š‡–”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘” ‘ˆ–Š‡ Ǥ

(9) – ‹• ‘•‡”˜‡† ˆ”‘ Ž‘™‡” ‡—ƒ”›‘–‡• Ž‹‡ ›‡ƒ•–•ǡ –‘ Š‹‰Š‡” ‡—ƒ”›‘–‡• Ž‹‡ ƒƒŽ‹ƒ ‡ŽŽ•ƒ†‡†‹ƒ–‡†„›

(10) ”‡ͳ’Ȁ

(11) ”‡ǡƒ–”ƒ•‡„”ƒ‡‹ƒ•‡Ȁƒ•‡”‡•‹†‹‰‹ –Š‡‡„”ƒ‡Ǥ‹–Š‹–•Ž—‹ƒŽ†‘ƒ‹

(12) ”‡ͳ’Ȁ

(13) ”‡•‡•‡•–Š‡Ǯˆ‘Ž†‹‰•–ƒ–‡ǯ‘ˆ’”‘–‡‹• ‹–Š‡ǡƒ†™Š‡‡ ‡••ƒ”›ƒ•‹‰ƒŽ‹•–”ƒ•‹––‡†‘˜‡”–Š‡‡„”ƒ‡„›ƒ –‹˜ƒ–‹‘ ‘ˆ–Š‡ƒ•‡†‘ƒ‹™Š‹ Š‹–—”’”‘ ‡••‡•–Š‡‡••‡‰‡”‡ ‘†‹‰–Š‡–”ƒ• ”‹’–‹‘ƒŽ ƒ –‹˜ƒ–‘”‘ˆ–Š‡Ǥ

(14)  S. cerevisiae,

(15) ”‡ͳ’Ǧ‡†‹ƒ–‡† •’Ž‹ ‹‰ ‘ˆ ƒ ʹͷʹ – — ‘˜‡–‹‘ƒŽ ‹–”‘ ˆ”‘ HAC1”‡Ž‹‡˜‡•–Š‡–”ƒ• ”‹’–ˆ”‘ƒ–”ƒ•Žƒ–‹‘ƒŽ„Ž‘  ͸͸ǡͳ͵͹ǤHAC1‡ ‘†‡•ƒ„ƒ•‹  Ž‡— ‹‡Ǧœ‹’’‡” –›’‡ –”ƒ• ”‹’–‹‘ ˆƒ –‘” –Šƒ– ƒ –‹˜ƒ–‡• ‹–• –ƒ”‰‡– ‰‡‡• „› ƒ †‹”‡ – ‹–‡”ƒ –‹‘ ™‹–Š —’•–”‡ƒ ƒ –‹˜ƒ–‹‰ •‡“—‡ ‡• ȋȌǤ

(16)  ƒƒŽ‹ƒ ‡ŽŽ•ǡ –Š‡ HAC1 Š‘‘Ž‘‰—‡ XBP1 ‹• ƒ –‹˜ƒ–‡† ‹ ƒ •‹‹Žƒ” ™ƒ› „› –Š‡ •’Ž‹ ‹‰ ‘ˆ ƒ ʹ͸ – — ‘˜‡–‹‘ƒŽ ‹–”‘ ͺͳǡ™Š‡”‡ƒ•–Š‡Trichodermareeseihac1ǡ–Š‡Aspergillusnidulansƒ†A.nigerhacA ƒ”‡ ƒ –‹˜ƒ–‡† „› –Š‡ •’Ž‹ ‹‰ ‘ˆ ƒ ʹͲ – — ‘˜‡–‹‘ƒŽ ‹–”‘ ͳͲͳǡͳͶͲǤ ‘ †‹•–‹‰—‹•Š –Š‡ —•’Ž‹ ‡†hacAˆ‘”–Š‡•’Ž‹ ‡†ˆ‘”ǡ–Š‡›ƒ”‡”‡ˆ‡””‡†–‘ƒ•hacAuȋ—‹†— ‡†Ȍƒ† hacAiȋ‹†— ‡†Ȍ”‡•’‡ –‹˜‡Ž›Ǥ ƒ‹‰ ‹–‘ ƒ ‘—– –Š‡ ƒ‰‹–—†‡ ‘ˆ –Š‡ ǡ ƒ† –Š‡ ”‹• –Šƒ– ƒ ——Žƒ–‹‘ ‘ˆ —ˆ‘Ž†‡†’”‘–‡‹•‹–Š‡’‘•‡‘–‘–Š‡ ‡ŽŽǡ‹–•‡‡•’Žƒ—•‹„Ž‡–Šƒ–ƒ–—”ƒŽ•‡Ž‡ –‹‘Šƒ• ˆƒ˜‘—”‡†•–”‹ – ‘–”‘ŽŽƒ„‹Ž‹–›‘ˆ–Š‡’ƒ–Š™ƒ›ǤŽ–Š‘—‰Š–Š‡ƒ†‹–• ‡–”ƒŽƒ –‹˜ƒ–‹‘ •–‡’ ƒ”‡ ‘•‡”˜‡† ‹ ƒŽŽ ‡—ƒ”›‘–‹  ‘”‰ƒ‹••ǡ –Š‡”‡ ƒ”‡ †‹ˆˆ‡”‡ ‡• ‹ –Š‡ ‘Ž‡ —Žƒ” ‡ Šƒ‹••—†‡”Ž›‹‰–Š‡•‹‰ƒŽŽ‹‰’ƒ–Š™ƒ›ǡ–Šƒ–‰‹˜‡–Š‡†‹ˆˆ‡”‡–‘”‰ƒ‹••†‹ˆˆ‡”‡– ™ƒ›• ‘ˆ ‘–”‘Ž ‘˜‡” –Š‡ ”‡•’‘•‡Ǥ

(17)  ƒƒŽ‹ƒ ‡ŽŽ• –Š‡  ‘•‹•–• ‘ˆ –Š”‡‡ †‹•–‹ – ’ƒ–Š™ƒ›•–Šƒ–‘’‡”ƒ–‡‹’ƒ”ƒŽŽ‡Žǡƒ†‹ Ž—†‡‰‡‡”ƒŽƒ––‡—ƒ–‹‘‘ˆ–”ƒ•Žƒ–‹‘ǡƒ’‘’–‘•‹•ǡ ƒ† ‹ ”‡ƒ•‡† ˆ‘Ž†‹‰ ƒ† †‡‰”ƒ†ƒ–‹‘ ‘ˆ —ˆ‘Ž†‡† ’”‘–‡‹• ͷͲǤ Š‡ ƒƒŽ‹ƒ  ‹• –”‹‰‰‡”‡†„›ƒ–Ž‡ƒ•––Š”‡‡†‹ˆˆ‡”‡–•–”‡•••‡•‘”•ǡƒŽŽ–”ƒ•‡„”ƒ‡’”‘–‡‹•ǤŠ‹• ‘ ‡”•ǣͳȌ

(18) ͳǡƒ— Ž‡ƒ•‡™Š‹ Š•’Ž‹ ‡•–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘XBP1ͳͺ͸Ǥ ʹȌ  ͸ǡ –Šƒ– ‹‰”ƒ–‡• –‘ –Š‡ ‘Ž‰‹ ‹ ”‡•’‘•‡ –‘  •–”‡••Ǥ

(19)  –Š‡ ‘Ž‰‹ǡ ‹–• ›–‘•‘Ž‹  †‘ƒ‹ǡ–Šƒ–‡ ‘†‡•ƒ–”ƒ• ”‹’–‹‘ˆƒ –‘”ǡ‹•’”‘–‡‘Ž›–‹ ƒŽŽ›”‡Ž‡ƒ•‡†ƒ†”‡Ž‘ ƒ–‡†–‘–Š‡ — Ž‡—•™Š‡”‡‹–—’Ǧ”‡‰—Žƒ–‡•‹–•–ƒ”‰‡–‰‡‡•ͷͶǤ͵Ȍǡƒ’”‘–‡‹‹ƒ•‡–Šƒ–‹”‡•’‘•‡ –‘  •–”‡•• ’Š‘•’Š‘”›Žƒ–‡• –Š‡ –”ƒ•Žƒ–‹‘ ‹‹–‹ƒ–‹‘ ˆƒ –‘”ǦʹDǡ ”‡•—Ž–‹‰ ‹ ‰‡‡”ƒŽ ‹Š‹„‹–‹‘‘ˆ–”ƒ•Žƒ–‹‘ͷʹǤ

(20)  ‘–”ƒ•––‘ƒƒŽ‹ƒ ‡ŽŽ•ǡ‹S.cerevisiae–Š‡‘Ž›‘™•‡•‘”‘ˆ–Š‡‹•

(21) ”‡ͳ’Ǥ

(22)  ƒ††‹–‹‘ –‘ –Š‡ ‘Ȁ‘ˆˆ •™‹– Š ‘ˆ –Š‡ ›‡ƒ•– ǡ •‡˜‡”ƒŽ ‘–Š‡” ˆƒ –‘”• ‹ˆŽ—‡ ‹‰ –Š‡  ‹” —‹– Šƒ˜‡ „‡‡ †‡• ”‹„‡†Ǥ Š‡ –”ƒ• ”‹’–‹‘ ‘ˆ HAC1 ™ƒ• •Š‘™ –‘ „‡ ƒ—–‘Ǧ ”‡‰—Žƒ–‡† ͳͳʹǡ ƒ† –”ƒ•Žƒ–‹‘ ‘ˆ –Š‡ —•’Ž‹ ‡† HAC1u  „Ž‘ ‡† „› Ž‘‰Ȃ”ƒ‰‡ „ƒ•‡ ’ƒ‹”‹‰ ͳ͵͹Ǥ —”–Š‡”‘”‡ǡ ƒ •‡ ‘† Ǧ–‘Ǧ— Ž‡—• •‹‰ƒŽŽ‹‰ ’ƒ–Š™ƒ› ™ƒ• ˆ‘—†ǡ ™Š‹ Š Ž‡ƒ†• –‘ ƒ ‹ ”‡ƒ•‡† ’”‘†— –‹‘ ‘ˆ ƒ ͳ’ ‘„‹‡† ™‹–Š ’”‘†— –‹‘ ‘” ƒ –‹˜ƒ–‹‘ ‘ˆ ƒ ’—–ƒ–‹˜‡‘†—Žƒ–‹‰ˆƒ –‘” ͹ͻǤ‘–Š•‹‰ƒŽ•ƒ”‡”‡“—‹”‡†ˆ‘”–Š‡•—’‡”Ǧƒ –‹˜ƒ–‹‘Ǥ ThehacATranscriptionalSwitch85.

(23) Chapter4 ‹ƒŽŽ›ǡ Ͷ’ƒ†‹–•ƒ –‹˜ƒ–‘” ʹ’™‡”‡ˆ‘—†–‘„‡”‡“—‹”‡†ˆ‘”–Š‡‹†— –‹‘‘ˆ–Š‡ ƒŒ‘”‹–› ‘ˆ –Š‡  –ƒ”‰‡– ‰‡‡• ͳʹͶǤ

(24) –‡‰”ƒ–‡† ™‹–Š –Š‡

(25) ”‡ͳ’ ’ƒ–Š™ƒ›ǡ –Š‡•‡ ‡Ž‡‡–• –—”–Š‡ˆ”‘ƒ•‹’Ž‡•™‹– Š‹–‘ƒ‘”‡ ‘’Ž‡š”‡•’‘•‡ǡƒ†‰‹˜‡–Š‡›‡ƒ•– ‡ŽŽƒ ‘”‡ˆ‹‡Ǧ–—‡† ‘–”‘Ž‘˜‡”–Š‡Ǥ Š‡‹A.niger•Šƒ”‡••‘‡ Šƒ”ƒ –‡”‹•–‹ •™‹–Š–Š‡S.cerevisiae”‡•’‘•‡ǡ„—– –Š‡”‡ ƒ”‡ •‘‡ †‹ˆˆ‡”‡ ‡•Ǥ • •ƒ’”‘’Š›–‹  ‘”‰ƒ‹••ǡ ˆ‹Žƒ‡–‘—• ˆ—‰‹ ƒ”‡ ƒ’ƒ„Ž‡ ‘ˆ •‡ ”‡–‹‰ Žƒ”‰‡ ƒ‘—–• ‘ˆ ’”‘–‡‹• –‘ ”‡ ”—‹– —–”‹‡–•Ǥ Š‹• Šƒ”ƒ –‡”‹•–‹  ‹‰Š– Šƒ˜‡ •Šƒ’‡†–Š‡•‡ ”‡–‘”›’ƒ–Š™ƒ›ƒ†Šƒ˜‡•‡–•’‡ ‹ˆ‹ †‡ƒ†•–‘–Š‡•–”‡••”‡•’‘•‡•ǡ™Š‹ Š ‹‰Š– ‡š’Žƒ‹ –Š‡ †‹ˆˆ‡”‡ ‡• ™‹–Š –Š‡ S. cerevisiae Ǥ ‹”•–Ž›ǡ ‹ ƒ††‹–‹‘ –‘ –Š‡ — ‘˜‡–‹‘ƒŽ‹–”‘•’Ž‹ ‹‰ǡ–Š‡ͷǯǦ—–”ƒ•Žƒ–‡†”‡‰‹‘ȋͷǯȌ‘ˆ–Š‡hacA–”ƒ• ”‹’–‘ˆ A. nigerǡ A. nidulans, ƒ† T. reesei ‹• –”— ƒ–‡† —’‘  •–”‡•• ͳͲͳǡͳͶͲǤ Š‹• Šƒ• ‘– „‡‡ ”‡’‘”–‡†ˆ‘”–Š‡›‡ƒ•–HAC1‘”–Š‡ƒƒŽ‹ƒXBP1Š‘‘Ž‘‰—‡•Ǥ‡ ‘†Ž›ǡƒ‘˜‡Ž–›’‡‘ˆ –”ƒ• ”‹’–‹‘ƒŽ†‘™”‡‰—Žƒ–‹‘‡ Šƒ‹•Šƒ•„‡‡†‡• ”‹„‡†‹ˆ‹Žƒ‡–‘—•ˆ—‰‹ǡ™Š‹ Š ™ƒ• –‡”‡† ”‡’”‡••‹‘ —†‡” •‡ ”‡–‹‘ •–”‡•• ȋȌ. ͳǡͳͳͺǤ. ’‘  •–”‡••ǡ ‹– †‘™. ”‡‰—Žƒ–‡•–Š‡–”ƒ• ”‹’–‹‘‘ˆ•‘‡‰‡‡•‡ ‘†‹‰•‡ ”‡–‡†’”‘–‡‹•ǡ–Š‡”‡„›Ž‘™‡”‹‰–Š‡ „—”†‡ ‘ –Š‡ Ǥ Š‹”†Ž›ǡ –Š”‡‡• ƒ”‡ ’”‡•‡– ‹ –Š‡hacA ’”‘‘–‡”• ‘ˆ A. niger, A. nidulansƒ†A.fumigatus ͳͲͲǤŠ‡•‡‡Ž‡‡–• ‘—Ž†’Žƒ›ƒ”‘Ž‡‹–Š‡—’”‡‰—Žƒ–‹‘‘ˆhacA „›–Š‡ƒ –‹˜ƒ–‘”’”‘–‡‹‹–•‡Žˆǡ™Š‹ Š™ƒ•‡š’‡”‹‡–ƒŽŽ›•Š‘™‹A.nigerͳͲͳǤŠ‡’‘•‹–‹‘ ‘ˆ–Š‡•‹–Š‡hacA’”‘‘–‡”‹”‡Žƒ–‹‘–‘•–ƒ”–•‹–‡•‘ˆ–Š‡ˆ—ŽŽǦŽ‡‰–Šƒ†–”— ƒ–‡† hacA ‘—Ž†ƒŽ•‘‹’Ž‹ ƒ–‡ƒ”‘Ž‡ˆ‘”–Š‡‹–Š‡–”— ƒ–‹‘‘ˆ–Š‡hacA–”ƒ• ”‹’–Ǥ ‡”‡ ™‡ •Š‘™ –Š‡ ‡ Šƒ‹• „‡Š‹† –Š‡ –”— ƒ–‹‘ ‘ˆ –Š‡ ͷǯ ‘ˆ –Š‡ A. niger hacAǡƒ†–Š‡‹˜‘Ž˜‡‡–‘ˆ–Š‡•‹–Š‡”‡‰—Žƒ–‹‘‘ˆ–Š‡”‡•’‘•‡Ǥ.   MaterialandMethods  StrainsandTransformations A. niger ͶǤͳ ȋcspA1, pyrGȌǡ ƒ† –”ƒ•ˆ‘”ƒ–• –Š‡”‡‘ˆ ™‡”‡ ‰”‘™ ‘ ‹‹ƒŽ ‡†‹ƒ ’Žƒ–‡• ‘–ƒ‹‹‰’‡”Ž‹–”‡ǣ͸‰ƒ͵ǡͳǤͷ‰ ʹͶǡͲǤͷ‰‰Ͷ˜͹ ʹǡͲǤͷ‰Žǡ–”ƒ ‡ ‡Ž‡‡–• ͳ͹ʹǡͳǤͷΨƒ‰ƒ”ǡƒ†ʹͲ‰ˆ”— –‘•‡ƒ•ƒ ƒ”„‘•‘—” ‡ȋ’ ͸ǤͲȌǤͷ—”‹†‹‡™ƒ• ƒ††‡† ‹ ƒ•‡ ‘ˆ ƒ—š‘–”‘’Š›Ǥ ”‘–‘’Žƒ•–• ™‡”‡ –”ƒ•ˆ‘”‡† ƒ ‘”†‹‰ –‘ •–ƒ†ƒ”† ’”‘ ‡†—”‡•. ͹͸Ǥ. Agrobacterium tumefaciens ‡†‹ƒ–‡† –”ƒ•ˆ‘”ƒ–‹‘ ‘ˆ A. niger ™ƒ•. ’‡”ˆ‘”‡† ƒ ‘”†‹‰ –‘ ’”‡˜‹‘—•Ž› †‡• ”‹„‡† ‡–Š‘†• ʹͷǤ ”ƒ•ˆ‘”ƒ–‹‘ ’Žƒ–‡• ™‡”‡ ‹ —„ƒ–‡†ƒ–ͳͺ‘Ǥ  . 86ThehacATranscriptionalSwitch.

(26) Chapter4 ConstructionofanA.nigerhacAStrain ͶǤͷ„ˆ”ƒ‰‡–ǡ ‘–ƒ‹‹‰hacA™‹–Šʹ„‘ˆ‹–•ͷǯˆŽƒ‹‰”‡‰‹‘ƒ†ͳǤͳ„͵ǯˆŽƒ‹‰ ”‡‰‹‘ǡ™ƒ•ƒ’Ž‹ˆ‹‡†ˆ”‘A.niger‰‡‘‹ —•‹‰’”‹‡”•ͳƒ†ʹǡƒ† Ž‘‡† ‹–‘ ’ǦŽ—–

(27)

(28) Ǧ ȋ

(29) ˜‹–”‘‰‡ȌǤ Š‡ ”‡•—Ž–‹‰ ’Žƒ•‹† ’ ͷͺ ™ƒ• —– ™‹–Š BglIIǡ ˆ‹ŽŽ‡†Ǧ‹™‹–ŠŽ‡‘™ǡƒ† —–™‹–ŠSalI.Š‹•”‡‘˜‡†–Š‡ƒŒ‘”’ƒ”–‘ˆ–Š‡hacA‰‡‡ˆ”‘ –Š‡’Žƒ•‹†ǡ™Š‹ Š™ƒ•”‡’Žƒ ‡†™‹–Š–Š‡hygB ƒ••‡––‡ˆ”‘’͹Ǧͳȋƒ ‡••‹‘—„‡”ǣ ͵ʹ͸ͻͺȌǤ ‘”–Š‹•’—”’‘•‡ǡ’͹Ǧͳ™ƒ• —–™‹–Š XbaIǡˆ‹ŽŽ‡†Ǧ‹™‹–ŠŽ‡‘™ƒ† —–™‹–Š XhoIǤ ”‘–Š‡”‡•—Ž–‹‰’Žƒ•‹†ǡƒ͸ǡ͸„KpnIXhoIˆ”ƒ‰‡–™ƒ•‹•‘Žƒ–‡†ǡ ‘–ƒ‹‹‰–Š‡ hygB ƒ••‡––‡ˆŽƒ‡†„›–Š‡—’•–”‡ƒƒ††‘™•–”‡ƒ”‡‰‹‘‘ˆhacAǤŠ‡ˆ”ƒ‰‡–™ƒ• Ž‘‡†„‡–™‡‡–Š‡Ž‡ˆ–Ǧƒ†”‹‰Š–„‘”†‡”‘ˆ–Š‡A.tumefaciens’Žƒ•‹†’ͳͶ ͳͳͳǤ Š‹• ”‡•—Ž–‡†‹–Š‡’Žƒ•‹†’ ͸Ͳǡ–Šƒ–™ƒ•—•‡†ˆ‘”–Š‡”‡’Žƒ ‡‡–‘ˆhacA‹A.niger„›A. tumefaciens‡†‹ƒ–‡†–”ƒ•ˆ‘”ƒ–‹‘Ǥ   ConstructionComplementationPlasmid ͵ǡʹ„FspIBamHIˆ”ƒ‰‡–ˆ”‘’Žƒ•‹†’ ͷͺ™ƒ•Ž‹‰ƒ–‡†‹–‘–Š‡HincIIBamHI•‹–‡• ‘ˆ’Ž—‡• ”‹’–Ϊǡ”‡•—Ž–‹‰‹’Žƒ•‹†’ ƒ Ǥ ‘”•‡Ž‡ –‹˜‡–ƒ”‰‡–‹‰‘ˆ–Š‡’Žƒ•‹†–‘–Š‡ pyrG Ž‘ —• ‘ˆ A. nigerǡ –Š‡ ͵Ǥͻ „ XbaI ˆ”ƒ‰‡– ‘ˆ ’’›” ȗ‘– ȋ‹†Ž› ’”‘˜‹†‡† „› ”Ǥ ‡–‡”—–ǡ‡‹•–ǡŠ‡‡–Š‡”Žƒ†•Ȍ™ƒ•‹•‡”–‡†‹–‘–Š‡SpeI•‹–‡‘ˆ’ ƒ ǡ”‡•—Ž–‹‰ ‹’Žƒ•‹†’ ƒ Ǧ’›” ȗǤŠ‡pyrG*‰‡‡ ‘–ƒ‹•ƒˆ‹ŽŽ‡†Ǧ‹BamHI•‹–‡†‹•”—’–‹‰–Š‡ Ǥ Ž› Š‘‘Ž‘‰‘—• ‹–‡‰”ƒ–‹‘ ‘ˆ –Š‡ ’Žƒ•‹† ƒ– –Š‡ pyrG Ž‘ —• ™‹ŽŽ ”‡•–‘”‡ –Š‡ —”‹†‹‡ ’”‘–‘–”‘’Š›ǡ‡ƒ„Ž‹‰•‡Ž‡ –‹˜‡–ƒ”‰‡–‹‰‘ˆ–Š‡ ‘•–”— ––‘–Š‡pyrGŽ‘ —•Ǥ   SiteDirectedMutagenesis ‘”—–ƒ‰‡‡•‹•’—”’‘•‡•‘ˆ–Š‡‡Ž‡‡–•’”‡•‡–‹–Š‡hacA’”‘‘–‡”ƒ†ͷǯ‘ˆ–Š‡ hacAǡƒ͸ʹ͵„’ˆ”ƒ‰‡– ‘–ƒ‹‹‰–Š‡•‡‡Ž‡‡–•™ƒ•ƒ’Ž‹ˆ‹‡†ˆ”‘’ ƒ  ™‹–Š ’”‹‡” ’ƒ‹” ͵ ƒ† Ͷǡ ƒ† Ž‘‡† ‹–‘ ’ǦŽ—–

(30)

(31) Ǧ ȋ

(32) ˜‹–”‘‰‡Ȍǡ ”‡•—Ž–‹‰ ‹ ’ǦŽ—–ǦŠƒ Ǥ‹–‡Ǧ•’‡ ‹ˆ‹ —–ƒ–‹‘•™‡”‡‹–”‘†— ‡†‹–‘–Š‡˜ƒ”‹‘—•‡Ž‡‡–•—•‹‰ –Š‡ —‹  Šƒ‰‡ •‹–‡ †‹”‡ –‡† —–ƒ‰‡‡•‹• ‹– ȋ–”ƒ–ƒ‰‡‡ȌǤ Š‡ ’”‹‡”• ƒ”‡ Ž‹•–‡† ‹ ƒ„Ž‡ͳǤŠ‡—–ƒ–‹‘•™‡”‡˜‡”‹ˆ‹‡†„›•‡“—‡ ‹‰’‡”ˆ‘”‡†™‹–Š–Š‡‹‰†›‡•‡“—‡ ‹‰ ‹–ȋ’’Ž‹‡†‹‘•›•–‡•Ȍƒ†ƒƒŽ›•‡†™‹–Š–Š‡

(33) ”‹•͵ͳͲͲ ‡‡–‹ ƒŽ›•‡”ȋ’’Ž‹‡† ‹‘•›•–‡•ȌǤͶͻ͵„’BstEIIAspIˆ”ƒ‰‡–‘ˆ’ ƒ ™ƒ•”‡’Žƒ ‡†™‹–Š–Š‡ ‘””‡•’‘†‹‰ „—–—–ƒ–‡†ˆ”ƒ‰‡–ˆ”‘–Š‡’ǦŽ—–ǦŠƒ ’Žƒ•‹†Ǥ Š‡— ‘˜‡–‹‘ƒŽ‹–”‘™ƒ•”‡‘˜‡†„›†‹‰‡•–‹‰’ ƒ ™‹–ŠPstIˆ‘ŽŽ‘™‡†„› ”‡ǦŽ‹‰ƒ–‹‘ǤŠ‡hacA˜ƒ”‹ƒ–™‹–Š–Š‡—•’Ž‹ ‡ƒ„Ž‡— ‘˜‡–‹‘ƒŽ‹–”‘™ƒ• ‘•–”— –‡† „› Ž‹‰ƒ–‹‘ ‘ˆ –Š‡ ƒ‡ƒŽ‡† ‘Ž‹‰‘— Ž‡‘–‹†‡ ’ƒ‹” ͳͲǦͳͳ ‹–‘ –Š‡ PstI †‹‰‡•–‡† ’ ƒ  ’Žƒ•‹†Ǥ ‘” —–ƒ–‹‘ ‘ˆ –Š‡ Ž‘‰ ”ƒ‰‡ ‹˜‡”–‡† ”‡’‡ƒ–•ǡ ƒ ͳ͹ͷ͸ „’  ˆ”ƒ‰‡– ™ƒ• ThehacATranscriptionalSwitch87.

(34) Chapter4 ‰‡‡”ƒ–‡† ‹ •‡˜‡”ƒŽ •— ‡••‹˜‡  ƒ† ƒ••‡„Ž› •–‡’•ǡ ™Š‹ Š ‹˜‘Ž˜‡† ’”‹‡”• ͵ǡ Ͷǡ ͳʹǡ ͳ͵ǡ ͳͶǡ ƒ† ͳͷ ȋƒ„Ž‡ ͳȌǤ Š‡ ”‡•—Ž–‹‰ ˆ”ƒ‰‡– ™ƒ• Ž‘‡† ‹–‘ ’ǦŽ—–

(35)

(36) Ǧ ǡƒ†ƒˆ–‡”•‡“—‡ ‹‰ƒͳ͵ͷͳ„’BstEIIBglIIˆ”ƒ‰‡– Ž‘‡†‹–‘’ ƒ Ǥ ˆ–‡”‹–”‘†— –‹‘‘ˆ–Š‡—–ƒ–‹‘•‹’ ƒ ǡ–Š‡XbaIpyrG*ˆ”ƒ‰‡–™ƒ• Ž‘‡†‹–‘–Š‡ SpeI •‹–‡ ‘ˆ ’ ƒ Ǥ ‘•–”— –• Šƒ˜‹‰ –Š‡ pyrGȗ ‰‡‡ ‹ –Š‡ •ƒ‡ ‘”‹‡–ƒ–‹‘ ƒ• –Š‡ ™‹Ž† –›’‡’ ƒ Ǧ’›” ȗ’Žƒ•‹†™‡”‡•‡Ž‡ –‡†ˆ‘”–”ƒ•ˆ‘”ƒ–‹‘–‘A.nigerhacAǤ   EMSA ͳͲͲ ’‘Ž ‘Ž‹‰‘— Ž‡‘–‹†‡ ’”‹‡”• ™‡”‡ ƒ‡ƒŽ‡† ‹ ͳͲͲ ɊŽ ͳͲ  

(37) Ǧ Ž ’  ͹Ǥͷ ‘–ƒ‹‹‰ͳͲͲƒŽǡ„›Š‡ƒ–‹‰–Š‡‹š–—”‡•–‘ͻͷιˆ‘”ͷ‹ƒ† ‘‘Ž‹‰–Š‡†‘™ –‘”‘‘–‡’‡”ƒ–—”‡„›’Žƒ ‹‰–Š‡–—„‡•‘–Š‡™‘”„‡ ŠǤͳ’‘Ž‘ˆƒ‡ƒŽ‡†‘Ž‹‰‘™ƒ• ‡†ǦŽƒ„‡ŽŽ‡† ‹ ƒ ʹͲ ɊŽ ”‡ƒ –‹‘ ™‹–Š ͷ ɊŽ ɀǦ͵ʹ ƒ† ʹͲ —‹–• Ͷ ‘Ž›— Ž‡‘–‹†‡ ‹ƒ•‡ ȋ‡™‰Žƒ†‹‘Žƒ„•ȌǤŠ‡”‡ƒ –‹‘™ƒ•‹ —„ƒ–‡†ˆ‘”ͳŠƒ–͵͹ιǡƒ†–Š‡”ƒ†‹‘ǦŽƒ„‡ŽŽ‡† ‘Ž‹‰‘— Ž‡‘–‹†‡ ’—”‹ˆ‹‡† ‘ ‹‘‰‡Ž ͵Ͳ •’‹ ‘Ž—• ȋ‹‘ƒ†ȌǤ ‹†‹‰ ”‡ƒ –‹‘• ȋʹͲ ɊŽȌ ™‡”‡’‡”ˆ‘”‡†‹–Š‡ˆ‘ŽŽ‘™‹‰„—ˆˆ‡”ǣʹͷ

(38) Ǧ Žȋ’ ͺǤͲȌǡͷ†‹–Š‹‘–Š”‡‹–‘ŽǡͳͲͲ  Žǡ Ͷ  •’‡”‹†‹‡ǡ ͲǤͳ Ɋ‰ȀɊŽ ’‘Ž›ȏ†ȋ

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

(40) Ǧ Žǡͺͻ ‘”‹ ƒ ‹†ǡʹͲǡ’ ͺǤ͵ȌǤ   SouthernBlotAnalysis. ‡‘‹ ˆ‘”‘—–Š‡”„Ž‘–ƒƒŽ›•‹•™ƒ•‹•‘Žƒ–‡†„›ƒ††‹‰ͺͲͲɊŽ„—ˆˆ‡”ȋͳͲͲ”‹• ’ ͺǤͲǡͷͲǡͳǡ͵ͷȌ–‘Ͳǡͳ‰‘ˆ‰”‘—†› ‡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†‹‰‡•–ͳͲɊ‰‘ˆ‰‡‘‹ ™ƒ•‹ —„ƒ–‡†͸Šƒ–͵͹ι™‹–ŠʹͲ ‘ˆƒƒ’’”‘’”‹ƒ–‡”‡•–”‹ –‹‘‡œ›‡ǤŠ‡”‡ƒ –‹‘’”‘†— –•™‡”‡•‡’ƒ”ƒ–‡†‘ƒͲǤͺΨ ƒ‰ƒ”‘•‡‰‡ŽǤŽ‘––‹‰ƒ†Š›„”‹†‹•ƒ–‹‘™ƒ•†‘‡ƒ ‘”†‹‰–‘•–ƒ†ƒ”†’”‘ ‡†—”‡•ͳͶͳǤ. 88ThehacATranscriptionalSwitch.

(41) Chapter4 Table1.‘Ž‹‰‘— Ž‡‘–‹†‡•—•‡†‹–Š‹••–—†›Ǥ Oligo ͳ ʹ ͵ Ͷ ͷ ͸ ͹ ͺ ͻ ͳͲ ͳͳ ͳʹ ͳ͵ ͳͶ ͳͷ ͳ͸ ͳ͹ ͳͺ ͳͻ ʹͲ. Sequence ͷ̵Ǧ‰‰ƒ‰ƒƒ ƒƒ‰‰–‰ –ƒ––‰ ͷ̵Ǧ‰–––ƒ‰‰ƒ‰ –––ƒ –  ͷ̵Ǧƒ–––ƒ ‰–ƒ ‰‰– ƒƒ––‰‰‰‰  ͷ̵Ǧ –‰ –‰–ƒ –‰ƒ ‰ƒ‰– ƒ–  ͷ̵Ǧ‰‰ –‰ƒ– –‰ƒa ƒ ‰gac‰ ––––ƒƒƒ‰ƒ‰– ͷ̵Ǧ– ‰––ƒ–‰ƒ ƒ ‰‰ac‰ –‰–‰–– –‰– ͷ̵Ǧƒ–‰‰–– ––ƒƒ‰‰ƒ ƒ ac– –– ––‰‰ – ͷ̵Ǧ– –––––ƒ––‰–– –c–‰‰–– ––ƒƒ‰‰ƒ ƒ  ͷ̵Ǧ ‰ƒ –ƒ ƒ– ƒ ‰– – aacgtca‰ ‰‰––ƒƒ‰ƒ–ƒƒ‰‰  – ƒ–ƒ‰–ƒƒƒ– ‰ƒ––‰ ͷ̵Ǧ–agt‰ c‰–cc–c–‰ ‰ƒt –t ƒ ͷ̵Ǧ–‰aƒ‰a– ‰ ƒgƒggƒ g‰ actƒ ͷ̵Ǧ‰ t‰‰gt–a‰ g t‰ aag ‰––ƒ–‰ƒ ƒ ‰‰–‰‰ –‰–‰––  ͷ̵Ǧ‰ƒ a‰‰tƒƒ–– –‰c ƒ–‰ƒ ––– – –– –– ƒ ƒ‰‰ ͷ̵Ǧ– ‰––ƒ–‰ƒ ƒ ‰‰–‰‰ –‰–‰–– –‰– ͷ̵Ǧ– ƒƒƒ ‰ – ƒƒ‰ƒ–– ‰––– ͷ̵Ǧ ‰– ‰ƒ‰ƒƒ ‰– ƒƒƒ‰‰ ‰ƒƒ ‰–  ͷ̵Ǧ–– ‰ƒ–‰ƒ ƒƒ‰‰ƒ–‰– ƒ‰ƒ ͷ̵Ǧ‰‰‰‰ƒ–‰ƒ‰ ––‰‰– ƒ–‰ƒ ͷ̵Ǧƒ–––ƒƒƒ–ƒ – – ƒ– ‰– –  ͷ̵Ǧ – – ‰‰ ‰‰–‰ ƒ‰– ƒƒ –– ƒ . Remark ǦʹͲ͹ͳ„’hacA•–ƒ”– ‘†‘ Ϊͳͳͷʹ„’hacA•–‘’ ‘†‘ ǦͷͲ͵„’hacA•–ƒ”– ‘†‘ ΪͳʹͲ„’hacA•–ƒ”– ‘†‘ ͳ ʹ ͵  —  ͷǯ•’Ž‹ ‡Ǧ•‹–‡ —•’Ž‹ ‡ƒ„Ž‡‹–”‘—• —•’Ž‹ ‡ƒ„Ž‡‹–”‘Ž•

(42) ˜‡”–‡†”‡’‡ƒ–ͷǯ

(43) ˜‡”–‡†”‡’‡ƒ–hacA‰‡‡ Ǧͳ͸ͺ„’hacA•–ƒ”– ‘†‘ Ϊͳʹͷ͵„’hacA•–ƒ”– ‘†‘ ‘”ͷǯhacA ‘”ǦbipA ‘”ǦbipA ‘”ǦpdiA ‘”ǦpdiA. —–ƒ–‹‘•ƒ”‡”‡’”‡•‡–‡†‹boldface–›’‡    NorthernBlotAnalysis ‘” ‘”–Š‡” „Ž‘– ƒƒŽ›•‹• —Ž–—”‡• ™‡”‡ ‰”‘™ Ȁ ƒ– ͵Ͳι ‘ ‹‹ƒŽ ‡†‹ƒ •—’’Ž‡‡–‡† ™‹–Š ͲǤʹΨ ™Ȁ˜ ›‡ƒ•– ‡š–”ƒ –Ǥ Ž‹“—‘–• ‘ˆ ͲǤͷ ‰ ™‡– › ‡Ž‹— ™‡”‡ –”ƒ•ˆ‡””‡† –‘ ͷͲ Ž ˆ”‡•Š ‡†‹— ƒ† –Š‡ —Ž–—”‡• ™‡”‡ ‹ —„ƒ–‡† ˆ‘” ͵ Š „‡ˆ‘”‡ ’”‘˜‘‹‰Ǧ•–”‡••„›–Š‡ƒ††‹–‹‘‘ˆ‡‹–Š‡”ʹͲɊ‰ȀŽ–—‹ ƒ› ‹‘”ʹͲǤ‘–Š‡ ‘–”‘Ž —Ž–—”‡• ‡“—ƒŽ ƒ‘—–• ‘ˆ ‡‹–Š‡”  ‘ˆ ™ƒ–‡” ™ƒ• ƒ††‡†Ǥ ƒ’Ž‡• ˆ”‘ –Š‡ •–”‡••‡† ƒ† —•–”‡••‡† —Ž–—”‡• ™‡”‡ –ƒ‡ ͵ Š ƒˆ–‡” ƒ’’Ž›‹‰ –Š‡ •–”‡•• ƒ‰‡–•Ǥ ‹ ‡ –—‹ ƒ› ‹‹•ƒ‘”‡•’‡ ‹ˆ‹ ƒ‰‡––‘‹†— ‡•–”‡••ǡ‘”–Š‡”„Ž‘–•‘ˆ–Š‡•‡–”‡ƒ–‡–• ƒ”‡•Š‘™™Š‡”‡’‘••‹„Ž‡Ǥ ‘”–Š‡ƒƒŽ›•‹•‘ˆ–Š‡•ƒ†hacA’”‘ ‡••‹‰ǡ–Š‡„Ž‘– ‹••Š‘™ǡƒ•”‡•—Ž–‡†‹— Š‘”‡’”‘‘— ‡†hacA•’Ž‹ ‹‰Ǥ ‘”‹•‘Žƒ–‹‘ǡ› ‡Ž‹—™ƒ•‰”‘—†™‹–Šͳ‰Žƒ••„‡ƒ†•—•‹‰–Š‡ ƒ•–”‡’ ͳʹͲȋ

(44) ͳͲͳƒ˜ƒ–Ȍǡƒ†–‘–ƒŽ™ƒ•‹•‘Žƒ–‡†—•‹‰–Š‡‡ƒ•›’Žƒ––‘–ƒŽ‹– ȋ‹ƒ‰‡ȌǤ

(45) ‘”†‡”–‘’”‡˜‡–†‡‰”ƒ†ƒ–‹‘‘ˆ–Š‡†—”‹‰–Š‡’—”‹ˆ‹ ƒ–‹‘’”‘ ‡†—”‡ǡͳǤͷ Ž• ”‡™Ǧ ƒ’–—„‡•™‡”‡ˆ‹ŽŽ‡†™‹–Šƒ’’”‘š‹ƒ–‡Ž›ͲǤͷŽͳ‰Žƒ••„‡ƒ†•ƒ†͹ͲͲɊŽ „—ˆˆ‡” ™Š‹ Š ™ƒ• ’”‘˜‹†‡† ™‹–Š –Š‡ ‡ƒ•› ’Žƒ– –‘–ƒŽ  ‹–Ǥ Š‡ ˆ‹ŽŽ‡† –—„‡• ™‡”‡ ˆ”‘œ‡ ‹ Ž‹“—‹† ‹–”‘‰‡ ’”‹‘” –‘ –Š‡ Šƒ”˜‡•–‹‰ ‘ˆ –Š‡ › ‡Ž‹ƒǤ Š‡ › ‡Ž‹— ™ƒ• Šƒ”˜‡•–‡† ”ƒ’‹†Ž› ƒ† †”‹‡† –Š‘”‘—‰ŠŽ› „‡–™‡‡ –‹••—‡ ’ƒ’‡”ǡ ƒ† ƒ •ƒŽŽ ’‹‡ ‡ ‘ˆ ThehacATranscriptionalSwitch89.

(46) Chapter4 › ‡Ž‹— ȋͳͲͲǦʹͲͲ ‰Ȍ ™ƒ• ƒ††‡† –‘ ƒ ’”‡Ǧˆ”‘œ‡ –—„‡ ƒ† ”‡ˆ”‘œ‡ ‹ Ž‹“—‹† ‹–”‘‰‡Ǥ Š‡ ˆ”‘œ‡ –—„‡• ™‡”‡ –Šƒ ’Žƒ ‡† ‹ –Š‡ ƒ•– ”‡’ ͳʹͲǡ ƒ† ”— ‘ Ͷ › Ž‡• ‘ˆ ʹͲ •‡ ‘†• ‘ •’‡‡† ͸Ǥͷǡ ™Š‹ Š ƒŽŽ‘™‡† –Šƒ™‹‰ ƒ† •‹—Ž–ƒ‡‘—• ‰”‹†‹‰ ‘ˆ –Š‡ › ‡Ž‹ƒǤ ‘” ‘”–Š‡” ƒƒŽ›•‹• ͳͲ Ɋ‰ ‘ˆ –‘–ƒŽ  ™ƒ• •‡’ƒ”ƒ–‡† ‘ ƒ ͳǤͷΨ ȋ™Ȁ˜Ȍ ƒ‰ƒ”‘•‡ ‰‡Ž ‘–ƒ‹‹‰ ͳͲ  ’Š‘•’Šƒ–‡ „—ˆˆ‡” ȋ’  ͹Ȍǡ ƒ† „Ž‘––‡† ‘–‘ ƒ ›„‘†  ‡„”ƒ‡ ȋ‡”•Šƒ Šƒ”ƒ ‹ƒ ‹‘–‡ ŠȌǤ Ž‘––‹‰ ƒ† Š›„”‹†‹•ƒ–‹‘ ™ƒ• †‘‡ ƒ ‘”†‹‰ –‘ •–ƒ†ƒ”† ’”‘ ‡†—”‡•. ͳͶͳǤ. ”‘„‡• ™‡”‡ ‰‡‡”ƒ–‡† „› Žƒ„‡ŽŽ‹‰  ˆ”ƒ‰‡–• ™‹–Š. †‡‘š› ›–‹†‹‡ ͷǯǦȏDǦ͵ʹȐǦ–”‹’Š‘•’Šƒ–‡ —•‹‰ –Š‡ ”‹‡Ǧ

(47) –

(48)

(49)  Žƒ„‡ŽŽ‹‰ ‹– ȋ–”ƒ–ƒ‰‡‡Ȍǡ ƒ† ’—”‹ˆ‹‡†‘‹‘‰‡Ž͵Ͳ•’‹ ‘Ž—•ȋ‹‘”ƒ†ȌǤš’‘•—”‡•™‡”‡ƒ†‡‘—”‹š ǦŽ—•šǦ ”ƒ›ˆ‹Ž•ȋ‰ˆƒȌǤ—ƒ–‹ˆ‹ ƒ–‹‘‘ˆ–Š‡‘”–Š‡”„Ž‘–•‹‰ƒŽ•™ƒ•†‘‡„›’Š‘•’Š‘”Ǧ‹ƒ‰‡” ƒƒŽ›•‹•ȋƒ ƒ”†

(50) •–ƒ–

(51) ƒ‰‡”ȌǤ   5’RACEandRTPCR ‘”ͷǯƒ†”‡˜‡”•‡–”ƒ• ”‹’–ƒ•‡ȋǦȌˆ‹”•–•–”ƒ† ™ƒ•ƒ†‡ˆ”‘ͳɊ‰ ‘ˆ–‘–ƒŽ—•‹‰–Š‡ ’Ž‹ˆ‹ ƒ–‹‘‹–ȋŽ‘‡–‡ ŠȌǤŠ‡ͷƲǦ‡†‘ˆ–Š‡ hacA –”ƒ• ”‹’– ™ƒ• ƒ’Ž‹ˆ‹‡† ‹ ƒ  ™‹–Š ’”‹‡” ͳ͸ —•‹‰ › Ž‹‰ ‘†‹–‹‘• ”‡ ‘‡†‡†„›–Š‡‹–ǯ••—’’Ž‹‡”Ǥ ‘”–Š‡–”ƒ• ”‹’–‹‘ƒƒŽ›•‹•‘ˆbipAƒ†pdiAǡ  ™ƒ•ƒ’Ž‹ˆ‹‡†—•‹‰’”‹‡”’ƒ‹”•ͳ͹Ǧͳͺƒ†ͳͻǦʹͲ”‡•’‡ –‹˜‡Ž›Ǥ   Microscopy ‘” ‹ ”‘• ‘’›ǡ › ‡Ž‹ƒ ™ƒ• ‰”‘™ „‡–™‡‡ –™‘ ‰Žƒ•• •Ž‹†‡• ‹ ‹‹ƒŽ ‡†‹ƒ •—’’Ž‡‡–‡† ™‹–Š ͲǤʹΨ ›‡ƒ•– ‡š–”ƒ –ǡ ™‹–Š ƒ† ™‹–Š‘—– –Š‡ ƒ††‹–‹‘ ‘ˆ ͳͲ Ɋ‰ȀŽ –—‹ ƒ› ‹Ǥ

(52) ƒ‰‡• ™‡”‡ –ƒ‡ —•‹‰ ƒ Ž›’—• ͷͳ ‹ ”‘• ‘’‡ ‡“—‹’’‡† ™‹–Š ƒ Ž›’—•ͳͳ†‹‰‹–ƒŽ ƒ‡”ƒǤ.    Results  TheA.nigerhacAStrain ‘ƒ••‡••–Š‡”‘Ž‡‘ˆ–Š‡hacA‰‡‡ƒ†–Š‡ ‘–”‘Ž‡ Šƒ‹••‹–Š‡‘ˆA.nigerǡ™‡ ƒ‹‡† –‘ ‘„–ƒ‹ ƒ ‘””‡•’‘†‹‰ ‘ ‘—– •–”ƒ‹Ǥ ‡ ‡–Ž›ǡ A. tumefaciens ‡†‹ƒ–‡† –”ƒ•ˆ‘”ƒ–‹‘Šƒ•„‡‡•Š‘™–‘„‡ƒ‡ˆˆ‹ ‹‡––‘‘Žˆ‘”‰‡‡”‡’Žƒ ‡‡–‹Aspergillus awamoriͻʹǤ•‹‰–Š‹•–”ƒ•ˆ‘”ƒ–‹‘’”‘ ‡†—”‡ǡ–Š‡hacA‰‡‡‘ˆA.niger™ƒ••— ‡••ˆ—ŽŽ› ”‡’Žƒ ‡† ™‹–Š –Š‡ Š›‰”‘› ‹ ”‡•‹•–ƒ ‡ ƒ••‡––‡Ǥ Š‡ ‘•–”— – ‘•‹•–‡† ‘ˆ –Š‡ 90ThehacATranscriptionalSwitch.

(53) Chapter4 Š›‰”‘› ‹ ƒ••‡––‡ ˆ”‘ ’͹Ǧͳ ˆŽƒ‡† „› ʹ „ ‘ˆ –Š‡ hacA ͷǯǦ—’•–”‡ƒ ƒ† ͳǤͷ „ ‘ˆ –Š‡͵ǯǦ†‘™•–”‡ƒ”‡‰‹‘•”‡•’‡ –‹˜‡Ž›Ǥ ™‘ –›’‡• ‘ˆ ‘Ž‘‹‡• ™‡”‡ ’”‡•‡– ‘ –Š‡ –”ƒ•ˆ‘”ƒ–‹‘ ’Žƒ–‡•Ǥ Š‡ ƒŒ‘”‹–› ‘ˆ –Š‡ ‘Ž‘‹‡•Šƒ†ƒ”‡‰—Žƒ”™‹Ž†–›’‡’Š‡‘–›’‡ǡ™Š‡”‡ƒ•ƒ’’”‘š‹ƒ–‡Ž›͵ͲΨ ‘•‹•–‡†‘ˆ •ƒŽŽ ‘Ǧ•’‘”—Žƒ–‹‰ ›‡ŽŽ‘™ ‘Ž‘‹‡•Ǥ ‘—–Š‡” ƒƒŽ›•‡• ’‡”ˆ‘”‡† ‘ „‘–Š –›’‡ ‘ˆ ‘Ž‘‹‡•ǡ •Š‘™‡† –Š‡ ƒ„•‡ ‡ ‘ˆ –Š‡ hacA ‰‡‡ ‹ –Š‡ ƒ„‡””ƒ– ‘Ž‘‹‡•ǡ ™Š‡”‡ƒ• –Š‡ ‘•–”— –™ƒ•‡ –‘’‹ ƒŽŽ›‹–‡‰”ƒ–‡†‹–”ƒ•ˆ‘”ƒ–•Šƒ˜‹‰ƒ™‹Ž†–›’‡’Š‡‘–›’‡ǤŠ—•ǡ †‡Ž‡–‹‘ ‘ˆ hacA ”‡•—Ž–• ‹ ƒ †‹•–‹ – ’Š‡‘–›’‡Ǥ ”‘™–Š ‹• •‡˜‡”‡Ž› ‹’ƒ‹”‡†ǡ ƒ† •’‘”—Žƒ–‹‘‹•ƒŽ‘•–ƒ„•‡– ‘’ƒ”‡†–‘–Š‡™‹Ž†–›’‡•–”ƒ‹ȋ ‹‰ǤͳȌǤ‹‹ƒŽ‡†‹ƒ ’Žƒ–‡•ǡ ƒ• ™‡ŽŽ ƒ• ‘ ’Žƒ–‡• ™‹–Š ”‹ Š ‡†‹— •— Š ƒ• ‘’Ž‡–‡ ‡†‹ƒ ȋȌ ƒ† ’‘–ƒ–‘ †‡š–”‘•‡ ƒ‰ƒ” ȋȌǡ –Š‡ hacA •–”ƒ‹ ˆ‘”‡† •ƒŽŽ ‘’ƒ – ‘Ž‘‹‡• ™‹–Š ‘Ž› ˆ‡™ ‘‹†‹ƒǤ Š‡ ‰”‘™–Š †‹ˆˆ‡”‡ ‡• ‘„•‡”˜‡† ™‡”‡ ƒƒŽ›•‡† ˆ—”–Š‡” ‹ ”‘• ‘’‹ ƒŽŽ›Ǥ †‡” ‘”ƒŽ‰”‘™–Š ‘†‹–‹‘•ǡ’ƒ”–‘ˆ–Š‡Š›’Šƒ‡‘ˆ–Š‡hacA•–”ƒ‹ƒ”‡•™‘ŽŽ‡ ‘’ƒ”‡†–‘ –Š‡™‹Ž†–›’‡ǡƒ†‘ˆ–‡–Š‡•‡•™‘ŽŽ‡Š›’Šƒ‡ƒ”‡„”ƒ Š‡†ƒ––Š‡–‹’•ǡˆ‘”‹‰ˆ‹‰‡”ǦŽ‹‡ ‡š–‡•‹‘• ƒ• •Š‘™ ‹ ‹‰—”‡ ͶǤ

(54)  –Š‡ ’”‡•‡ ‡ ‘ˆ –—‹ ƒ› ‹ǡ ‰”‘™–Š ‘ˆ –Š‡ hacA •–”ƒ‹™ƒ•‡ƒ”Ž›ƒ„‘Ž‹•Š‡†ǤŠ‡Š›’Šƒ‡ƒ”‡•Š‘”–‡”–Šƒ‹–Š‡™‹Ž†–›’‡•–”ƒ‹ǡƒ†Šƒ˜‡ •™‘ŽŽ‡ ‹””‡‰—Žƒ” •Šƒ’‡•Ǥ Š—•ǡ ƒ’’Ž‹ ƒ–‹‘ ‘ˆ Ǧ•–”‡•• ‹ –Š‡ ˆ‘” ‘ˆ ͳͲ Ɋ‰ȀŽ –—‹ ƒ› ‹•‡˜‡”‡Ž›ƒˆˆ‡ –‡†‰”‘™–Š‘ˆ–Š‡hacA•–”ƒ‹ǡ‹†‹ ƒ–‹‰–Šƒ–ƒˆ— –‹‘ƒŽ ‹•‡ ‡••ƒ”›ˆ‘” ‘’‹‰™‹–ŠǦ•–”‡••Ǥ ‘”–Š‡”ƒƒŽ›•‹••Š‘™‡†–Š‡‹ƒ„‹Ž‹–›‘ˆ–Š‡hacA•–”ƒ‹–‘ƒ –‹˜ƒ–‡–Š‡ȋ ‹‰Ǥ ͳȌǤ

(55) ƒ™‹Ž†–›’‡•–”ƒ‹–Š‡—’Ǧ”‡‰—Žƒ–‹‘‘ˆ–Š‡ Šƒ’‡”‘‡bipAƒ†–Š‡ˆ‘Ž†ƒ•‡•cypBƒ† pdiA, †—‡ –‘ Ǧ•–”‡••ǡ ‘‹ ‹†‡• ™‹–Š –Š‡ ’”‘ ‡••‹‰ ‘ˆ hacA Ǥ

(56)  –Š‡ hacA •–”ƒ‹ǡ Š‘™‡˜‡”ǡǦ•–”‡••†‘‡•‘––”‹‰‰‡”ƒ ‘’ƒ”ƒ„Ž‡”‡•’‘•‡ǤŠ‡„ƒ•ƒŽ–”ƒ• ”‹’–Ž‡˜‡Ž•‘ˆ bipAǡcypBƒ†pdiAƒ”‡Ž‘™ƒ†‹”‡•’‘•‡–‘–—‹ ƒ› ‹–”‡ƒ–‡–‘Ž›ƒ•Ž‹‰Š–‹ˆƒ› —’”‡‰—Žƒ–‹‘™ƒ•‘„•‡”˜‡†Ǥ

(57)  ‘–”ƒ•–ǡ”‡–”ƒ•ˆ‘”ƒ–‹‘‘ˆ–Š‡hacA•–”ƒ‹™‹–Š–Š‡™‹Ž† –›’‡ hacA ‰‡‡ ˆ—ŽŽ› ”‡•–‘”‡† –Š‡ –”ƒ• ”‹’–‹‘ƒŽ ’ƒ––‡” ‘ˆ ”‡‰—Žƒ–‹‘ǡ –›’‹ ƒŽ ˆ‘” ƒ ™‹Ž† –›’‡•–”ƒ‹ǤŠ‡hacA•–”ƒ‹™ƒ•—•‡†ˆ—”–Š‡”ƒ•ƒ„ƒ ‰”‘—†ˆ‘”•–—†›‹‰–Š‡hacA‰‡‡ ‡š’”‡••‹‘‡ Šƒ‹•Ǥ. ThehacATranscriptionalSwitch91.

(58) Chapter4                . Figure 1. . Š‡‘–›’‹  ƒ† –”ƒ• ”‹’–‹‘ƒŽ ƒƒŽ›•‹• ‘ˆ –Š‡ A. niger hacA •–”ƒ‹Ǥ (A) ”‘™–Š ‘ˆ  –Š‡hacA•–”ƒ‹ ‘’ƒ”‡†–‘–Š‡™‹Ž†–›’‡ͶǤͳ•–”ƒ‹‘†‹ˆˆ‡”‡–ƒ‰ƒ”‡†‹ƒǤǡ‹‹ƒŽ  ǡ ‘’Ž‡–‡ ‡†‹— •—’’Ž‡‡–‡† ™‹–Š ͲǤͷΨ ›‡ƒ•– ‡š–”ƒ –Ǣ ǡ ’‘–ƒ–‘ †‡š–”‘•‡ ‡†‹—Ǣ ƒ‰ƒ”Ǣ–ǡͳͲɊ‰ȀŽ–—‹ ƒ› ‹Ǥ(B)‘”–Š‡”„Ž‘–ƒƒŽ›•‹••Š‘™‹‰–Š‡Žƒ ‘ˆƒˆ— –‹‘ƒŽ  ‹–Š‡hacA•–”ƒ‹ǡƒ†–Š‡”‡•–‘”ƒ–‹‘‘ˆ–Š‡”‡•’‘•‡„›–”ƒ•ˆ‘”‹‰–Š‡hacA•–”ƒ‹™‹–Š  ’Žƒ•‹†’ ƒ Ǧ’›” ȗǤ”‘„‡•–Šƒ–™‡”‡—•‡†ˆ‘”Š›„”‹†‹•ƒ–‹‘•ƒ”‡•Š‘™–‘–Š‡”‹‰Š–Ǥ.  . TheRoleoftheSmallORFandthePutative5’Splicesite Š‡ͷǯ‘ˆˆ—ŽŽǦŽ‡‰–Š–”ƒ• ”‹’–‘ˆhacA ‘–ƒ‹•ƒ•ƒŽŽ‘’‡”‡ƒ†‹‰ˆ”ƒ‡ȋ— Ȍ‘ˆ ͶͶ ‘†‘• ȋ ‹‰Ǥ ʹȌǡ ™Š‹ Š ‹• ‘– ’”‡•‡– ‹ –Š‡ –”— ƒ–‡† hacA Ǥ ‡ ’”‡˜‹‘—•Ž› Š›’‘–Š‡•‹•‡† –Šƒ– –Š‹• ”‡‰‹‘ ‘—Ž† „‡ ‹˜‘Ž˜‡† ‹ ƒ –”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž ‡ Šƒ‹• ‘ˆ hacAuǡ•‹ ‡–”ƒ•Žƒ–‹‘ƒŽ ‘–”‘Ž‹•‘ˆ–‡ƒ••‘ ‹ƒ–‡†™‹–Š•Š‘”– •ǡ’”‡•‡–‹–Š‡ ͷǯ ‘ˆ • ͻͲǤ Š‡ S. cerevisiae –”ƒ• ”‹’–‹‘ ˆƒ –‘” Ͷ’ ƒ† –Š‡ ƒƒŽ‹ƒ –”ƒ• ”‹’–‹‘ ˆƒ –‘”  Ͷ ƒ”‡ –”ƒ•Žƒ–‹‘ƒŽŽ› ‘–”‘ŽŽ‡† „› •ƒŽŽ — • ͷͳǡͷͻǤ ‘ –‡•– –Š‹• Š›’‘–Š‡•‹•ǡ –Š‡   •–ƒ”– ‘†‘ ‘ˆ –Š‡ —  ™ƒ• —–ƒ–‡† –‘  ǡ ƒ† –Š‡ —–ƒ–‡† hacA ‘•–”— –™ƒ•‹–”‘†— ‡†‹–‘–Š‡hacA•–”ƒ‹Ǥ ‘™‡˜‡”ǡ‘”–Š‡”ƒƒŽ›•‡•‘ˆ–Š‡—  •–”ƒ‹ ƒ†•—„•‡“—‡– •‹‰ƒŽ “—ƒ–‹ˆ‹ ƒ–‹‘†‹† ‘– •Š‘™ Ž‡ƒ” ‡ˆˆ‡ –• ‘ˆ –Š‡ —–ƒ–‹‘ ‘ –Š‡ –”ƒ• ”‹’–‹‘ Ž‡˜‡Ž• ‘ˆ –Š‡ ˆ‘Ž†ƒ•‡• ƒ†Ȁ‘” –Š‡ –”— ƒ–‹‘ ‘ˆ hacA  —’‘ ƒ’’Ž‹ ƒ–‹‘‘ˆǦ•–”‡••ǡ™Š‡ ‘’ƒ”‡†–‘™‹Ž†–›’‡ȋ ‹‰Ǥʹƒ†ȌǤ

(59) –Šƒ•–‘„‡‘–‡†–Šƒ– ƒ •‡ ‘†   ‹• ’”‡•‡– ‹ –Š‡ ”‡ƒ†‹‰ ˆ”ƒ‡ ‘ˆ –Š‡ ͶͶǦ ‘†‘ —  ‘ˆ hacAǤ Š‹•  ǡ Š‘™‡˜‡”ǡ‹•Ž‘ ƒ–‡†͹–—’•–”‡ƒ‘ˆ–Š‡ ‡–”ƒŽ•’ƒ ‡”— Ž‡‘–‹†‡‘ˆʹȋ ˆǤƒ„Ž‡

(60) Ȍǡ ƒ†™ƒ•–Š‡”‡ˆ‘”‡‘–—–ƒ–‡†Ǥ•ƒ”‡•—Ž–ƒ‡‹‰Š–Ǧ ‘†‘Ž‘‰— ”‡ƒ‹•‹–ƒ –‹–Š‡ ͷǯ‘ˆ–Š‡hacAu‘ˆ–Š‹••–”ƒ‹Ǥ  92ThehacATranscriptionalSwitch.

(61) Chapter4   . Figure2.—–ƒ–‹‘ƒŽƒƒŽ›•‹•‘ˆ–Š‡— ƒ†–Š‡’—–ƒ–‹˜‡ͷǯ•’Ž‹ ‡Ǧ•‹–‡ǡ–™‘‡Ž‡‡–•’”‡•‡–‹ –Š‡ ͷǯ ‘ˆ –Š‡ hacAu Ǥ (A)  Š‡ƒ–‹  ‘˜‡”˜‹‡™ ‘ˆ –Š‡ ͷǯ ‘ˆ –Š‡ hacA ‰‡‡Ǥ   ‹†‹ ƒ–‡• –Š‡ •–ƒ”– ‘†‘ ‘ˆ hacAǡ • ƒ”‡ ‹†‹ ƒ–‡† „› ‘’‡ „‘š‡•ǡ –Š‡ •–ƒ”– ‘ˆ hacAu ƒ† hacAi  „› „Žƒ  „‘š‡•ǡ ƒ† –Š‡ ͶͶǦ ‘†‘ —  „› ƒ ‘’‡ ƒ””‘™Ǥ Š‡ —–ƒ–‹‘• ‹–”‘†— ‡† –‘ †‹•”—’– –Š‡ —  ƒ† –Š‡ ’—–ƒ–‹˜‡ ͷǯ•’Ž‹ ‡Ǧ•‹–‡ ƒ”‡ ‹†‹ ƒ–‡† —†‡”‡ƒ–Š –Š‡ ˆ‹‰—”‡Ǥ Š‡ ’—–ƒ–‹˜‡

(62) ”‡ —––‹‰ •‹–‡ ‹• ‹†‹ ƒ–‡† „› ƒ ƒ””‘™Ǥ (B) ‘”–Š‡” „Ž‘– ƒƒŽ›•‹• •Š‘™‹‰–Š‡‡ˆˆ‡ –‘ˆ•–”‡••‹’‘•‡†„›–—‹ ƒ› ‹ȋ–Ȍ‘–Š‡–”ƒ• ”‹’–‹‘‘ˆhacAǡbipA ƒ†pdiAǤͳͺ”™ƒ•ƒ•ƒŽ‘ƒ†‹‰ ‘–”‘ŽǤ(C)‡Žƒ–‹˜‡bipAŽ‡˜‡Ž•‹–Š‡™–ǡ–Š‡ȟ— •–”ƒ‹ ƒ†–Š‡—–Ǧͷǯ•’Ž‹ ‡Ǧ•‹–‡•–”ƒ‹‹–Š‡ƒ„•‡ ‡ȋ„Žƒ „ƒ”•Ȍƒ†’”‡•‡ ‡ȋ‰”‡›„ƒ”•Ȍ‘ˆʹͲɊ‰ȀŽ –—‹ ƒ› ‹Ǥ. ThehacATranscriptionalSwitch93.

(63) Chapter4

(64) –‡”‡•–‹‰Ž›ǡ –Š‹• —  •Š‘™• ”‡ƒ”ƒ„Ž‡ •‡“—‡ ‡ ‹†‡–‹–› ™‹–Š –™‘ •ƒŽŽ — • ’”‡•‡–‹–Š‡hacA–”ƒ• ”‹’–•‘ˆA.nidulansƒ†A.fumigatusǤŠ‡–”ƒ•Žƒ–‡†•‡“—‡ ‡•‘ˆ –Š‡•‡ — • ‘ˆ A. nigerǡ A. nidulans ƒ† A. fumigatus ƒ”‡  ǡ ǡ ƒ†  ǡ ”‡•’‡ –‹˜‡Ž›Ǥ Ž–Š‘—‰Š –Š‹• ‘•‡”˜ƒ–‹‘ ‘—Ž† „‡ •‘Ž‡Ž› –Š‡ ”‡•—Ž– ‘ˆ –Š‡ ˆƒ – –Šƒ–ƒŽŽ–Š”‡‡— •‘˜‡”Žƒ’™‹–Š–Š‡ʹ•‡“—‡ ‡‹‡ƒ Š’”‘‘–‡”ǡƒˆ— –‹‘‘ˆ–Š‡ — •‹–”ƒ•Žƒ–‹‘ƒŽƒ––‡—ƒ–‹‘ ƒ‘–„‡‡š Ž—†‡†Ǥ ”‡˜‹‘—• ‹•’‡ –‹‘ ‘ˆ –Š‡ •‡“—‡ ‡ ƒ”‘—† –Š‡ •–ƒ”– •‹–‡ ‘ˆ hacAi  ”‡˜‡ƒŽ‡† •–”‘‰•‡“—‡ ‡•‹‹Žƒ”‹–›™‹–Š–Š‡‹–”‘„‘”†‡”•‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘ ͳͲͳǤ

(65) ˆ–Š‹• ™‘—Ž† Ž‡ƒ† –‘ ƒ •‹‹Žƒ” •‡ ‘†ƒ”› •–”— –—”‡ ƒ• ‹• –Š‡ ƒ•‡ ˆ‘” –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ „‘”†‡”•ǡ –Š‹• •–”— –—”‡ ‘—Ž† „‡ ƒ ’‘–‡–‹ƒŽ –ƒ”‰‡– ˆ‘”

(66) ”‡ǡ ƒ† ‡š’Žƒ‹ –Š‡ ‡ Šƒ‹• „‡Š‹† –Š‡ –”— ƒ–‹‘ ‘ˆ hacAǤ

(67)  ‘–”ƒ•– –‘ –Š‡ •’Ž‹ ‹‰ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ™Š‹ Š‹˜‘Ž˜‡•ƒ

(68) ”‡‡†‹ƒ–‡† —–‹‡ƒ Š‘ˆ–Š‡–™‘Ž‘‘’•ˆ‘ŽŽ‘™‡†„›”‡ǦŽ‹‰ƒ–‹‘‘ˆ–Š‡ –™‘‡š‘•„›–Ž‹‰ƒ•‡ ͳͷͳǡ–Š‡Š›’‘–Š‡–‹ ƒŽ

(69) ”‡Ǧ‡†‹ƒ–‡†ͷǯǦ–”— ƒ–‹‘‘ˆhacA ™‘—Ž† ‹˜‘Ž˜‡ ‘Ž› ƒ —– ƒ– –Š‡ ”‡ ‘‰‹–‹‘ •‹–‡Ǥ Ž–Š‘—‰Š ‘  •‡ ‘†ƒ”› •–”— –—”‡ ‹ ‘”’‘”ƒ–‹‰–Š‡’—–ƒ–‹˜‡ͷǯ•’Ž‹ ‡Ǧ•‹–‡‹ƒ•–ƒ„Ž‡•–‡ǦŽ‘‘’•–”— –—”‡ ‘—Ž†„‡’”‡†‹ –‡†ǡ —–ƒ–‹‘ƒŽƒƒŽ›•‹•™ƒ•’‡”ˆ‘”‡†–‘–‡•–™Š‡–Š‡”‘”‘––Š‹••‹–‡™‘—Ž†„‡ƒ•—„•–”ƒ–‡ˆ‘”

(70) ”‡ǤŠ‡•‡˜‡‘Ǧ’ƒ‹”‡†— Ž‡‘–‹†‡•‹–Š‡’—–ƒ–‹˜‡Ž‘‘’™‡”‡—–ƒ–‡†ˆ”‘’—”‹‡•–‘ ’›”‹‹†‹‡•ǡ‘”˜‹ ‡˜‡”•ƒǡ‹‘”†‡”–‘†‡•–”‘›ƒ’—–ƒ–‹˜‡”‡ ‘‰‹–‹‘•‹–‡ˆ‘”

(71) ”‡ȋ ‹‰ǤʹȌǤ

(72) ˆ

(73) ”‡ ‹†‡‡† ™‘—Ž† „‡ ‹˜‘Ž˜‡†ǡ –Š‹• •–”ƒ–‡‰› •Š‘—Ž† Ž‡ƒ† –‘ –Š‡ ‹ƒ„‹Ž‹–› –‘ ˆ‘” –Š‡ –”— ƒ–‡†hacA‡••‡‰‡”. ‘™‡˜‡”ǡ‘”–Š‡”ƒƒŽ›•‹•‘ˆƒ•–”ƒ‹„‡ƒ”‹‰•— Š—–ƒ–‹‘•ǡ †‹†‘–•Š‘™ƒ›‡ˆˆ‡ –‘ˆ–Š‡—–ƒ–‹‘•‹–Š‡’—–ƒ–‹˜‡ͷǯ•’Ž‹ ‡Ǧ•‹–‡‘–Š‡’”‘ ‡••‹‰‘ˆ –Š‡hacAȋ ‹‰ǤʹȌǤŠ”‡‡Š‘—”•ƒˆ–‡”–—‹ ƒ› ‹Ǧ‹†— ‡†•–”‡••ǡ–Š‡–”— ƒ–‡†hacA ™ƒ•’”‡•‡–ƒ–Ž‡˜‡Ž• ‘’ƒ”ƒ„Ž‡–‘–Š‡™‹Ž†–›’‡•–”ƒ‹ǤŠ‡—’Ǧ”‡‰—Žƒ–‹‘‘ˆpdiA ƒ†bipAŽ‡˜‡Ž•—’‘•–”‡••™ƒ•ƒŽ•‘•‹‹Žƒ”–‘–Š‡™‹Ž†–›’‡•‹–—ƒ–‹‘Ǥ.   TheRoleoftheUPREsinthehacAPromoter ”‡˜‹‘—•Ž›ǡ ™‡ ‹†‡–‹ˆ‹‡† „›  –Š”‡‡ • ‹ –Š‡ hacA ’”‘‘–‡”Ǥ Š‡ ’”‡•‡ ‡ ƒ† ‘•‡”˜ƒ–‹‘ ‘ˆ –Š‡•‡ –Š”‡‡ „‘š‡• ‹ –Š‡ hacA ’”‘‘–‡”• ‘ˆ A. niger, A. nidulans, and A. fumigatusǡ •—‰‰‡•–• ƒ ˆ— –‹‘ ˆ‘” ƒŽŽ ‘ˆ –Š‡ ͳͲͲǤ —–ƒ–‹‘ƒŽ ƒƒŽ›•‹• ™ƒ• ƒ””‹‡† ‘—– –‘ †‡–‡”‹‡ –Š‡ ‘–”‹„—–‹‘ ‘ˆ ‡ƒ Š ‘ˆ –Š‡•‡ ‡Ž‡‡–• –‘ –Š‡ –”ƒ• ”‹’–‹‘ƒŽ ”‡‰—Žƒ–‹‘ ‘ˆ hacAǤ —–ƒ–‹‘• ™‡”‡ ‹–”‘†— ‡† ‹ –Š‡ ‘”‡ •‡“—‡ ‡• ‘ˆ –Š‡ hacA •Ǥ Š‡ Ž‘•• ‘ˆ „‹†‹‰†—‡–‘–Š‡—–ƒ–‹‘•™ƒ• ‘ˆ‹”‡†invitro„›•™‹–Š ƒ ’”‘–‡‹ƒ†͵Ͷ „’ †‘—„Ž‡ •–”ƒ†‡† ‘Ž‹‰‘• ‘’”‹•‹‰ –Š‡ †‹ˆˆ‡”‡– —–ƒ–‡† •Ǥ Š‡”‡ƒ• –™‘ —–ƒ–‹‘• ȋ ‹‰Ǥ ͵Ȍ ™‡”‡ •—ˆˆ‹ ‹‡– –‘ ‘’Ž‡–‡Ž› ƒ„‘Ž‹•Š „‹†‹‰ ‘ˆ ƒ  –‘ ʹ ƒ† ͵ǡ–Š‡ ‘””‡•’‘†‹‰ʹ—–ƒ–‹‘•‹–”‘†— ‡†‹ͳ†‹†‘–Ž‡ƒ†–‘ ‘’Ž‡–‡Ž‘•• ‘ˆ„‹†‹‰Ǥ•ƒ”‡•—Ž–ǡ–™‘ƒ††‹–‹‘ƒŽ—–ƒ–‹‘•™‡”‡‹–”‘†— ‡†‹–Š‡ͳǤ‘‡‘ˆ–Š‡ •–”ƒ‹• ƒ””›‹‰ —–ƒ–‹‘• ‹ –Š‡ hacA • •Š‘™‡† ’Š‡‘–›’‹  ‰”‘™–Š Šƒ‰‡• ‘ ƒ. 94ThehacATranscriptionalSwitch.

(74) Chapter4 ’Žƒ–‡–‡•–ȋ ‹‰ǤͶȌǡƒ†–Š‡•‡”‡•—Ž–•™‡”‡ ‘ˆ‹”‡†„›Ž‹‰Š–‹ ”‘• ‘’›ȋ†ƒ–ƒ‘–•Š‘™ȌǤ ‘™‡˜‡”ǡ‘”–Š‡” ƒƒŽ›•‡• ’‡”ˆ‘”‡† ‘ –Š‡ •–”ƒ‹• ”‡˜‡ƒŽ‡† ƒƒŽ–‡”‡† –”ƒ• ”‹’–‹‘ƒŽ ’ƒ––‡”ˆ‘”hacAƒ†–Š‡ˆ‘Ž†ƒ•‡•Ǥ Š‡ ʹ ™ƒ• —–ƒ–‡† •‡’ƒ”ƒ–‡Ž›ǡ ‹ ‘„‹ƒ–‹‘ ™‹–Š ͵ǡ ‘” ‹ ‘„‹ƒ–‹‘™‹–Šͳƒ†͵ǡ–Š‡–”— ƒ–‹‘‘ˆhacA†‹†‘–‘ —”ƒ›‘”‡ ‹”‡•’‘•‡–‘Ǧ•–”‡••ȋ ‹‰Ǥ͵ȌǤŽ–Š‘—‰Š ‘Ž›–Š‡ˆ—ŽŽǦŽ‡‰–Š–”ƒ• ”‹’–™ƒ•ˆ‘”‡†‹ –Š‡ ʹ —–ƒ–• –Š”‡‡ Š‘—”• ƒˆ–‡”  –”‡ƒ–‡–ǡ bipA ƒ† pdiA –”ƒ• ”‹’–• ™‡”‡ —’”‡‰—Žƒ–‡†–‘–Š‡•ƒ‡‡š–‡–ƒ•‹–Š‡™‹Ž†–›’‡•–”ƒ‹ǡ‹†‹ ƒ–‹‰–Šƒ––”— ƒ–‹‘‘ˆhacA ‹•‘–ƒ‡ ‡••‹–›ˆ‘”‹†— –‹‘‘ˆ–Š‡’ƒ–Š™ƒ›Ǥ

(75)  ‘–”ƒ•–ǡ—–ƒ–‹‘‘ˆͳ‘”͵ •‡’ƒ”ƒ–‡Ž›ǡ†‹†‘–Ž‡ƒ†–‘ƒ’’ƒ”‡– Šƒ‰‡•‹–Š‡•’Ž‹ ‹‰‘ˆhacA—†‡”Ǧ•–”‡•• ‘†‹–‹‘•Ǥ ‘™‡˜‡”ǡ —–ƒ–‹‘ ‘ˆ ͳ †‹† ”‡•—Ž– ‹ Ž‘™‡” „ƒ•ƒŽ Ž‡˜‡Ž• ‘ˆ hacA  ȋ ‹‰Ǥ͵ȌǤŽŽ–Š”‡‡•–”ƒ‹• ƒ””›‹‰—–ƒ–‹‘•‹ͳŠƒ†ƒ’’”‘š‹ƒ–‡Ž›ŠƒŽˆ‘ˆ–Š‡hacA Ž‡˜‡Ž• ‘’ƒ”‡†–‘–Š‡™‹Ž†–›’‡ƒ†–Š‡‘–Š‡”—–ƒ–•–”ƒ‹•ǤŠ‹•‹•‹Ž‹‡™‹–Š –Š‡ ˆ‹†‹‰ –Šƒ– hacA ‹• •—„Œ‡ – –‘ ƒ—–‘Ǧ”‡‰—Žƒ–‹‘ ͳͲͳǤ ‘–Š‡” ‘„•‡”˜ƒ–‹‘ ‹• Š‘™‡˜‡” ‘”‡ ’—œœŽ‹‰Ǥ

(76)  –Š‡™‹Ž† –›’‡ •–”ƒ‹ǡ –Š‡ƒ‘—– ‘ˆ –Š‡ ˆ—ŽŽǦŽ‡‰–Š hacA ‡••‡‰‡” Šƒ† †”‘’’‡†•‹‰‹ˆ‹ ƒ–Ž›–Š”‡‡Š‘—”•ƒˆ–‡”ƒ††‹–‹‘‘ˆ–‘–Š‡ —Ž–—”‡ǤŠ‹•‡ˆˆ‡ –™ƒ•ƒŽ•‘ ‘„•‡”˜‡† ˆ‘” –Š‡ —–ƒ–• — ǡ —–Ǧͷǯ•’Ž‹ ‡ ƒ† 

(77)  ȋ—–ƒ–‡†

(78) ˜‡”–‡† ‡’‡ƒ–ǡ •‡‡ „‡Ž‘™Ȍ—’‘•–”‡••ȋ†ƒ–ƒ‘–•Š‘™ȌǤ ‘™‡˜‡”ǡ‡š ‡’–ˆ‘”ʹǡ–Š‹•‡ˆˆ‡ –™ƒ•‘– ‘„•‡”˜‡†ˆ‘”–Š‡‘–Š‡”—–ƒ–•Ǥ–Š‡ ‘–”ƒ”›ǡˆ—ŽŽǦŽ‡‰–ŠhacAŽ‡˜‡Ž•™‡”‡•Ž‹‰Š–Ž› ‹†— ‡† „› Ǧ•–”‡•• ‹ •–”ƒ‹• ™‹–Š ‡‹–Š‡” ƒ —–ƒ–‹‘ ‹ ͳǡ ͵ ‘” ‹ „‘–Š ‡Ž‡‡–•Ǥ Š‹• ‘„•‡”˜ƒ–‹‘ –Šƒ– ͳ •‡‡• –‘ ”‡’”‡•• –”ƒ• ”‹’–‹‘ ‘ˆ –Š‡ ˆ—ŽŽ Ž‡‰–Š hacA  ™Š‡ ƒ ‹ ‹• ƒ„—†ƒ–Ž› ’”‡•‡–ǡ ™‘—Ž† ‹’Ž› ƒ †—ƒŽ ˆ— –‹‘ ˆ‘” ͳǤ †‡”‘”ƒŽ ‘†‹–‹‘•™Š‡–Š‡ ‘ ‡–”ƒ–‹‘‘ˆ ƒ ‹‹•Ž‘™ǡ–Š‡ͳˆ— –‹‘•ƒ• ƒƒ –‹˜ƒ–‘”•‹–‡ǡ™Š‡”‡ƒ•‹–ˆ— –‹‘•ƒ•ƒ”‡’”‡••‘”•‹–‡†—”‹‰•–”‡••ǤŠ‡—†‡”Ž›‹‰ ‡ Šƒ‹•‘ˆ–Š‹•’‘••‹„Ž‡†—ƒŽˆ— –‹‘‘ˆͳ‹•— Ž‡ƒ”ƒ†™‹ŽŽ„‡•—„Œ‡ ––‘ˆ—”–Š‡” ‹˜‡•–‹‰ƒ–‹‘Ǥ Š‡–”ƒ• ”‹’–‹‘•–ƒ”–•‹–‡•‘ˆ–Š‡–Š”‡‡•‹‰Ž‡—–ƒ–•™‡”‡†‡–‡”‹‡†„› ͷǯ ‘  •ƒ’Ž‡• ‘ˆ ‘Ǧ‹†— ‡† ƒ†  ‹†— ‡† —Ž–—”‡•ǡ –‘ Š‡  ™Š‡–Š‡” —–ƒ–‹‘•‹–Š‡•Šƒ†ƒ‡ˆˆ‡ –‘–Š‡‡šƒ –•–ƒ”–•‹–‡‘ˆhacAuƒ†hacAiǤ‹‡ ‹ ™‹Ž† –›’‡ǡ ‹ ƒŽŽ –Š”‡‡ —–ƒ–• ȋͳǡ ʹ ƒ† ͵Ȍ –Š‡ ˆ—ŽŽ Ž‡‰–Š hacA •–ƒ”–‡† ƒ– ͵Ͳ͵ – —’•–”‡ƒ ‘ˆ –Š‡  Ǥ

(79)   •–”‡••‡† —Ž–—”‡• ‘ˆ –Š‡ ͳ ƒ† ͵—–ƒ–ǡ–Š‡–”ƒ• ”‹’–‹‘•–ƒ”–•‹–‡‘ˆ–Š‡–”— ƒ–‡†hacA™ƒ•ˆ‘—†͹͵– —’•–”‡ƒ‘ˆ–Š‡ ǡƒ•‹™‹Ž†–›’‡Ǥ. ThehacATranscriptionalSwitch95.

(80) Chapter4. 96ThehacATranscriptionalSwitch.

(81) Chapter4        Figure  3. Š‡ ”‘Ž‡ ‘ˆ –Š‡ • ‘ –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ hacAǤ (A)  Š‡ƒ–‹  ‘˜‡”˜‹‡™ ‘ˆ –Š‡ ’”‘‘–‡””‡‰‹‘‘ˆhacAǤ

(82) †‹ ƒ–‡†„›„Žƒ „‘š‡•ƒ”‡–Š‡–”ƒ• ”‹’–‹‘•–ƒ”–’‘‹–•‘ˆhacAuƒ†  hacAiǤ‡’”‡•‡–‡†„›‘’‡„‘š‡•ƒ”‡–Š‡•ǤŠ‡„‹†‹‰ ‘•–ƒ–‘ˆ‡ƒ Š‹••Š‘™ —†‡”‡ƒ–Š–Š‡ˆ‹‰—”‡Ǥ(B)••Š‘™‹‰–Š‡‹ƒ„‹Ž‹–›‘ˆ ƒ –‘„‹†–‘–Š‡—–ƒ–‡†•Ǥ Š‡•‡“—‡ ‡•‘ˆ–Š‡͵Ͷ„’‘Ž‹‰‘•”‡’”‡•‡–‹‰–Š‡•ƒ”‡•Š‘™ǡƒ†—–ƒ–‹‘•‹–”‘†— ‡† ‹‡ƒ Š–‘ƒ„‘Ž‹•Š„‹†‹‰„› ƒ ƒ”‡‹†‹ ƒ–‡†‹italicsƒ†ƒ”‡underlinedǤ(C)‘”–Š‡” ƒƒŽ›•‹••Š‘™‹‰–Š‡‡ˆˆ‡ –‘ˆ—–ƒ–‡†•‘–”ƒ• ”‹’–‹‘Ž‡˜‡Ž•‘ˆhacAǡbipAƒ†pdiAǤ •–”‡••™ƒ•‹’‘•‡†„›–Š‡ƒ††‹–‹‘‘ˆʹͲ–‘–Š‡‡†‹ƒǤ(D)‡Žƒ–‹˜‡hacAŽ‡˜‡Ž• ‹–Š‡†‹ˆˆ‡”‡–—–ƒ–•†—”‹‰‘Ǧ•–”‡•• ‘†‹–‹‘•Ǥ. ThehacATranscriptionalSwitch97.

(83) Chapter4  . Figure4.Š‡‘–›’‡•‘ˆ–Š‡•–”ƒ‹•—•‡†‹–Š‹••–—†›Ǥ(A) ”‘™–Š‘ˆ–Š‡™‹Ž†–›’‡ƒ†—–ƒ– •–”ƒ‹• ‘ ‘’Ž‡–‡ ȋȌ ‡†‹ƒ ’Žƒ–‡• ‹ –Š‡ ƒ„•‡ ‡ ȋ—’’‡” ”‘™Ȍ ‘” ‹ –Š‡ ’”‡•‡ ‡ ȋŽ‘™‡” ”‘™Ȍ ‘ˆ ͳͲ Ɋ‰ȀŽ –—‹ ƒ› ‹Ǥ (B) ‹ ”‘• ‘’‡ ‹ƒ‰‡• ‘ˆ –Š‡ ™‹Ž† –›’‡ •–”ƒ‹ ȋ™–Ȍǡ –Š‡ hacA †‡Ž‡–‹‘ •–”ƒ‹ ȋhacAȌǡ –Š‡ •–”ƒ‹ „‡ƒ”‹‰ –Š‡ ‹–”‘ǦŽ‡•• hacA ‰‡‡ ȋȟ‹–”‘Ȍǡ ƒ† –Š‡ •–”ƒ‹ „‡ƒ”‹‰–Š‡—•’Ž‹ ƒ„Ž‡hacA˜ƒ”‹ƒ–ȋ—–Ǧ‹–”‘ȌǤŠ‡„ƒ”‹†‹ ƒ–‡•ͳɊǤ. 98ThehacATranscriptionalSwitch.

(84) Chapter4 LongrangeBasePairingandTranslationalBlock ‘‰Ǧ”ƒ‰‡ „ƒ•‡ ’ƒ‹”‹‰ ™ƒ• •Š‘™ –‘ ’”‡˜‡– –Š‡ —‹†— ‡† ˆ‘” ‘ˆ –Š‡ ›‡ƒ•– HAC1 Š‘‘Ž‘‰—‡ˆ”‘„‡‹‰‡ˆˆ‹ ‹‡–Ž›–”ƒ•Žƒ–‡†ͳ͵͹Ǥƒ”–‘ˆ–Š‡ʹͷʹ–‹–”‘‘ˆ–Š‡›‡ƒ•–HAC1u  ˆ‘”• ƒ •–ƒ„Ž‡ †‘—„Ž‡Ǧ•–”ƒ†‡† •–”— –—”‡ „› „ƒ•‡ ’ƒ‹”‹‰ ™‹–Š –Š‡ ͷǯǡ –Š‡”‡„› ’”‡˜‡–‹‰ –Š‡ ”‹„‘•‘‡• ˆ”‘ ”‡ƒ†‹‰ –Š”‘—‰ŠǤ  ‘˜‡–‹‘ƒŽ ‹–”‘ •’Ž‹ ‹‰ ”‡‘˜‡• –Š‡ʹͷʹ–‹–”‘ǡƒ†™‹–Š‹––Š‡”‹‰Š–ŠƒŽˆ‘ˆ–Š‡‹˜‡”–‡†”‡’‡ƒ–ǡ–Š‡”‡„›”‡Ž‡ƒ•‹‰HAC1u ˆ”‘‹–•–”ƒ•Žƒ–‹‘„Ž‘ Ǥ  ‹†‡–‹ ƒŽ ‡ Šƒ‹• ‹• ‘– ’‘••‹„Ž‡ ˆ‘” –Š‡ A. niger hacAu Ǥ Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ‘ˆ hacA ‹• ‘Ž› ʹͲ – Ž‘‰ǡ ƒ† ƒ •–ƒ„Ž‡ •–‡ǦŽ‘‘’ •–”— –—”‡ ‹• ’”‡†‹ –‡†ˆ‘”–Š‹••‡“—‡ ‡ǡ™Š‹ ŠŽ‡ƒ˜‡•‘— Ž‡‘–‹†‡•ƒ˜ƒ‹Žƒ„Ž‡ˆ‘”ƒ’—–ƒ–‹˜‡Ž‘‰Ǧ”ƒ‰‡ „ƒ•‡ ’ƒ‹”‹‰ ™‹–Š –Š‡ ͷǯǤ ‘™‡˜‡”ǡ •‡“—‡ ‡ ƒƒŽ›•‡• ”‡˜‡ƒŽ‡† ƒ ʹ͸ – Ž‘‰ ‹˜‡”–‡† ”‡’‡ƒ–‹–Š‡hacAuȋ ‹‰ǤͷȌǤŠ‹•”‡’‡ƒ– ‘–ƒ‹•ͳͺ„ƒ•‡’ƒ‹”•ǡ‘ˆ™Š‹ Šͳʹƒ”‡  ’ƒ‹”•Ǥ

(85)  ‘’ƒ”‹•‘ǡ–Š‡›‡ƒ•–HAC1‹˜‡”–‡†”‡’‡ƒ–•–”‡– Š‡•‘—–‘˜‡”ͳͻ–ǡƒ†Šƒ•ͳ͸ „ƒ•‡’ƒ‹”•‘ˆ™Š‹ Šͳͳƒ”‡ ’ƒ‹”•ǤŠ‡Ž‡ˆ–ŠƒŽˆ‘ˆ–Š‡‹˜‡”–‡†”‡’‡ƒ–‹–Š‡A.nigerhacA ‹• Ž‘ ƒ–‡† ‹ –Š‡ ͶͶǦ ‘†‘ — ǡ ƒ† –Š‡ ”‹‰Š– ŠƒŽˆ ‹ –Š‡ hacA   Ž‘•‡ –‘ –Š‡  Ǥ ”— ƒ–‹‘ —’‘ Ǧ•–”‡•• ”‡‘˜‡• ƒ’’”‘š‹ƒ–‡Ž› ʹ͵Ͳ – ‘ˆ –Š‡ ͷǯ ‘ˆ hacA  ‹ Ž—†‹‰–Š‡— ƒ†–Š‡”‡„›–Š‡Ž‡ˆ–ŠƒŽˆ‘ˆ–Š‡‹˜‡”–‡†”‡’‡ƒ–ǡ’‘••‹„Ž›”‡Ž‡ƒ•‹‰hacA ˆ”‘ƒ–”ƒ•Žƒ–‹‘ƒŽ„Ž‘ ƒƒŽ‘‰‘—•–‘–Š‡›‡ƒ•–HAC1‡ Šƒ‹•Ǥ ‘†‡–‡”‹‡–Š‡‡ˆˆ‡ –‘ˆ–Š‡’—–ƒ–‹˜‡„ƒ•‡’ƒ‹”‹‰–‘–Š‡”‡‰—Žƒ–‹‘‘ˆhacAǡ„‘–Š ŠƒŽˆǦ•‹–‡•‘ˆ–Š‡‹˜‡”–‡†”‡’‡ƒ–™‡”‡—–ƒ–‡†Ǥ‹ ‡„‘–ŠŠƒŽˆǦ•‹–‡•ƒ”‡Ž‘ ƒ–‡†‹ ‘†‹‰ ”‡‰‹‘•ǡ ‘Ž› •‹Ž‡– —–ƒ–‹‘• ™‡”‡ ‹–”‘†— ‡† –‘ †‹•”—’– –Š‡ ’—–ƒ–‹˜‡ „ƒ•‡ ’ƒ‹”‹‰ ȋ ‹‰Ǥ ͷȌǤŽ–Š‘—‰Š–Š‹••–”ƒ–‡‰›Ž‡ˆ–͸„ƒ•‡’ƒ‹”•‹–ƒ –ǡ‹–‹•Ž‹‡Ž›–Šƒ–ƒ•–ƒ„Ž‡„ƒ•‡’ƒ‹”‹‰‘ˆ „‘–Š ŠƒŽˆǦ•‹–‡• ‹• ‘ Ž‘‰‡” ’‘••‹„Ž‡Ǥ ƒŽ›•‹• ‘ˆ –Š‡ 

(86)  •–”ƒ‹ „› ‘”–Š‡” „Ž‘–• •Š‘™‡† –Šƒ–—†‡”‘”ƒŽ‰”‘™–Š ‘†‹–‹‘•ƒ’’”‘š‹ƒ–‡Ž›͵–‹‡•‘”‡bipAƒ†pdiA–”ƒ• ”‹’– ™‡”‡’”‡•‡– ‘’ƒ”‡†–‘™‹Ž†Ǧ–›’‡ȋ ‹‰ǤͷȌǡ‹†‹ ƒ–‹‰Š‹‰Š‡”„ƒ•ƒŽŽ‡˜‡Ž•‘ˆ ƒ ‹–Š‹• •–”ƒ‹Ǥ†‡”•–”‡••–Š‡–”ƒ• ”‹’–‹‘ƒŽ’ƒ––‡”‘ˆhacAǡbipAƒ†pdiA™ƒ••‹‹Žƒ”ƒ•‹ ™‹Ž†–›’‡•–”ƒ‹Ǥ   TheUnconventionalIntron

(87)  ‘”†‡” –‘ •–—†› –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ •’Ž‹ ‹‰ ™‡ ƒ‹‡† ƒ– ‘•–”— –‹‰ ƒ ireA •–”ƒ‹Ǥ ‘™‡˜‡”ǡ —Ž–‹’Ž‡ ƒ––‡’–• ƒ† –‡ Š‹“—‡• –‘ ‘•–”— – •— Š ƒ •–”ƒ‹ ƒŽŽ ˆƒ‹Ž‡† ’”‡•—ƒ„Ž› „‡ ƒ—•‡ ‘ˆ –Š‡ Ž‡–ŠƒŽ‹–› ‘ˆ –Š‡ †‡Ž‡–‹‘Ǥ Š‡”‡ˆ‘”‡ǡ –Š‡ ‹’‘”–ƒ ‡ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ‹–”‘ˆ‘”–Š‡™ƒ•†‡–‡”‹‡†‹–Š‡ˆ‘ŽŽ‘™‹‰–™‘™ƒ›•Ǣ„›ƒƒŽ›•‹• ‘ˆƒ•–”ƒ‹ ƒ””›‹‰–Š‡hacA‰‡‡Žƒ ‹‰–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘ǡƒ†„›ƒ•–”ƒ‹Šƒ˜‹‰ ƒhacA˜ƒ”‹ƒ–™‹–Šƒ—•’Ž‹ ‡ƒ„Ž‡— ‘˜‡–‹‘ƒŽ‹–”‘Ǥ‡‘˜ƒŽ‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ ‹–”‘ˆ”‘–Š‡hacA‰‡‡”‡•—Ž–‡†‹ƒ ‘•–‹–—–‹˜‡ȋ ‹‰ǤͷȌǤ. ThehacATranscriptionalSwitch99.

(88) Chapter4. 100ThehacATranscriptionalSwitch.

(89) Chapter4 Figure 5. Š‡ ˆ— –‹‘ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ǡ ƒ† Ž‘‰Ǧ”ƒ‰‡ „ƒ•‡ ’ƒ‹”‹‰ ‹ –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ –Š‡ Ǥ (A)  Š‡ƒ–‹  ‘˜‡”˜‹‡™ ‹†‹ ƒ–‹‰ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ƒ† –Š‡ Ž‘ ƒ–‹‘‘ˆ–Š‡‹˜‡”–‡†”‡’‡ƒ–•‹”‡Žƒ–‹‘–‘‘–Š‡”‡Ž‡‡–•’”‡•‡–‹hacAǤŠ‡hacA ‹• ‹†‹ ƒ–‡† „› ƒ •‘Ž‹† „Žƒ  ƒ””‘™ǡ ƒ† –Š‡ ͷǯ „› ƒ ‰”‡› „‘šǤ Š‡ ‹˜‡”–‡† ”‡’‡ƒ–• ȋ

(90) Ȍ ƒ”‡ †‡’‹ –‡† „› ‘’‡ „‘š‡•ǡ ƒ† –Š‡ „ƒ•‡ ’ƒ‹”‹‰ ‘ˆ –Š‡ ‹˜‡”–‡† ”‡’‡ƒ–• —†‡”‡ƒ–ŠǤ Š‡ –Š‡‘”‡–‹ ƒŽŽ›”‡ƒ‹‹‰„ƒ•‡’ƒ‹”‹‰•ƒˆ–‡”‹–”‘†— –‹‘‘ˆ•‹Ž‡–—–ƒ–‹‘•‹•ƒŽ•‘‹†‹ ƒ–‡†ǤŠ‡ •–”— –—”‡ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ ‹• †‡’‹ –‡† „› –Š‡ •–‡ǦŽ‘‘’ •–”— –—”‡ǡ ƒ† —–ƒ–‹‘• ‹–”‘†— ‡†–‘’”‡˜‡–•’Ž‹ ‹‰„›†‹•”—’–‹‰–Š‡•–”— –—”‡ƒ”‡‹†‹ ƒ–‡†ǤŠ‡‹–”‘•‡“—‡ ‡‹• •Š‘™‹Ž‘™‡” ƒ•‡ƒ† Ž‡ƒ˜ƒ‰‡•‹–‡•‹†‹ ƒ–‡†„›ƒ””‘™•Ǥ(B)‘”–Š‡”„Ž‘–ƒƒŽ›•‹••Š‘™‹‰ –Š‡‡ˆˆ‡ –‘ˆ–Š‡†‹ˆˆ‡”‡–—–ƒ–‹‘•‘”†‡Ž‡–‹‘•‘–Š‡‡š’”‡••‹‘ƒ†–”— ƒ–‹‘‘ˆhacAǡƒ† ‘–Š‡–”ƒ• ”‹’–Ž‡˜‡Ž•‘ˆbipAƒ†pdiAǤ•–”‡••™ƒ•‹’‘•‡†„›–”‡ƒ–‡–™‹–Š–—‹ ƒ› ‹ǡ ƒ†–Š‡‰‡‡ ‘†‹‰ˆ‘”ͳͺ”™ƒ•—•‡†ƒ• ‘–”‘Ž’”‘„‡Ǥ(C)Ǧ•Š‘™‹‰–Š‡–”ƒ• ”‹’– Ž‡˜‡Ž•‘ˆbipAƒ†pdiA‹–Š‡™‹Ž†–›’‡ƒ†–Š‡—–Ǧ‹–”‘•–”ƒ‹ǤŠ‡ͶŽƒ‡•‹‡ƒ Š ‘†‹–‹‘ ‘””‡•’‘†–‘ͳͲǡͳͷǡʹͲǡƒ†ʹͷ › Ž‡•Ǥ.    ‘–Šǡ –Š‡ ˆ—ŽŽǦŽ‡‰–Š ƒ† –Š‡ –”— ƒ–‡† hacA –”ƒ• ”‹’–• ™‡”‡ ƒ„—†ƒ–Ž› ’”‡•‡– —†‡” ‘”ƒŽ ‰”‘™–Š ‘†‹–‹‘•ǡ ƒ† –Š‡ Ž‡˜‡Ž• ‘ˆ bipA ƒ† pdiA ™‡”‡ ”‡•’‡ –‹˜‡Ž› ͳͻǦˆ‘Ž† ƒ† ͳ͵Ǧˆ‘Ž†Š‹‰Š‡” ‘’ƒ”‡†–‘–Š‡Ž‡˜‡Ž•‡ƒ•—”‡†‹–Š‡™‹Ž†Ǧ–›’‡•–”ƒ‹†—”‹‰‘Ǧ•–”‡•• ‘†‹–‹‘•Ǥ

(91) †— –‹‘‘ˆǦ•–”‡••„›–Š‡ƒ††‹–‹‘‘ˆ–—‹ ƒ› ‹Šƒ†‘ƒ††‹–‹‘ƒŽ‡ˆˆ‡ –‘–Š‡ ”‡•’‘•‡‹–Š‹••–”ƒ‹ǡ‹†‹ ƒ–‹‰–Šƒ–•’Ž‹ ‹‰‘ˆ–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘‹••—ˆˆ‹ ‹‡––‘ ˆ—ŽŽ›‹†— ‡–Š‡’ƒ–Š™ƒ›Ǥ‘”–Š‡”„Ž‘–ƒƒŽ›•‹•‘ˆƒ•–”ƒ‹ ‘–ƒ‹‹‰ƒ—•’Ž‹ ‡ƒ„Ž‡hacA ˜ƒ”‹ƒ– ȋ—– ‹–”‘Ȍ ”‡˜‡ƒŽ‡† –Šƒ– •’Ž‹ ‹‰ ‘ˆ –Š‡ — ‘˜‡–‹‘ƒŽ ‹–”‘ǡ ƒ† ƒ• ƒ ”‡•—Ž– ƒ ‹ǡ ‹• ‡ ‡••ƒ”› –‘ ƒ –‹˜ƒ–‡ –Š‡ †‘™•–”‡ƒ ’ƒ–Š™ƒ› ȋ ‹‰Ǥ ͷȌǤ ”— ƒ–‹‘ ‘ˆ hacA ‹ ”‡•’‘•‡ –‘ –—‹ ƒ› ‹ ‹†— ‡† •–”‡•• ƒ† –Š‡ ƒ ‘’ƒ‹‡† —’”‡‰—Žƒ–‹‘ ‘ˆ bipA ƒ† pdiA†‹†‘––ƒ‡’Žƒ ‡‹–Š‹••–”ƒ‹ǤbipAƒ†pdiA–”ƒ• ”‹’–• ‘—Ž†‘–‡˜‡„‡†‡–‡ –‡† ‘–Š‡‘”–Š‡”„Ž‘–‹„‘–Š ‘†‹–‹‘•Ǥ

(92) –Šƒ•„‡‡”‡’‘”–‡†–Šƒ–†‡Ž‡–‹‘‘ˆbipA‹•Ž‡–ŠƒŽ ˆ‘”Aspergillusͳ͵ͲǤŽ•‘ǡƒ––‡’–•–‘†‡Ž‡–‡pdiA‹A.niger™‡”‡—•— ‡••ˆ—ŽͳͲͷǤŠ‡”‡ˆ‘”‡ǡ –Š‡ƒ’’ƒ”‡–ƒ„•‡ ‡‘ˆƒbipAƒ†pdiA–”ƒ• ”‹’–‘–Š‡‘”–Š‡”„Ž‘–‘ˆ–Š‡—–Ǧ‹–”‘ •–”ƒ‹‹•‘•–Ž‹‡Ž›ƒ”‡•—Ž–‘ˆ–Š‡Ž‹‹–‡†‡š’‘•—”‡–‹‡‘ˆ–Š‡Ǧ”ƒ›ˆ‹ŽǤ‘’”‘˜‡–Š‹•ǡ Ǧ’‡”ˆ‘”‡†‘ˆ”‘–Š‡—–Ǧ‹–”‘•–”ƒ‹™Š‹ Š”‡˜‡ƒŽ‡†–Šƒ–„‘–ŠbipAƒ† pdiA–”ƒ• ”‹’–•™‡”‡‹†‡‡†’”‡•‡–‹–Š‹••–”ƒ‹ƒŽ„‡‹–ƒ–Ž‘™‡”Ž‡˜‡Ž• ‘’ƒ”‡†–‘–Š‡ ™‹Ž†–›’‡•–”ƒ‹ȋ ‹‰ǤͷȌǤ ‡ƒ”ƒ„Ž›ǡ „‘–Š –Š‡ ‹–”‘ ƒ† —–Ǧ‹–”‘ •–”ƒ‹ †‹•’Žƒ›‡† Ž‡ƒ” ’Š‡‘–›’‡•Ǥ Š‡‰”‘™‘ƒ‰ƒ”’Žƒ–‡•–Š‡‹–”‘•–”ƒ‹’”‘†— ‡†ˆ‡™‡” ‘‹†‹ƒ–Šƒ–Š‡™‹Ž†–›’‡ •–”ƒ‹ǡ ™Š‡”‡ƒ• •’‘”—Žƒ–‹‘ ™ƒ• ƒŽ‘•– ƒ„•‡– ‹ –Š‡ —–Ǧ‹–”‘ •–”ƒ‹ ȋ ‹‰Ǥ ͶȌǤ ‹ ”‘• ‘’‹  ‹•’‡ –‹‘ ‘ˆ –Š‡ —–Ǧ‹–”‘ •–”ƒ‹ ‰”‘™ ‹ –Š‡ ’”‡•‡ ‡ ‘ˆ –—‹ ƒ› ‹. ThehacATranscriptionalSwitch101.

(93) Chapter4 ”‡˜‡ƒŽ‡† ƒ ’Š‡‘–›’‡ •Ž‹‰Š–Ž› ‘’ƒ”ƒ„Ž‡ –‘ –Š‡ hacA •–”ƒ‹Ǣ ’‘‘” ‰”‘™–Š ƒ† •™‘ŽŽ‡ Š›’Šƒ‡ȋ ‹‰ǤͶȌǤ . ’’ƒ”‡–Ž›ǡ„‘–Š ƒ ‹ƒ† ƒ —Šƒ˜‡’Š›•‹‘Ž‘‰‹ ƒŽˆ— –‹‘•‹A.nigerǡƒ†–Š‡. Žƒ  ‘ˆ ‡‹–Š‡” ‘‡ ”‡•—Ž–• ‹ –Š‡ ‘„•‡”˜‡† ’Š‡‘–›’‡Ǥ Š‡ ’Š‡‘–›’‡ ‘ˆ –Š‡ •–”ƒ‹ Šƒ˜‹‰ –Š‡ ‹–”‘ǦŽ‡•• ˜ƒ”‹ƒ– ‘ˆ –Š‡ hacA ‰‡‡ǡ ƒ† –Š‡”‡ˆ‘”‡ ‘Ž› ƒ’ƒ„Ž‡ ‘ˆ ’”‘†— ‹‰ ƒ ‹ ’”‘–‡‹ǡ‹•‘–‡”‡Ž›ƒ”‡•—Ž–‘ˆ’‡”ƒ‡–ƒ –‹˜ƒ–‹‘‘ˆ–Š‡Ǥ˜‡”‡š’”‡••‹‘‘ˆhacAi ‹ƒ™‹Ž†–›’‡„ƒ ‰”‘—†ǡŽ‡ƒ†‹‰–‘ƒ•–”ƒ‹™Š‹ Š ‘•–‹–—–‹˜‡Ž›’”‘†— ‡• ƒ ‹’”‘–‡‹ „—–ƒŽ•‘ ƒ’ƒ„Ž‡‘ˆ’”‘†— ‹‰ ƒ —’”‘–‡‹ˆ”‘‹–•‡†‘‰‡‘—•hacA‰‡‡ǡ†‹†‘–Ž‡ƒ†–‘ •‹‹Žƒ”’Š‡‘–›’‹ ƒŽ Šƒ‰‡•ȋ”‡•—Ž–•‘–•Š‘™ȌǤŠ‹•‹•‹ƒ‰”‡‡‡–™‹–Š–Š‡’”‡˜‹‘—• ‘„•‡”˜ƒ–‹‘–Šƒ––Š‡ƒ‹‘ƒ ‹†•‡“—‡ ‡‘ˆ–Š‡Ǧ–‡”‹ƒŽ’ƒ”–‘ˆ–Š‡ ƒ —ˆ”‘†‹ˆˆ‡”‡– Aspergilli ‹• Š‹‰ŠŽ› ‘•‡”˜‡† ͳͲͳǤ Š‡ ’”‡ ‹•‡ ”‘Ž‡ ‘ˆ ƒ — ‹ –Š‡ ˆ—‰—•ǡ Š‘™‡˜‡”ǡ ‹• •—„Œ‡ –‘ˆˆ—”–Š‡”‹˜‡•–‹‰ƒ–‹‘Ǥ  . Discussion 

(94) ‡—ƒ”›‘–‡•ǡ•‡ ”‡–‹‘•–”‡••Ž‡ƒ†•–‘˜ƒ”‹‘—•”‡•’‘•‡•™Š‹ Šƒ‹ƒ–Š‘‡‘•–ƒ•‹•‘ˆ ˆ— –‹‘•Ǥ ‡ ‘ˆ –Š‡ •‡ ”‡–‹‘ •–”‡•• ”‡•’‘•‡• ‹• –Š‡ ǡ ™Š‹ Š ‹• ‘‘ –‘ ƒŽŽ ‡—ƒ”›‘–‹  ‡ŽŽ•Ǥ Š‡”‡ ƒ”‡ Š‘™‡˜‡” †‹ˆˆ‡”‡ ‡• ‹ –Š‡ ‘Ž‡ —Žƒ” ‡ Šƒ‹•• —†‡”Ž›‹‰ –Š‡ƒ‘‰†‹ˆˆ‡”‡–‘”‰ƒ‹••Ǥ

(95) –Š‹••–—†›ǡ™‡†‡• ”‹„‡•‘‡—‹“—‡ˆ‡ƒ–—”‡•‘ˆ–Š‡ A.nigerǡ‘–”‡’‘”–‡†ˆ‘”–Š‡S.cerevisiaeƒ†ƒƒŽ‹ƒǤ

(96) „‘–ŠŽ‘™‡”ƒ†Š‹‰Š‡”‡—ƒ”›‘–‡•ǡƒ –‹˜ƒ–‹‘‘ˆ–Š‡•–ƒ”–•™‹–Š–Š‡•’Ž‹ ‹‰‘ˆ ƒ— ‘˜‡–‹‘ƒŽ‹–”‘ˆ”‘–Š‡hacA–”ƒ• ”‹’–ǡ‡†‹ƒ–‡†„›

(97) ”‡Ǥ’Ž‹ ‹‰‘ˆ–Š‡ʹͲ– ‹–”‘ˆ”‘–Š‡A.nigerhacA‹•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡––‘ƒ –‹˜ƒ–‡–Š‡Ǥ •–”ƒ‹ ‘–ƒ‹‹‰ƒ—•’Ž‹ ‡ƒ„Ž‡˜ƒ”‹ƒ–‘ˆhacA†‹•’Žƒ›‡†’‘‘”‰”‘™–Šƒ†™ƒ•—ƒ„Ž‡–‘ ƒ –‹˜ƒ–‡–Š‡’ƒ–Š™ƒ›ƒ†—’Ǧ”‡‰—Žƒ–‡–Š‡ˆ‘Ž†ƒ•‡•‹”‡•’‘•‡–‘•–”‡••ǡ™Š‡”‡ƒ•ƒ•–”ƒ‹ ‘–ƒ‹‹‰ƒ‹–”‘ǦŽ‡••˜ƒ”‹ƒ–‘ˆ–Š‡hacA‰‡‡†‹•’Žƒ›‡†ƒ ‘•–‹–—–‹˜‡ȋ ‹‰ǤͷȌǤ

(98)  ƒ††‹–‹‘ –‘ –Š‡ ”ƒ–Š‡” †”ƒ•–‹  ‘Ȁ‘ˆˆ •™‹– Š ‘ˆ — ‘˜‡–‹‘ƒŽ ‹–”‘ •’Ž‹ ‹‰ǡ ‘–Š‡” ”‡‰—Žƒ–‘”› ‡Ž‡‡–• ƒ”‡ ’”‡•‡– ‹ –Š‡ ’”‘‘–‡” ‘ˆ –Š‡ hacA ‰‡‡ ƒ† ‹ –Š‡ hacAu –”ƒ• ”‹’–ǡ™Š‹ Š’”‘˜‹†‡•–Š‡ˆ—‰—•ƒ††‹–‹‘ƒŽ”‡‰—Žƒ–‘”› ‘–”‘Ž‘˜‡”–Š‡”‡•’‘•‡Ǥ ‘‰Ǧ”ƒ‰‡„ƒ•‡’ƒ‹”‹‰‹–Š‡hacA‡••‡‰‡”’”‡˜‡–•‡ˆˆ‹ ‹‡––”ƒ•Žƒ–‹‘‘ˆ–Š‡ ˆ—ŽŽŽ‡‰–Š–”ƒ• ”‹’–ȋ ‹‰ǤͷȌǤ’’‘•‡†–‘–Š‡•‹–—ƒ–‹‘‹S.cerevisiaeǡ™Š‡”‡’ƒ‹”‹‰–ƒ‡• ’Žƒ ‡„‡–™‡‡–Š‡ͷǯƒ†–Š‡— ‘˜‡–‹‘ƒŽ‹–”‘ǡ–Š‡ͷǯ‘ˆ–Š‡AspergillushacA ˆ‘”•ƒ†‘—„Ž‡•–”ƒ†‡†•–”— –—”‡™‹–Š–Š‡ ‘†‹‰”‡‰‹‘‘ˆ–Š‡–”ƒ• ”‹’–ǡ”‡•—Ž–‹‰ ‹–”ƒ•Žƒ–‹‘ƒŽƒ––‡—ƒ–‹‘ȋ ‹‰ǤͷȌǤ

(99) –Š‡ ƒ•‡‘ˆA.nigerǡ–Š‡”‡Ž‡ƒ•‡‘ˆ–Š‡–”ƒ•Žƒ–‹‘ƒŽ „Ž‘  —’‘  •–”‡•• ‹• ‡š‡”–‡† ˜‹ƒ ƒ •™‹– Š –‘ ƒ ƒŽ–‡”ƒ–‹˜‡ –”ƒ• ”‹’–‹‘ •–ƒ”– Ž‘ ƒ–‡† ƒ’’”‘š‹ƒ–‡Ž› ʹ͵Ͳ „’ †‘™•–”‡ƒ ‘ˆ –Š‡ —•—ƒŽ –”ƒ• ”‹’–‹‘ •–ƒ”– •‹–‡Ǥ Š‹• •™‹– Š ‹• 102ThehacATranscriptionalSwitch.

(100) Chapter4 †‡’‡†‡–‘ –Š‡ ʹ ƒ† ‘ ƒ  ‹–•‡Žˆǡ –Š—• ‘ˆ‹”‹‰ –Š‡ ƒ—–‘Ǧ”‡‰—Žƒ–‹‘ ‘ˆ hacA –”ƒ• ”‹’–‹‘ǡ†‡‘•–”ƒ–‡†’”‡˜‹‘—•Ž› ͳͲͳǤʹ‹•–Š‡ ƒ –ƒ”‰‡–‘•–’”‘š‹ƒŽ–‘–Š‡  ƒ†Ž‘ ƒ–‡†ͺͳ„’—’•–”‡ƒ‘ˆ–Š‡•–ƒ”–•‹–‡‘ˆhacAiǤ—–ƒ–‹‘‘ˆʹǡ‡‹–Š‡” ‹†‹˜‹†—ƒŽŽ›‘”‹ ‘„‹ƒ–‹‘™‹–Šͳƒ†͵ǡƒ„‘Ž‹•Š‡†–Š‡ƒ„‹Ž‹–›–‘•›–Š‡•‹•‡ –Š‡ •Š‘”–‡” –”ƒ• ”‹’– †—”‹‰  •–”‡•• ȋ ‹‰Ǥ ͵Ȍǡ ‹†‹ ƒ–‹‰ ‹–• ‹’‘”–ƒ ‡ ˆ‘” –Š‡ –”ƒ• ”‹’–‹‘ƒŽ•™‹– ŠǤŠ‡ˆƒ ––Šƒ––Š‡–”ƒ• ”‹’–‹‘ƒŽ•™‹– Š†‡’‡†•‘ ƒ ‹–•‡Žˆ™ƒ• •Š‘™ „› –™‘ ‡š’‡”‹‡–•Ǥ ‹”•–Ž›ǡ ƒ •–”ƒ‹ ‘–ƒ‹‹‰ –Š‡ hacA ‰‡‡ ™‹–Š‘—– –Š‡ — ‘˜‡–‹‘ƒŽ‹–”‘ǡƒ†–Š‡”‡ˆ‘”‡‘Ž› ƒ’ƒ„Ž‡‘ˆ’”‘†— ‹‰–Š‡ƒ –‹˜‡ˆ‘”‘ˆ–Š‡ ƒ  ’”‘–‡‹ǡ’”‘†— ‡†„‘–Š–Š‡ˆ—ŽŽŽ‡‰–Šƒ†–Š‡–”— ƒ–‡†hacA†—”‹‰‘”ƒŽ‰”‘™–Š ‘†‹–‹‘•ȋ ‹‰ǤͷȌǤ‡ ‘†Ž›ǡƒ•–”ƒ‹ ƒ””›‹‰ƒ—•’Ž‹ ‡ƒ„Ž‡˜‡”•‹‘‘ˆ–Š‡hacA‰‡‡ȋ ˆǤ —–Ǧ‹–”‘ •–”ƒ‹ ‹‰Ǥ ͷȌǡ ƒ† –Š‡”‡ˆ‘”‡ —ƒ„Ž‡ –‘ ’”‘†— ‡ –Š‡ ƒ –‹˜‡ ˆ‘” ‘ˆ –Š‡ ƒ  ’”‘–‡‹ǡ™ƒ•—ƒ„Ž‡–‘•›–Š‡•‹•‡–Š‡–”— ƒ–‡†‡••‡‰‡”‹”‡•’‘•‡–‘•–”‡••Ǥ Š‡”‡ƒ• ƒ Ž‡ƒ” ˆ— –‹‘ ƒ „‡ ƒ••‹‰‡† –‘ –Š‡ ʹǡ –Š‡ ”‘Ž‡• ‘ˆ ͳ ƒ† ͵‹–Š‡”‡‰—Žƒ–‹‘‘ˆhacAƒ”‡•‘‡™Šƒ–‘”‡’—œœŽ‹‰Ǥ–Š‡™Š‘Ž‡ǡ—–ƒ–‹‘‘ˆ ͳ ƒ† ͵ ‹†‹˜‹†—ƒŽŽ› ‘” –‘‰‡–Š‡” Ž‡ˆ– –Š‡ ”‡•’‘•‡ ƒ† –Š‡ ƒ ‘’ƒ›‹‰ –”— ƒ–‹‘ ‘ˆ hacA ‹–ƒ –Ǥ ‘™‡˜‡”ǡ –Š‡•‡ •‹–‡• ‘—Ž† ’”‘˜‹†‡ ƒ ƒ††‹–‹‘ƒŽ ˆ‹‡ –—‹‰ ‡ Šƒ‹• ˆ‘” –Š‡ hacA –”ƒ• ”‹’–‹‘Ǥ Š‡ –Š‡ ͳ ™ƒ• ‹ƒ –‹˜ƒ–‡† ƒŽ‘‡ ‘” ‹ ‘„‹ƒ–‹‘ ™‹–Š ‘–Š‡” •‹–‡• ȋͳǡ ͳΪ͵ǡ ƒ† ͳΪʹΪ͵Ȍǡ –Š‡ „ƒ•ƒŽ Ž‡˜‡Ž ‘ˆ hacA –”ƒ• ”‹’–‹‘ †”‘’’‡† –‘ ƒ’’”‘š‹ƒ–‡Ž› ͷͲΨ ‘’ƒ”‡† –‘ ™‹Ž† –›’‡ ȋ ‹‰Ǥ ͵ȌǤ

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