DE102004047409A1 - Use of laccases as reporter genes - Google Patents

Abstract

A method of determining regulatory properties of a genetic element comprising the steps of: DOLLAR A (a) introducing into a cell a DNA molecule having a DNA I sequence whose regulatory properties are to be determined, wherein the DNA Sequence is linked to another DNA II sequence coding for an Aspergillus nidulans laccase in such a way that the regulatory elements of the DNA-I sequence control the expression of the laccase, DOLLAR A (b) growing under (a) cells under conditions which allow the expression of the laccase, DOLLAR A (c) assigning the conditions chosen under (b) to the regulatory properties of the DNA-I sequence.

Description

The This invention relates to the use of laccases from Aspergillus nidulans as reporter systems in qualitative and quantitative processes Determination of gene expression, especially for qualitative and quantitative determination of promoter strengths, in process of localization of proteins in a cell, as well as methods for the selection of certain recombinant cells as well as in methods for heterologous gene expression.

laccases (EC 1.10.3.2) are blue multi-hybrid enzymes, those of fungi and Plants are produced. They oxidize a number of aromatic ones Compounds such as mono-, di-, and polyphenols, aromatic amines and Methoxy-phenols. During oxidation, the substrate becomes electrons withdrawn, "collected" in the enzyme and then each transfer four electrons to oxygen and water formed. (Messerschmidt & Huber, 1990).

mushrooms usually contain several laccases, which are probably different exercise biological functions. In the Basidiomycenten Coprinus cinereus eight laccases have been described, while in the Ascomycete A. nidulans six laccases occur (Wagner, 2003; Hoegger et al., 2004). The exact biological role of the fungal Laccases is largely unknown. They usually have an N-terminal Secretion signal and are secreted. The best is the function of basidiomycete laccases in lignin degradation (Crestini et al., 2003). In other mushrooms, a function in melanin biosynthesis assumed or in the degradation of phenolic substances (Hermann et al., 1983; Fernández-Larrea & Stahl, 1996).

reporter Gene Such as beta-galactosidase or glucuronidase become diverse in molecular biology and biotechnology. You have, however the disadvantage that the substrates are introduced into the cells have to. In addition, many fungi have endogenous enzymes that are high in basal levels Lead enzyme activities.

Other frequently used reporter genes make the fluorescent proteins (Green Fluorescent Protein GFP, and Yellow Fluorescent Protein, for the formation of fluorescence and are widely used for the localization of proteins but also for the measurement of promoter strengths ( US 5,491,084 ). However, they can not be used well for genetic screens, since the fluorescence signal can only be read relatively insensitive to agar plates.

It It was therefore the task of new methods for genetic screening such as detection of the expression of a particular gene, measurement of the expression level of a genetic regulation signal, detection of the localization of a Protein in the cell (cell compartments), which provide not the o.g. the o.g. Have restrictions and disadvantages.

description the invention

• (a) Einführen eines DNA-Moleküls mit einer DNA-I-Sequenz, deren regulatorische Eigenschaften bestimmt werden sollen, in eine Zelle, wobei die DNA-I-Sequenz mit einer weiteren DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente der DNA-I-Sequenz die Expression der Laccase kontrollieren,
• (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression der Laccase erlauben,
• (c) Zuordnen der unter (b) gewählten Bedingungen zu den regulatorischen Eigenschaften der DNA-I-Sequenz.

The present invention relates, in a first embodiment, to a method for determining regulatory properties of a genetic element comprising the steps:

• (A) introducing into a cell a DNA molecule having a DNA I sequence whose regulatory properties are to be determined, wherein the DNA I sequence is linked to another DNA II sequence coding for a laccase from Aspergillus nidulans is linked so that the regulatory elements of the DNA I sequence control the expression of the laccase,
• (b) culturing the cells obtained under (a) under conditions which allow the expression of the laccase,
• (c) assigning the conditions chosen under (b) to the regulatory properties of the DNA I sequence.

• (a) Einführen eines DNA-Moleküls, das eine DNA Sequenz besitzt, die für das gewünschte Protein codiert, und die so mit einer weiteren DNA-II-Sequenz verknüpft ist, die für eine Laccase aus Aspergillus nidulans codiert, das das Protein, das von dem DNA-Molekül codiert wird, eine Fusion aus dem gewünschten Protein mit der Laccase darstellt, in die Zellen,
• (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des fusionierten Proteins erlauben,
• (c) Nachweis der Lokalisation der Laccase in einem Zellkompartiment und damit der Lokalisation des gewünschten Proteins in diesem Zellkompartiment, insbe sondere durch Zellfraktionierung und anschliessende Bestimmung der Laccaseaktivität in den verschiedenen Fraktionen.

In a second embodiment, the invention relates to a method for the localization of a desired protein in a cell compartment comprising the steps:

• (a) introducing a DNA molecule having a DNA sequence coding for the desired protein and thus linked to another DNA II sequence coding for an Aspergillus nidulans laccase containing the protein, the is encoded by the DNA molecule, represents a fusion of the desired protein with the laccase into the cells,
• (b) culturing the cells obtained under (a) under conditions which allow the expression of the fused protein,
• (c) Detection of the localization of the laccase in a cell compartment and thus the localization of the desired protein in this cell compartment, in particular by cell fractionation and subsequent determination of laccase activity in the various fractions.

• (a) Einführen eines DNA-Moleküls, das eine DNA Sequenz des gewünschten Gens besitzt, in eine Zelle, wobei die DNA-Sequenz mit einer weiteren DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente des gewünschten Gens die Expression der Laccase kontrollieren,
• (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des gewünschten Gens erlauben,
• (c) Nachweis der Expression der Laccase in der Zelle und damit Nachweis der Expression des gewünschten Gens.

In a third embodiment, the invention relates to a method for determining expression of a desired gene in a cell comprising the steps of:

• (A) introducing a DNA molecule having a DNA sequence of the desired gene into a cell, wherein the DNA sequence is linked to a further DNA II sequence coding for an Aspergillus nidulans laccase such that the regulatory elements of the desired gene control the expression of the laccase,
• (b) cultivating the cells obtained under (a) under conditions which allow the expression of the desired gene,
• (c) detecting the expression of the laccase in the cell and thus detecting the expression of the desired gene.

• (a) Einführen eines DNA-Moleküls, das eine DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, besitzt und mit deren C-terminalem Ende eine weitere DNA-I-Sequenz, die für das gewünschte Genprodukt codiert, so verknüft ist, das die Expression als Fusionsprotein aus Laccase und gewünschtem Genprodukt erfolgt,
• (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des gewünschten Gens erlauben,
• (c) Isolierung des Fusionsproteins aus Laccase und gewünschtem Genprodukt.

In a fourth embodiment, the invention relates to a method for expressing a desired gene in a cell comprising the steps of:

• (A) introducing a DNA molecule having a DNA II sequence coding for an Aspergillus nidulans laccase, and having at its C-terminal end a further DNA I sequence coding for the desired gene product, as described above linked, the expression takes place as a fusion protein of laccase and the desired gene product,
• (b) cultivating the cells obtained under (a) under conditions which allow the expression of the desired gene,
• (c) isolating the fusion protein from laccase and desired gene product.

in this connection becomes laccase as reporter gene or reporter protein for heterologous used expressed proteins. Many industrially important proteins become filamentous Mushrooms produced. An attempt is made to secrete the proteins, to facilitate the purification. Laccases can be used to non-secreted To secrete proteins. A C-terminal fusion of a desired Protein with laccase leads to secretion of the protein and, due to the laccase activity, allows the light Detection of the secreted protein. By introducing a protease interface between the two proteins, the desired protein can be released become.

Suitable cells for the above-mentioned methods are herbal, for example carrot roots, animal and human, for example Hela or HEK293, and in particular fungal cells. The processes can be carried out particularly well in cells of the following types:
Alternaria alternata, Clathrospora diplospora, Alternaria brassicae, Alternaria brassicicola, Alternaria raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria microscopica, Ophiobolus herpotrichus, Cucurbitaria elongata, Phoma sp. Leptosphaeria Doliolum, Cucurbitaria Berberidis, Paraphaeosphaeria agavensis Paraphaeosphaeria nolinae, Paraphaeosphaeria obtusispora, Paraphaeosphaeria conglomerata, Paraphaeosphaeria quadriseptat, Paraphaeosphaeria filamentosa, Melanomma Sanguinarium, Paraphaeosphaeria glauco-punct, Septoria nodorum, Phoma herbarum Pleospora rudis, Helminthosporium solani, velutinum Helminthosporium, Leptosphaeria bicolor, Paraphaeosphaeria michotii, Paraphaeosphaeria pilleata, Cucurbidothis pityophila, Bimuria novae-zelandiae, Letendraea helminthicola, Kirschsteiniothelia elaterascu, Helicascus kanaloanus, Herpotrichia diffusa Mycosphaerella mycopappi, Herpotrichia juniperi, Byssothecium circinans, Lophiostoma crenatum, Trematosphaeria heterospora, Kirschsteiniothelia maritima, Sporomia lignicola, Westerdykella cylindrica, Westerdykella dispersa, Delichia didyma, Dendryphiopsis atra, Kirschsteiniothelia aethiops, Sarcinomyces petricola, Phaeosclera dematioides, Sarcinomyces crustaceus, Scytalidium dimidiatum, Scytalidium hyalinum, S cytalidium dimidiatum, Botryosphaeria ribis, Capnobotryella sp., bartschii Capnobotryella renispora, Coccodinium, Hortaea werneckii, Phaeotheca Fissurella, Acrospermum compressum, Acrospermum ramineum, Phaeotrichum benjaminii, Aureobasidium pullulans, Discosphaerina fagi, dothidea hippophaeos, dothidea insculpta, Stylodothis puccinioides, dothidea ribesia, Delphi Ella strobiligena, Myriangium duriaei, Myriangium duriaei, Piedraia hortae, Raciborskiomyces longisetosum, Capnodium citri, Euascomycetes sp., Thecotheus holmskjoldii, Lasallia rossica, Baeomyces rufus, Anamylopsora pulcherrima, Placopsis gelida, Trapelia involuta, Trapelia placodioides, Pertusaria trachytallina, Ochrolechia parella, Coccotrema cucurbitula , Lepolichen coccophorus, Diploschistes rampoddensis, Diploschistes thunbergianus, Graphis scripta, Conotrema populorum, Gyalecta ulmi, Dibaeis, Stenocybe pullatula, Sphinctrina turbinata, Cladonia sulphuri na, Cladonia bellidiflora, Cladonia subcervicornis, Metus conglomeratus, Pilophorus robustus, Pilophorus acicularis, Pilophorus cereolus, Pilophorus strumaticus, Cladia retipora, Stereocaulon paschale, Stereocaulon taeniarum, Stereocaulon vesuvianum, Stereocaulon ramulosum, Lecidella meiococca, Squamarina lentigera, Lecanora intumescens, Alectoria sarmentosa, Letharia columbiana, Vulpicida pinastri, Cetraria slandica, Cornicularia normoerica, Pleurosticta acetabulum, Xanthoparmelia conspersa, Evernia prunastri, Parmelia saxatilis, Hypogymnia physodes, Bunodophoron scrobicu latum, Leifidium tenerum, Sphaerophorus globosus, Sphaerophorus stereocauloides, Neophyllis melacarpa, Exogenous glareosum, Lecania cyrtella, Psora decipiens, Cliostomum griffithii, Toninia sedifolia, Bacidia rosella, Caloplaca flavorubescens, Xanthoria elegans, Xanthoria parietina, Tephromela atra, Thelomma mammosum, Texosporium sancti-jacobi, Cyphelium inquinans, Buellia disciformis, Physcia aipolia, Cyphelium tigillare, Lecanora dispersa, Leprocaulon sp. Lec Aspergillus parasiticus, Aspergillus tamarii, Aspergillus flavus, Aspergillus sojae, Aspergillus nomius, Aspergillus avenaceus, Eurotium herbariorum, Eurotium rubrum, Aspergillus restrictus, Aspergillus clavatus, Aspergillus fumigatus, Neosartorya fischeri, Aspergillus versicolor, Aspergillus oryzae, Aspergillus parasiticus , Aspergillus terreus, Fennellia flavipes, Aspergillus wentii, Chaetosartorya cremea, Aspergillus ochraceus, Aspergillus awamori, Aspergillus niger, Aspergillus candidus, Aspergillus cervinus, Penicillium allii, Penicillium expansum, Penicillium commune, Penicillium freii, Penicillium notatum, Eupenicillium crustaceum, Geosmithia namyslowskii, Penicillium namyslowskii, Eupenicillium javanicum, Chromocleista malachitea, Monascus purpureus, Hamigera avellanea, Merimbla ingelheimense, Hamigera striata, Hemicarpenteles ornatus, Penicilliopsis clavariiformis, Aspergillus zonatus, Aspergillus sparsus, Chromocleista cinnabarina, Chromocleista cinnabarina, Talaromyces flavus, Talaromyces b acillisporus, Talaromyces luteus, Talaromyces luteus, Elaphomyces leveillei, Elaphomyces maculatus, Trichocoma paradoxa, Geosmithia cylindrospora, Talaromyces eburneus, Talaromyces emersonii, Byssochlamys nivea, Thermoascus crustaceus, Malbranchea dendritica, Malbranchea filamentosa, Auxarthron zuffianum, Renispora flavissima, Malbranchea albolutea, Coccidioides immitis, Uncinocarpus reesii, Coccidioides immitis, Onygena equina, Pseudoamauroascus australiensi, Castanedomyces australiensis, Trichophyton rubrum, Malbranchea gypsea, Ascosphaera apis 2, Ascosphaera apis, Eremascus albus, Ajellomyces capsulatus, Histoplasma capsulatum Eukaryo, Histoplasma capsulatum, Paracoccidioides brasiliensis, Lacazia loboi, Lacazia loboi, Exophiala dermatidis, Graphium calicioides, Capronia pilosella, Cladophialophora boppii, Cladophialophora arxii, Fonsecaea pedrosoi, Cladophialophora devriesii, Phialophora verrucosa, Capronia mansonii, Exophiala dermatitidis, Pullularia prototropha, Exophiala dermatitidis, Sarcinomyces phaeomuriformis, Capronia coronata, Capronia epimyces, Rhinocladiella phaeophora, Ramichloridium anceps, Capronia fungicola, Capronia pilosella, Exophiala jeanselmei, Phaeococcomyces exophialae, Phaeoannellomyces elegans, Nadsoniella nigra, Exophiala sp., Rhinocladiella atrovirens, Capronia dactylotricha, Capronia pulcherrima, Capronia acutiseta, Cladophialophora sp., Ceramothyrium linnaeae, Exophiala mansonii, Phialophora sp., Coniosporium perforans, Coniosporium apollinis, Coniosporium per forans, Glyphium elatum, Euascomycetes sp., Rhynchostoma minutum, Ceramothyrium carniolicum, Caliciopsis pineam, Lasioderma serricorne yeast-like, Halosarpheia marina, Halosarpheia viscosa, Phaeonectriella lignicola, Halosarpheia lotica, Halosarpheia retorquens, Nais inornata, Halosarpheia heteroguttulata, Halosarpheia cincinnatula, Halosarpheia fibrosa, Halosarpheia spartinae, Graphium putredinis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Microascus cirrosus, Graphium putredinis, Graphium tectonae, Graphium putredinis, Petriella setifera, Lomentospora prolificans, Ceratocystis fimbriata, Graphium penicillioides, Glomerella cingulata, Verticillium longisporum, dahliae Verticillium, Volutella colletotrichoides, Plectosphaerella cucumerina, Cordyceps yakusimensis, Cordyceps sobolifera, Cordyceps capitata, Cordyceps cochlidiicola, Cordyceps prolifica, Cordyceps inegoensis, inflatum Tolypocladium, Cordyceps paradoxa, Cordyceps japonica, Cordyceps heteropoda, Hamiltonaphis styraci yeast-li, Cordyceps ramosopulvinata, Paecilomyces sp., Cordyceps kanzashiana, Trichoderma koningii, Trichoderma viride, Taphrina deformans, Hypocrea lutea, Hypomyces chrysosperm, Volutella ciliata, Volutella ciliata, Chaetopsina fulva, Gibberella pulicaris, Gibberella moniliformis, Botryotinia Fattyelia, Botryotinia convoluta, Mycoarachis inversa, Cordyceps pruinosa, Cordyceps takaomontana, Botrytis cinerea, Cordyceps brongniartii, Cordyceps militaris, Paecilomyces tenuipes, Bionectria ochroleuca, Nectria ochroleuca, Nectria aureofulva, Nectria cinnabarina, Geosmithia putterillii, Geosmithia lavendula, Lu lworthia fucicola, Chaetomium elatum, Neurospora crassa, Sordaria firmicola, Podospora anserina, Ascovaginospora stellipala, Phyllachora graminis, Kionochaeta ramifera, Kionochaeta spissa, Kionochaeta spissa, Kionochaeta ivoriensis, Meliola juddiana, Meliola niessleana, Neurospora crassa, Ophioceras leptosporum, Magnaporthe grisea, Pseudohalonectria falcata , Cryphonectria cubensis, Cryphonectria havanensis, Endothia gyrosa, leucostoma persoonii, Ophiostoma penicillatum, Pesotum fragrans, Ophiostoma bicolor, piceae Ophiostoma, Ophiostoma floccosum, schenckii Ophiostoma piliferum, Ophiostoma ulmi, Ophiostoma, Ophiostoma stenoceras, Ophiostoma cucullatum, Ophiostoma ainoae europhioides, Ophiostoma, papulosa amerospora, Ascolacicola austriaca, Rhamphoria delicatula, sordariomycete sp., Poronia punctata, Rosellinia necatrix, Xylaria sp., Xylaria polymorpha, Muscodor sp., Barrmaelia oxyacanthae, Hypoxylon fragiforme, Monographella nivalis, Pestalosphaeria hansenii, Pestalosphaeria sp., Hyponectria buxi, Monilinia fructicola , Monilinia laxa , Sclerotinia sclerotiorum, dark septate endophyte DS16b, Euascomycetes sp., Neobulgaria premnophila, Blumeria graminis, Phyllactinia guttata, Spathularia flavida, Cyttaria darwinii, Geomyces pannorum var. pannoru, woolhopensis Thelebolus stercoreus, Ascozonus, Selenaspora guernisacii, Inermisia aggregata, Pyronema domesticum, Otidea onotica, Glaziella aurantiaca, Cephaliophora tropica, Cephaliophora irregularis, Scutellospora castanea, Geopyxis carbonaria Sarcoscypha austriaca, Pseudopithyella minuscula, Kompsoscypha chudei Pithya cupressina, Nanoscypha tetraspora , Phillipsia domingensis, Wynnea sp., Cookeina tricholoma, Microstoma floccosum, Wolfina aurantiopsis, Strobiloscypha keliae, Desmazierella acicola, Neournula pouchetii, Chorioactis geaster, Donadinia sp., Galiella rufa, Plectania rhytidia, Sarcosoma mexicanum, Pseudoplectania nigrella, Tuber borchii, Tuber gibbosum , Choiromyces meandriformis, Choiromyces venosus, Tuber excavatum, Tuber magnatum, Tuber panniferum, Dingleya verrucosa, Reddellomyces donkii, Labyrinthomyces varius, Helvella lacunosa, Wynnella silvicolor, Helvella terrestris, Balsamia vulgaris, Barssia oregonensis, Balsamia magnata, Underwoodia columnaris, Morchella elata 2, Morchella esculenta Verpa conica, Disciotis venosa, Fischerula subcaulis, Leucangium carthusianum, Verpa bohemica, Pseudorhizina californica, Gyromitra esculenta 2, Gyromitra melaleucoides, Discina macrospora, Gyromitra montana, Hydnotrya cerebriformis, Rhizina undulata, Pachyphloeus citrinus, Cazia flexiascus, Terfezia arenaria, Peziza echinospora, Terfezia terfezioides, Orbilia fimicola, Orbilia auricolor, Dactylella oxyspora, Cephaliophora longispora, Cephaliophora muscicola, Arxiozyma Telluris, Saccharomyces transvaalensis, Saccharomyces dairensis, Saccharomyces servazzii, Saccharomyces unisporus, Zygosaccharomyces bailii, Zygosaccharomyces lentus, Zygosaccharomyces bisporus, Zygosaccharomyces mellis, Zygosaccharomyces rouxii, Zygosaccharomyces rouxii 2, Torulaspora globosa, Zygosaccharomyces florentinus, Candida colliculosa, Torulaspora delbrueckii, Torulaspora pretoriensis, Zygosaccharomyces microellipso, Zygosaccharomyces mrakii, Torulaspora delbrueckii, Saccharomyces kudriavcevii, Saccharomyces mikatae, Saccharomyces cerevisiae, Saccharo myces cariocanus, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces pastorianus, Kluyveromyces polysporus, Kluyveromyces yarrowii, Saccharomyces dairensis 2, Kluyveromyces africanus, Kluyveromyces africanus, Saccharomyces rosinii, Kluyveromyces lodderae, Saccharomyces spencerorum, Candida holmii, Saccharomyces barnettii, Saccharomyces exiguus, Saccharomyces exiguus, Kazachstania viticola, Saccharomyces castellii, Candida glabrata gossypii ,, Kluyveromyces delphensis, Saccharomyces kluyveri, Eremothecium, Holleya sinecauda, Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, Zygosaccharomyces fermentati, Hanseniaspora uvarum, Saccharomycodes ludwigii, Kluyveromyces marxianus, Kluyveromyces wickerhamii, Kluyveromyces dobzhanskii Kluyveromyces lactis Kluyveromyces nonfermentati, Kluyveromyces aestuarii, Tetrapisispora nanseiensis, Tetrapisispora arboricola, Kluyveromyces phaffii, Tetrapisispora iriomotensis, Kluyveromyces blattae, Phaffomyces opuntiae, Phaffomyces op untiae, Phaffomyces antillensis, Phaffomyces thermotolerans, Candida insectalens, Candida silvatica, Pichia membranifaciens, Issatchenkia orientalis, Brettanomyces anomaly 2, Dekkera anomala, Brettanomyces anomalis, Brettanomyces bruxellensis, Dekkera bruxellensis, Dekkera custersiana, Dekkera naardenensis, Williopsis salicorniae, Candida haemulonii, Candida tsuchiyae , Candida oregonensis, Candida intermedia, Candida pseudointermedia, Candida intermedia, Candida akabanensis, Candida melibiosica, Clavispora lusitaniae, Candida torresii, Candida agrestis, Metschnikowia bicuspidata, Metschnikowia reuchnii, Metschnikowia pulcherrima, Candida mogii, Williopsis californica, Williopsis pratensis, Starmera amethionina var. Candida utilopis, Pichia anomala, Williopsis mucosa, Candida albicans, Candida dubliniensis, Candida maltosa, Candida viswanathii, Candida iodidea, Candida tropicalis, Candida sojae, Candida parapsilosis, Lodderomyces elongisporus, Candida fungicola, Candida sagamina, Candida candida mesenterica, Candida suecica, Candida rugosa, Candida savonica, Candida tanzawaensis, Candida austromarina, Candida sake, Candida kruisii, Candida insectamans, Candida lyxosophila, Candida glaebosa, Candida pseudoglaebosa, Candida saitoana, Candida fluviatilis, Candida palmioleophila, Candida palmioleophila, Candida shehatae var. Insectos, Candida shehatae var., Shehatae, Candida shehatae var. Lignosa, Candida coipomoensis, Candida ergastensis, Candida chilensis, Candida glucosophila, Debaryomyces hansenii, Debaryomyces castellii, Debaryomyces udenii, Candida psychrophila, Debaryomyces hansenii var. Fab, Candida fermenticarens, Candida oleophila, Candida zeylanoides, Candida boleticola, Candida schatavii, Candida ralunensis, Candida krissii, Candida laureliae, Candida santamariae var. membr, Candida santamariae var. santa, Candida beechii, Candida sophiae-reginae, Candida natalensis, Candida quercitrusa, Candida fragi, Candida xestobii, Candida fukuyamaensis, Taphrina farlowii, Pichia guilliermondii, Pichia mexica Candida conglobata, Candida insectorum, Candida aaseri, Candida butyri, Candida dendronema, Candida naeodendra, Candida tenuis, Candida diddensiae, Candida friedrichii, Candida membranifaciens, Candida buinensis, Candida atlantica, Candida atmospheraerica, Pichia triangularis, Candida multigemmis, Pichia farinosa, Candida cylindracea, Endomyces fibuliger, Saccharomycopsis fibuligera, Saccharomycopsis capsularis, Pachysolen tannophilus, Endomyces geotrichum, Galactomyces geotrichum, Galactomyces citri-aurantii, Galactomyces reessii "Dipodascus albidus" Dipodascus albidus, Dipodascus geniculatus, Dipodascus australiensis, Dipodascus armillariae, Dipodascus macrosporus, Geotrichum klebahnii, Dipodascus aggrega tus, Geotrichum eriense, Geotrichum fermentans, Candida apicola, Candida bombi, Starmerella bombicola, Candida geochares, Candida spandovensis, Zygoascus hellenicus, Candida valdiviana, Arxula terrestris, Candida chiropterorum, Candida drimydis, Dipodascopsis uninucleata, Waltomyces lipofer, Neolecta vitellina, Schizosacchar omyces pombe, Schizosaccharomyces japonicus, Pneumocystis carinii, Pneumocys Tis carinii, Taphrina mirabilis, Taphrina nana, Taphrina pruni, Taphrina ulmi, Taphrina communis, Taphrina flavorubra, Taphrina deformans 2, Taphrina prunisubcordatae, Taphrina populina, Taphrina wiesneri, Taphrina carnea, Taphrina virginica, Taphrina letifera, Taphrina robinsoniana, Protomyces inouyei, Protomyces lactucae, Protomyces macrosporus, Protomyces pachyderm, Saitoella complicata, Leucoagaricus gongylophorus, Dictyonema pavonia, Tulostoma macrocephala, Pluteus petasatus, Lentinula lateritia, Lentinula lateritia, Cyathus striatus, Calvatia gigantea, Lycoperdon sp.Agaricus bisporus, Amanita muscaria (fly agaric), Leucopaxillus albissimus , Tricholoma matsutake, Entoloma strictius, Stropharia rugosoannulata Basidiomycete symbiont of Apte, Calathella mangrovei, Nia vibrissa, Halocyphina villosa, Cyphellopsis anomala, Physalacria maipoensis, Favolaschia intermedia subsp. , Athelia bombacina, Cortinarius iodes, Inocybe geophylla, Lepiota procera (parasolus, Limnoperdon incarnatum, Panellus serotinus, Panellus stipticus, Hygrophorus sordidus, Humidicutis marginata, Typhula phacorrhiza, Crucibulum laeve, Laccaria amethystina, Laccaria pumila, Coprinus cinereus, Omphalina ericetorum, Pleurotus ostreatus, Fistulina hepatica, Fistulina hepatica, Schizophyllum commune, Pleurotus tuberregium, Scleroderma citrina, Xerocomus chrysenteron, Boletus satanas, Paxillus panuoides Peniophora nuda, Peniophora nuda, Scytinostroma alutum, Echinodontium tinctorium, Echinodontium tinctorium, Russula compacta, Russula compacta, Lentinellus omphalodes, Lentinellus omphalodes, Lentinellus ursinus, Lentinellus ursinus, Clavicorona pyxidata, Clavicorona pyxidata, Auriscalpium vulgare, Auriscalpium vulgare, Hericium coralloides, Hericium erinaceum, Hericium ramosum, Hericium ramosum, Stereum gausapatum, Stereum ostrea, Stereum hirsutum, Gloeocystidiellum leucoxantha, Aleurodiscus botryosus, Stereum annosum, Bondarzewia berk eleyi, Albatrellus skamanius, Heterobasidion annosum, Laxitextum bicolor, Trichaptum fusco-violaceum Trichaptum biforme, TTrichaptum abietinu, Trichaptum abietinum, Trichaptum laricinum, Basidioradulum radula, Hyphodontia alutaria, Inonotus hispidus, Phellinus igniarius, Coltricia perennis, Schizopora paradoxa, Fomitopsis pinicola, Daedalea quercina , Antrodia carbonica, Polyporus squamosus, Trametes suaveolens, Fomes fomentarius, Lentinus tigrinus, Ganoderma australe, Dentocorticium sulphurellum, Poria cocos, Cantharellus tubaeformis, Phaeolus schweinitzii, Laetiporus sulphureus, Sparassis spathulata, Bjerkandera adusta, Oxyporus latemarginatus, Trichaptum abietinum, Phlebia radiata, Phanerochaete chrysosporium, Rhizoctonia zeae, Tretopileus sphaerophorus Ceriporia purpurea, Termitomyces cartilagineus, Panus rudis, Steccherinum rhois, Meripilus giganteus, Albatrellus syringae, Spongipellis unicolor, Hydnellum sp., Thelephora sp., Gloeophyllum sepiarium, Geastrum saccatum, Sphaerobolus stellatus, Gomphus floccosus, Pseudocolus fusif ormis, Ramaria stricta, Clavariadelphus pistillaris, Multiclavula mucida, Clavulina cristata, Hydnum repandum, Thanatephorus cucumeris, Thanatephorus praticola, Auricularia auricula, Auricularia polytricha, Botryobasidium isabellinum, Botryobasidium subcoronatum, Oxyporus sp., Bullera grandispora, Mrakia frigida, Mrakia psychrophilia, Cystofilobasidium capitatum , Leucosporidium larimarini, Tilletiopsis cremea, Tilletiopsis lilacina, Tilletiopsis washingtonensis, vaccinii Exobasidium, Sympodiomycopsis paphiopedili, Tilletiopsis pallescens, Tilletia caries, Tilletiaria anomala, Tilletiopsis fulvescens, Tilletiopsis minor, Tilletiopsis albescens, rhododendri Graphiola cylindrica, Exobasidium, vaccinii Exobasidium, Dacrymyces chrysospermus, Dacrymyces Stillatus, Calocera cornea, Heterotextus alpinus, Trichosporon caseorum, Trichosporon lactis, Trichosporon cutaneum, Asterotremella parasitica, Cryptococcus humicolus, Cryptococcus albidus, Filobasidium floriforme, Holtermannia corniformis, Tremella foliacea, Bullera schimicola, Bullera walsii, Bullera huiaensis, Bullera boninensis, Bullera mrakii, Bullera coprosmaensis, Bullera oryzae, Bullera derxii, Bullera sinensis, Bullera armeniaca, Dioszegia crocea, Bullera variabilis, Filobasidiella neoformans, Tsuchiaea wingfieldii, Bullera dendrophila, Bullera miyagiana, Bullera globospora, Tremella globospora, Kockovaella imperatae, Kockovaella sacchari, Kockovaella thailandica, Kockovaella phaffii, Kockovaella phaffii, Fellomyces fuzhouensis, Kockovaella machilophila, Fellomyces distylii, Fellomyces ogasawarensis, Kockovaella schimae, Fellomyces penicillatus, Fellomyces polyborus, Fellomyces horovitziae, Sterigmatosporidium polymorphu, Tremella moriformis, Bullera hannae, Bullera penniseticola Bullera unica, Bulleromyces albus, Bullera pseudoalba, fungus sp., Sirobasidium magnum, Fibulobasidium inconspicuum, Taphrina california, Taphrina maculans, Bensingtonia yamatoana, Sporobolomyces inositophilus, Sporobolomyces singularis, Sporobolomyces falcatus, Sporobolomyces tsugae, Microbotryum violaceum, Leucosporidium scotti i, Bensingtonia intermedia, Sporidiobolus pararoseus, Sporidiobolus pararoseus, Sporobolomyces roseus, Sporidiobolus johnsonii, johnsonii Sporidiobolus, salmonicolor Sporidiobolus, Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodosporidium fluviale, ruineniae Sporidiobolus, mixiomycetes, Sporobolomyces sasicola, Sporobolomyces xanthus, Kurtzmanomyces nectairei, Bensingtonia sp., Sporobolomyces ruber, Sterigmatomyces halophilus, Bensingtonia ingoldii, Bensingtonia sae, Bensingtonia miscanthi, Bensingtonia subrosea, Kondoa malvinella, Bensingtonia yuccicola, Bensingtonia phylladus, Bensingtonia ciliata, Bensingtonia naganoensis, Sporobolomyces subbrunneus, Cronartium ribicola, Peridermium harknesseii, Hyalopsora polypodii, Gymnoconia nitens, Nyssopsora echinata, Helicobasidium purpureum, Helicobasidium corticioides, Helicogloea variabilis Sporobolomyces kluyveri-nielii, Sporobolomyces phyllomatis, Sporobolomyces salicinus, Rhodotorula minuta, Sporobolomyces foliicola, Spor obolomyces oryzicola, Sporobolomyces vermiculatus, Erythrobasidium hasegawianum, Sporobolomyces yunnanensis, Sporobolomyces elongatus, Rhodotorula lactosa, Archaeospora leptoticha, Paraglus brasilianum, Glomus verruculosum, Glomus intraradices, Glomus proliferum, Glomus etunicatum, Gigaspora margarita, Scutellospora projecturata, Scu tellospora castanea, Scutello spora cerradensis, Entrophospora sp., Glomus versiforme, Mucor circinelloides f. lusita, Mucor racemosus, Cepedea virguloidea, Mucor ramosissimus, Ellisomyces anomalus, Backusella ctenidia, Circinomucor circinelloides, Mucor racemosus, Opalina ranarum, Parasitella parasitica, Chaetocladium brefeldii, Chaetocladium jonesii, Pilaira anomala, Thamnidium elegans, Helicostylum elegans, Pirella circinans, Mucor mucedo, Mucor hiemalis f. hiemalis, Rhizomucor variabilis, Zygorhynchus heterogamus, Actinomucor elegans, Dicranophora fulva, Pilobolus umbonatus, Backusella circina, Mucor recurvus var., indicus, Blakeslea trispora, Poitrasia circinans, Choanephora cucurbitarum, Gilbertella persicaria, Hyphomucor assamensis, Rhizopus microsporus var. micr, Rhizopus microsporus rhiz, Rhizopus azygosporus, Rhizopus microsporus var. chin, Rhizopus microsporus var. olig, Amylomyces rouxii, Rhizopus arrhizus, Sporodiniella umbellata, Syzygites megalocarpus, Rhizopus stolonifer, Benjaminiella poitrasii, Cokeromyces recurvatus, Mycotypha africana, Mycotypha microspora, Kirkomyces cordense, Mucor amphibiorum, Mucor indicus, Apophysomyces elegans, Saksenaea vasiformis, Radiomyces spectabilis, Phycomyces blakesleeanus, Spinellus fusiger, Circinella umbellata, Phascolomyces articulosus, Zychaea mexicana, Fennellomyces linderi, Thamnostylum piriforme, Absidia corymbifera, Absidia blakesleeana, Dichotomocladium elegans, Rhizomucor miehei, Rhizomucor pusillus, Thermomucor indicae seudaticae, Protomycocladus faisalabadensi, Absidia glauca, Chlamydoabsidia padenii, Absidia coerulea, Absidia repens, Halteromyces radiatus, Cunninghamella bertholletiae, Cunninghamella polymorpha, Cunninghamella elegans, Cunninghamella echinulata, Gongronella butleri, Hesseltinella vesiculosa, Syncephalastrum monosporum var, Syncephalastrum racemosum (U.), Mucor ramannianus, Blastocladiella emersonii, Entomophthora muscae, Entomophthora Schizophora, Eryniopsis ptycopterae, aulicae Entomophaga, Pandora neoaphidis, Strongwellsea castrans, Zoophthora culisetae, Conidiobolus lamprauges, Conidiobolus thromboides, Conidiobolus coronatus, sp Chytridiomycetes., simulii Smittium, simulii Smittium commune, Smittium, Smittium cylindrosporum , Immittatum, Immitatum, Sodium tronadorium, Smittium orthocladii, Sutrum tipulidarum, Sodium dipterorum, Sodium megazygosporum, Sodium cf. dipterorum, Smittium cf. phytotelmatum, Smittium phytotelmatum, Furculomyces boomerangus, Sardinian angustum, Furculomyces boomerangus, Smit tium morbosum, culinarum, simulatum, culinarum, culinarisoidum, fecundum, annulatum, smittium coloradense, smittium mucronatum, caudate, genistelloides hibernus, capniomyces stellatus, culisetae, culisetae, culisetae, coemansia reversa, spirodactylon aureum , Coemansia braziliensis, Kickxella alabastrina, Linderina pennispora, Dipsacomyces acuminosporus, Martensiomyces pterosporus, Spiromyces aspiralis, Spiromyces minutus, Basidiobolus haptosporus, Basidiobolus ranarum, Chytridium confervae, sp Neocallimastix., Neocallimastix frontalis 2, Dissophora decumbens, Mortierella multidivaricata, transverse Echinosporangium, Mortierella verticillata , Mortierella chlamydospora, Mortierella wolfii, Mortierella polycephala, Monoblepharella elongata, Monoblepharella mexicana, Monoblepharis hypogyna, Monoblepharis insignis, Hyaloraphidium curvatum, Spizellomyces acuminatus, Filobasidiella neoformans 3, Candida floricola., Candida santjacobensis, dark septate , Metschnikowia agaves, Candida etchellsii., Pneumocystis carinii, Candida cantarellii., Candida stellata., Nadsoniella nigra, Candida paludigena., Candida versatilis., Candida otoi, Candida silvanorum., Candida entomophila., Candida sorbophila., Candida uringiensis, Candida antillancae , Candida tepae., Candida musae., Candida ishiwadae., Candida ncudensis, Candida castrensis., Candida vanderwaltii., Metschnikowia australis., Candida fructus, Metschnikowia pulcherrima., Candida bertae., Wangiella dermatitidis, Candida galacta., Candida salmanticensis. , Candida parapsilosis, Candida petrohuensis., Candida populi., Candida blankii., Ochrolechia parella, Candida rhagii., Candida homilentoma., Candida fennica., Bulgaria inquinans, Sporidiobolus sp., Wickerhamiella domercqiae, Zygosaccharomyces rouxii, Metschnikowia gruessii. Metschnikowia reukaufii., Metschnikowia bicuspidata., Metschnikowia krissii., Candida anatomiae, Arxula terrestris, Candida agrestis, Glomerobolus gelineus, Candida vinaria, Blastocladiella emersonii, Candida la ctis-condensi, Metschnikowia zobellii, Candida bondarzewiae, Candida sequanensis, Candida edax, Candida ernobii, Candida karawaiewii, Saccharomyces cerevisiae, Barrmaelia oxyacanthae, Arxula adeninivorans.

Especially suitable embodiments the inventive Methods are characterized in that the DNA II sequence is a DNA sequence is responsible for a polypeptide according to SEQ ID NO: 2,4,6 or 8 encodes or is a DNA sequence encoding a polypeptide coded, 80% identity to SEQ ID NO: 2,4,6 or 8 comprises.

Especially preferred are those DNA II sequences which code for a polypeptide with laccase activity, the 85, 90, 92, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO: 2,4,6 or 8 owns. The identity between two polypeptide sequences is determined using the GCG software, which has been developed by the University of Wisconsin, by means of of the GAP program using the program's default values Settings for gap penalty, determined.

Laccase activity is the ability to oxidize phenolic substances as described, for example, in Machuca et al., 1998, at page 218 (Determination of laccase type PO activity) becomes.

The Introduce of DNA molecules in cells, familiar cloning methods known to those skilled in the art are used. and transfection methods, such as co-precipitation, Protoplast fusion, electroporation, retroviral transfection and the like, used to bind said nucleic acids in respective expression system for expression. suitable Systems are used, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Eds., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Farther can the introduction from DNA into cells also by homologous recombination. This will be the DNA usually introduced into an appropriate vector for homologous recombination, as he is known, for example, Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503 is known.

The Breed The cells are known in the art depending on the host organism Wise. Microorganisms are usually in a liquid medium, the one carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, Magnesium salts and optionally contains vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C up to 60 ° C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid be kept at a fixed value, that is regulated during the cultivation be or not. The cultivation can be batchwise, semi-batchwise or continuous respectively. nutrient can presented at the beginning of the fermentation or semicontinuously or continuously fed in become.

Select from Cells that express a laccase from Aspergillus nidulans happens by laccase catalyzed conversion of a substrate, then as easily identifiable product, usually is present as a dye. Such colored cells or colonies, those in their environment to implement the substrate in agar, can easily from the non-colored Distinguish cells or colonies and then separated.

Especially suitable substrates are the compounds ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate), DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol). These connections will be hereinafter also referred to by the term "test substrates".

These Test substrates can converted from laccases in the presence of oxygen to a blue dye which enables rapid recognition of laccase activity. The color change can be recognized directly with the eye or - in particular for quantification - photometrically be measured.

One Advantage in some embodiments the inventive Method is the fact that some laccases from A. nidulans be secreted from the cell into the medium. This makes it possible for the Laccase outside the cell, for example directly on the agar plate to detect without the test substrates added to the cell Need to become.

A further preferred embodiment the inventive Method is that the individual laccases different specificities across from have the test substrates. Thus, it becomes possible to differential investigations to carry out with, for example, two laccases, wherein the first laccase the Test substrate 1 and not the test substrate 2 converts and the second Laccase the test substrate 2 and not the test substrate 1 converts. The substrate specificity of the laccases lccB given in the table below, lccC, and lccD allows the selection of suitable laccases and test substrates for a such a differential method.

For the inventive method suitable laccases are those in Aspergillus nidulans naturally occurring laccases including polymorphisms, alleles and sequence variants, as well as the derivable laccases, as far as They still have the essential biological properties of naturally occurring Lac casen, in particular the substrate specificity with respect to the test substrates mentioned above, exhibit.

Particularly suitable laccases are the laccases lccA, lccB, lccC and lccD from A. nidulans, whose DNA and polypeptide sequences are listed in the Sequence Listing. SEQ ID NO: 1 lccA DNA sequence SEQ ID NO: 2 lccA polypeptide sequence SEQ ID NO: 3 lccB DNA sequence SEQ ID NO: 4 lccB polypeptide sequence SEQ ID NO: 5 lccC DNA sequence 1 SEQ ID NO: 6 lccC polypeptide sequence SEQ ID NO: 7 lccD DNA sequence SEQ ID NO: 8 lccD polypeptide sequence

A particularly advantageous embodiment of the process according to the invention results from the fact that the A. nidulans laccases have different specificities compared to the test substrates. The following table shows the substrate specificities:

• +: sets the said substrate with oxygen to a blue dye around
• -: no conversion with oxygen to a colored compound

Further suitable dyes for laccase detection are those of Dias et al., 2003 and Johannes & Majcherezyk, 2000 described.

experimental part

insulation of laccases from Aspergillus nidulans

The Genome of A. nidulans was examined for the presence of laccases and next to YA and TilA four new laccases, lccA, lccB, lccC and lcc-D, discovered. The sequence of the laccases found is in the sequence listing shown.

The Laccases lccB, lccC and lccD were amplified by PCR and under the control of the alcohol-inducible promoter, alcA, as well as the xylA promoter, expressed in A. nidulans. (Waring et al. 1989, Zadra et al. 2000).

To the Test of enzyme activity were the tribes cultivated on threonine-containing or xylose agar plates and the laccase catalyzed oxidation of ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonates) studied.

In the case of LccC, a color change occurred ( 2 ). When grown with glucose as the carbon source, no blue staining occurred in the case of expression under the control of the αcA promoter. Under these conditions, the alcA promoter is repressed. In contrast, laccase D showed no blue color. Since another laccase test uses substances other than substrate, we chose this alternative approach (Hermann, et al., 1983). For this, the cultures were overlaid with the two substrates DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol) and incubated. These substrates were converted to a blue dye. LccC did not react with these substances ( 2 ). In the case of expression of the laccases under the control of the xylA promoter, LccC became blue in color when the colonies were incubated on xylose-containing plates. Analogous to the expression under control of the alcA promoter, no blue staining was detected on glucose-containing nutrient media, since under these conditions the xylA promoter is also repressed. The blue staining of the corresponding construct with LccD could also be achieved only by incubation with the above-described dyes. The staining occurred only when the colonies grew on xylose-containing media, on glucose also no coloration occurred here.

Laccases as Reporter genes for the determination of promoter activities

Of the open reading frame of laccase C can be fused with a promoter be - analog to the fusion with the alcA or xylA promoter - thereby determining its activity become. A fungus can be stored under different conditions (temperature, pH, medium composition) and thus the conditions the promoter activity be determined. The fungus can be cut on agar plates and the activity assessed visually or spectroscopically. In addition, the Mushroom in liquid culture cultured and the laccase activity in culture supernatant be quantified. Laccase B and D can be used with both other substrates are used.

Laccases to Localization of proteins

By a translational fusion of an N-terminal region of an testing protein with a laccase, in which the secretion signal has deleted the functionality of a predicted secretion signal be tested.

By a translational fusion of a protein to be tested with the laccase (with deleted secretion signal) may be the subcellular localization of the protein. Cell organelles can be fractionated and the laccase activity be tested in the different fractions.

Laccases as Reporter gene for genetic screens

to Isolation of promoters based on specific environmental parameters (temperature, pH, substrates), LccC can be fused with DNA fragments become. The generated gene bank can be transformed into a fungus strain and positive, i. Laccase-secreting strains can be identified the blue coloration be isolated. This approach can also be performed in microtiter plates, so that the "readout" by robots done can. Thus, the method is also for high-throughput experiments suitable.

laccases can also serve for the isolation of regulators defined promoters. For this purpose, a specific promoter is fused with the laccase gene and introduced into a mushroom strain. The trunk, then expression the laccase according to the promoter activity can be mutagenized to isolate mutants with altered regulation. Thereby can e.g. strains with reinforced promoter activity getting produced. When the same promoter in the strain for expression a desired gene is used, so the expression of this gene can be increased. It can also mutants with reduced prommotor strength can be isolated. This Effect may be due to the fact that positive regulators have been mutated. These mutants can be used to control the corresponding regulators e.g. through complementation to isolate with a gene bank.

Laccases lccB, lccC or lccD as reporter genes for the quantification of promoter activities

Of the open reading frame of the three laccases, in which the secretion signals could have been deleted be fused with promoters and transformed into fungi. These Constructs lead for the production of intracellular Laccase. The enzyme activity can be quantified exactly after cell disruption. Because the three Laccases can implement different substrates, allowing multiple promoter activities simultaneously be quantified. This system is similar to simultaneous use of beta-galactosidase and glucuronidase.

SEQUENCE LISTING

directory of the pictures

2 : Expression of LccC and LccD in RMS011. The laccase genes were introduced into RMS011 under the control of the xylA promoter and the colonies cultured on agar plates with glucose (A) or glycerol (B, C) for 2 days at 37 ° C. The medium was spiked with ABTS. The cultures in (C) were overlaid with ADBP and DMA after growth.

3 : Expression of LccC under the control of the xylA promoter. The lccC gene was introduced into RMS011 under the control of the xylA promoter. Depending on the number of copies, the strains express the gene to different extents, which can be seen in the different staining intensity.

4 : Quantification of LccC activity. The lccC gene was introduced into RMS011 under the control of the alcA promoter. Liquid medium was inoculated with spores and cultured with glucose on substrate for 20 h. Subsequently, the hyphae were harvested and induced in medium with 100 mM ethanol. At various times, equal amounts of mycelium were withdrawn and the enzyme was released from the hyphae by incubating for 2 h in laccase assay buffer (137 mM di-sodium hydrogenphosphate, 26 mM citric acid, pH 6.0) at 4 ° C. Subsequently, laccase activity was measured: to 900 μl of laccase assay buffer was added 100 μl of ABTS (50 mM in H ₂ O). The reaction was photometrically followed by addition of enzyme started at 405 nm. Reaction temperature was at 37 ° C. Samples 1-3 are independent cultures derived from independent precultures. The labels a and b result from the duplicate determination of the samples.

literature

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• Zadra, I., Abt, B., Parson, W. & Haas, H. (2000). XylP promoter-based expression system and its used for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE. Appl. Environ. Microbiol. 66, 4810-4816

Claims (12)

Method for determining regulatory properties a genetic element comprising the steps of: (A) Introduce a DNA molecule with a DNA I sequence whose regulatory properties are determined should, in a cell, whereby the DNA I sequence with another DNA II sequence responsible for a laccase encoded by Aspergillus nidulans is linked that the regulatory elements of the DNA I sequence control the expression of the laccase, (B) Breed the cells obtained under (a) under conditions involving expression to allow the laccase (c) assigning the conditions chosen under (b) on the regulatory properties of the DNA I sequence. Method according to claim 1, characterized in that that the cells are selected are from the group of fungal, plant and animal Cells. Method according to claim 1, characterized in that that the genetic element is a promoter. Method according to claim 1, characterized in that that it is a regulatable promoter. A method of localizing a desired protein in a cell compartment comprising the steps of: (a) introducing a DNA molecule having a DNA sequence encoding the desired protein and thus linked to another DNA II sequence which codes for a laccase from Aspergillus nidulans, which represents the protein encoded by the DNA molecule, a fusion of the desired protein with the laccase, into the cells, (b) growing the cells obtained under (a) Conditions that affect the expression of the fused protein allow, (c) detection of the localization of the laccase in a cell compartment and thus the localization of the desired protein in this cell compartment. Method according to claim 5, characterized in that that the cell compartments are fractionated and the laccase activity in the individual fractions is determined. Method for determining the expression of a desired Gene in a cell that includes the steps: (a) Introduce a DNA molecule, the one DNA sequence of the desired Gens possesses, in a cell, whereby the DNA sequence with another DNA II sequence responsible for a laccase encoded by Aspergillus nidulans is linked that the regulatory elements of the desired gene expression control the laccase, (b) growing the one obtained under (a) Cells under conditions that require the expression of the desired Allow gene (c) detection of the expression of the laccase in the Cell and thus proof of expression of the desired gene. Method for expressing a desired Protein as a fusion protein in a cell, or as a secreted Fusion protein comprising the steps: (a) Introduce a DNA molecule, the one DNA II sequence responsible for a laccase from Aspergillus nidulans coded, has and with their C-terminal end of another DNA I sequence responsible for the desired gene product coded, so linked is that expression as a fusion protein from laccase and desired Gene product occurs, (b) Breeding the cells obtained under (a) under conditions involving expression of the desired Allow gene (c) Isolation of the fusion protein from laccase and desired Gene product. Method according to claim 8, characterized in that that a fungal cell is used as the cell. Method according to claim 8, characterized in that that the fusion protein is secreted using as secretion sequence the endogenous leader sequence of the laccase used is used. Method according to claim 8, characterized in that that expression of the gene for the wished Fusion protein under the transcriptional control of alcA or the xylA promoter from A. nidulans. Method according to one of Claims 1, 5, 7, 8, characterized that the DNA II sequence is a DNA sequence coding for a polypeptide according to SEQ ID NO: 2,4,6 or 8 encodes or is a DNA sequence encoding a polypeptide coded, 80% identity to SEQ ID NO: 2,4,6 or 8 comprises.