molecular analysis of sexual sporulation in aspergillus...

Molecular Analysis of Sexual Sporulation

in Aspergillus nidulans

Dissertation zur

Erlangung des Doktorgrades

der Naturwissenschaften

(Dr. rer. nat.)

dem Fachbereich Biologie der

Philipps-Universität

Marburg/Lahn

vorgelegt von

Huijun Wei

aus

Tianjin / VR China

Marburg, März 2003

Vom Fachbereich Biologie der Philipps-Universität Marburg

Als Dissertation am 08. 05. 2003 angenommen

Disputation am 14. 05. 2003

Erstgutachter: HD Dr. R. Fischer

Zweitgutachter: Prof. Dr. R. K. Thauer

Die Untersuchungen zur vorliegenden Arbeit wurden von August 2000 bis März 2003 im

Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg

und am Max-Planck-Institut für terrestrische Mikrobiologie in Marburg unter der Leitung von

Prof. Dr. R. K. Thauer und der Betreuung von HD Dr. R. Fischer durchgeführt.

Hiermit versichere ich, daß ich die vorliegende Arbeit “Molecular Analysis of Sexual

Sporulation in Aspergillus nidulans“ selbständig verfaßt, keine anderen als die angegebenen

Hilfsmittel verwendet und sämtliche Stellen, die im Wortlaut oder dem Sinn nach anderen

Werken entnommen sind, mit Quellenangaben kenntlich gemacht habe.

Marburg, im März 2003

Im Zusammenhang mit der Thematik der vorliegenden Dissertation wurden bzw. werden

folgende Publikationen erstellt:

Wei, H., Scherer, M., Singh, A., Liese, R. and Fischer, R. (2001). Aspergillus nidulans �-

1,3 glucanase (mutanase), mutA, is expressed during sexual development and mobilizes

mutan. Fungal Genetics and Biology. 34(3), 217-227. (with Cover).

Scherer, M., Wei, H. and Fischer, R. (2002). Aspergillus nidulans catalase-peroxidase,

CpeA, is upregulated during sexual development through the APSES transcription factor

StuA. Eukaryotic Cell. 1(5), 725-735.

Wei, H., Requena, N. and Fischer, R. (2003). The MAPKK-kinase SteC regulates

conidiophore morphology and is essential for hyphal fusion and sexual development in the

homothallic fungus Aspergillus nidulans. Molecular Microbiology. 47(6), 1577-1588.

Wei, H., Weber, R., Bunting, S., Vienken, K., and Fischer, R. (2003). Molecular analysis

of a high affinity hexose transporter (hgtA) in the filamentous fungus Aspergillus nidulans. In

preparation.

Warmbold, J., Töws, M., Mertens, D., Konzack, S., Rischitor, P., Vienken, K., Wei, H.

and Fischer, R. (2003). Establishment of DsRed from the coral Discosoma as a fluorescent

marker in Aspergillus nidulans and construction of expression vectors for protein tagging

using recombination in Escherichia coli (GATEway). In preparation.

Abbreviations

APS ammonium perooxdisulfate

CM complete Medium

DAPI 4,6-diamidino-2-phenylindol

DEAE diethylaminoethyl

DEPC diethylpyrocarbonat

DTT 1,4 Dithiothreitol

GFP green fluorecent protein

IPTG isopropylthio-�-D-galactoside

LacZ �-galactosidase

LB Luria-Bertani medium

MM minimal medium

RT-PCR reverse transcriptase-polymerase chain reaction

SAP shrimp alkaline phosphatase

TAE Tris-acetate-EDTA

TE Tris-EDTA

TEMED N,N,N’,N’-tetramethylendiamine

X-Gal 5-bromo-4-chloro-3-indoxyl-�-D-galctoside

Content

Content

1 Summary........................................................................................... Zusammenfassung........................................................................... 摘要.......................................................

1

3

5

2 Introduction…………………………………………………….............. 7

2.1 Sexual development and fruiting body formation in A. nidulans………………… 7

2.2 Determinants influencing fruiting body formation in A. nidulans……………….. 10

2.2.1 Environmental factors affecting sexual development………………………….….. 10

2.2.2 Genetic determinants regulating fruiting body development……………………… 11

2.3 Objective of this study……………………………………..………………………… 15

2.3.1 Carbon cycle related to �-1,3 glucanase (mutanase) and high-affinity hexose

transporters in sexual development of A. nidulans…………………………………

15

2.3.2 MAP kinase cascade……………………………………………………………….. 17

3 Materials…………………………………………………………………. 20

3.1 Equipment and Chemicals…………………………………………………………... 20

3.2 Media…………………………………………………………………………….…… 21

3.3 A. nidulans and E. coli strains………………………………………………………. 22

3.4 Plasmids and Cosmids……………………..………………………………………… 24

3.5 Oligonucleotides………………………………………..……………………………. 26

4 Methods…………………………………………………………..……… 28

4.1 Growth condition and storage for transformed E. coli strains…………………… 28

4.2. Transformation of A. nidulans……………………………………………………... 28

4.2.1 Praparation of protoplast…………………………………………………………… 28

4.2.2 Protoplast transformation…………………………………………………………... 29

Content

4.3 DNA and RNA manipulations………………………………………………………. 29

4.3.1 Plasmid DNA preparation from E. coli cells………………………………………. 29

4.3.2 Genomic DNA preparation from A. nidulans……………………………………… 30

4.3.3 Precipitation of DNA……………………………………………………………… 30

4.3.4 DNA electrophoresis through agarose gel…………………………………………. 30

4.3.5 Digestion of DNA by restriction endonucleases…………………………………… 31

4.3.6 PCR…………………………………………………………………………….…... 31

4.3.7 DNA isolation from agarose gel…………………………………………………… 32

4.3.8 Dephosphorylation of digested DNA………………………………………………. 32

4.3.9 DNA ligation……………………………………………………………………….. 33

4.3.10 DNA sequencing…………………………………………………………………… 33

4.3.11 Transformation of E. coli…………………………………………………………... 33

4.3.12 DNA-DNA hybridization (Southern blot analysis)………………………………... 33

4.3.13 Isolation of total RNA from A. nidulans…………………………………………... 34

4.3.14 DNA-RNA hybridization (Northern blot analysis)………………………………… 35

4.3.15 Construction of DNA plasmids…………………………………………………….. 36

4. 4 Biochemical methods………………………………………..……………………….. 38

4.4.1 Isolation of protein from A. nidulans……………………………………………….. 38

4.4.2 Determination of protein concentration (Bradford Assay)…………………………. 39

4.4.3 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)……………………………. 39

4.4.4 Western blotting…………………………………………………………………….. 40

4.4.5 Preparation of A. nidulans nuclear extracts…………………………………………. 40

4.4.6 Purification of DNA-binding proteins……………………………………………… 41

4.5 Fluorescence microscopy………………………………………………..……….…… 42

4.6 Other methods………………………………………………………………………… 42

4.6.1 Quantification of mutan (alkali-soluble fraction)…………………………………... 42

4.6.2 Growth of �mutA, wildtype and mutA overexpression strains in mutan medium…. 43

Content

5 Results…………………………………………………………………… 44

5.1 Analysis of the carbon cycle during sexual development in A. nidulans………….. 44

5.1.1 Molecular cloning of the mutA gene………………………………………………... 44

5.1.2 mutA disruption and overexpression…………………………………………….…. 47

5.1.3 MutA is expressed in Hülle cells…………………………………………………… 50

5.1.4 Molecular cloning of the hgtA gene………………………………………………… 52

5.1.5 hgtA disruption……………………………………………………………………… 55

5.1.6 HgtA is expressed in ascogenous hyphae within the cleistotithia..………………… 56

5.1.7 Identification of regulatory regions in the upstream sequence of mutA………….…. 57

5.1.8 Binding protein isolation……………………………………………………………. 59

5.2 Signal transduction in sexual development of A. nidulans…………………………. 60

5.2.1 Molecular cloning of the steC gene……………………………………………….... 60

5.2.2 Complementation analysis of the steC mutant and steC overexpression…………… 64

5.2.3 steC deletion affects heterokaryon formation and conidiophore morphology……… 65

5.2.4 Deletion of steC inhibits hyphal fusion and sexual development……………….….. 69

5.2.5 steC-transcription is developmentally regulated and induced in metulae and

phialides…………………………………………………………………………….

70

5.2.5 SteC activates at least two MAP kinases………………………………………….... 73

6 Discussion………………………………………………………………. 75

6.1 The carbon cycle during sexual development in A. nidulans………………………. 75

6.1.1 MutA is expressed during sexual development in A. nidulans and mobilizes

mutan………………………………………………………………………………..

75

6.1.2 hgtA encodes a high affinity glucose transporter and is expressed in ascogenous

hyphae ………………………………………………………..…………………….

78

6.2 The MAPKK-kinase SteC regulates conidiophore morphology and is essential

for heterokaryon formation and sexual development in A. nidulans…………….

79

6.2.1 Hyphal extension, conidiophore and conidia development………………………… 80

Content

6.2.2 Heterokaryon and cleistothecium formation……………………………………….. 82

6.2.3 Identification of SteC targets……………………………………………………….. 83

6.3 Outlook………………………………………………………………………………… 84

7 Literature…………………………………………………………………. 87

8 Acknowledgement

Summary

1

1 Summary

Aspergillus nidulans is a filamentous fungus, able to reproduce with mitotically derived

conidiospores (conidia) and meiotic ascospores. Both spore forms are generated at or in

morphologically differentiated structures called conidiophores and cleistothecia (fruiting bodies),

respectively. Whereas the developmental program of conidiophore formation is well studied,

cleistothecium differentiation is only poorly understood. A. nidulans is especially attractive to

analyze fruiting body formation because it is a homothallic fungus and does not require a mating

partner to initiate the developmental program.

A subtractive cDNA library of A. nidulans was established in previous work to identify

differentially expressed genes during sexual development. Two clones from this substractive

hybridisation library (SSH) were further studied in this work. One displayed homology to fungal

�-1,3 glucanases (mutanase). Since �-1,3 glucan was considered as main reserve material

accumulated during vegetative growth as a cell wall component and consumed during sexual

development for the formation of cleistothecia, this gene was analysed in detail. The gene, mutA,

is disrupted by 3 introns and encodes a putative protein of 48 kDa molecular mass with a signal

peptide for secretion at the N-terminus. The deduced protein displays 24-42 % identical amino

acids to mutanases of other fungi. A proposed mutan binding domain characterised in e.g.

Penicillium is not present in A. nidulans. Expression analysis of the mutA promoter fused with

sgfp revealed specific induction of the gene during sexual development in Hülle cells. To study

the role of mutA during sexual differentiation, a mutA deletion strain was constructed. Although

degradation of mutan was affected in this strain, it was still able to form cleistothecia with a

similar number as wild type. These results suggest that additional carbon sources are available

during sexual development. In minimal medium supplemented with mutan fraction as a sole

carbon source, mutA wild type and overexpression strains showed much denser growth in

contrast to a sparse growth of mutA mutant strains. The progressive 5’ deletion of the mutA

promoter fused with sgfp led to identify three putative DNA-binding regions for regulatory

factors. One of those potential factors was characterized as a 40 kDa protein. Mass-spetrometric

analysis remains to be done to identify this protein.

Another gene found in the SSH library showed high homology to high-affinity hexose

transporter genes. The gene, hgtA, encodes an open reading frame of 2 kb interrupted by 6 introns

Summary

2

with 44-67 bp in length. The translated sequence of 531 amino acids has a molecular mass of 59

kDa. Hydrophobicity values showed the presence of 12 putative transmembrane (TM) domains, a

characteristic feature of the major facilitator superfamily. The HgtA protein displays 32-42 %

identical amino acids to other glucose transporter genes of other fungi. �hgtA strains show no

evident phenotype compared with a wild type strain. The expression of sgfp fused with the hgtA

promoter revealed a specific induction of hgtA in ascogenous hyphae within cleistothecia.

In addition to the investigation of the target genes that are involved in sexual development

of A. nidulans, one upstream factor was also studied. Extracellular signals can be transduced into

intracellular responses by the action of MAP kinase cascades. Sequential phosphorylation results

in the transient activation of a MAP kinase, which in turn activates certain transcription factors

and thus a set of pathway-specific genes. Many steps in this cascade are conserved, and

homologues have been discovered from yeast to human. The present work has characterized the

MAPKK kinase, SteC, a homologue of Saccharomyces cerevisiae Ste11, in A. nidulans. The 886-

amino-acid-long protein shares the highest similarity to Neurospora crassa Nrc-1. Deletion of the

gene in A. nidulans results in a slower growth rate, the formation of more branched hyphae,

altered conidiophore morphology, an inhibition of heterokaryon formation and a block of

cleistothecium development. The gene is transcriptionally activated during asexual development

and controls the phosphorylation of two putative kinases.

Zusammenfassung

Zusammenfassung

Aspergillus nidulans ist ein filamentöser Pilz, der sich mit mitotischen Conidiosporen

(Conidien) und meiotischen Ascosporen vermehren kann. Beide Sporenformen werden an

oder in morphologisch differenzierten Strukturen, den Conidiophoren bzw. den

Cleistothecien (Fruchtkörper) gebildet. Während das Entwicklungsprogramm der

Coniophorbildung sehr gut untersucht ist, wird die Cleistothecienbildung noch nicht gut

verstanden. A. nidulans ist zur Untersuchung der Fruchtkörperbildung besonders gut

geeignet, da er ein homothallischer Pilz ist, der keinen Kreuzungspartner zur Einleitung

des Entwicklungsprogramms benötigt.

In einer vorangegangenen Arbeit wurde eine subtraktive cDNA Genbank etabliert, um

während der sexuellen Entwicklung differenziell exprimierte Gene zu identifizieren. In der

vorliegenden Arbeit wurden zwei der Klone der subtraktiven Genbank (SSH) molekular

untersucht. Einer der Klone wies Homologie zu �-1,3 Glucanasen (Mutanase) auf. Da �-

1,3 Glucan während des vegetativen Wachstums als Reservematerial angehäuft wird, um

dann während der sexuellen Entwicklung zur Bildung der Cleistothecien verwendet zu

werden, wurde dieses Gen im Detail analysiert. Das Gen, mutA, wird durch 3 Introns

unterbrochen und kodiert für ein putatives Protein mit einer berechneten molekularen

Masse von 48 kDa. Das abgeleitete Protein enthält am N-Terminus eine Signalsequenz, die

auf eine Sekretion des Proteins hindeutet. MutA weist 24-42 % identische Aminosäuren im

Vergleich zu Mutanasen in anderen Pilzen auf. Eine in anderen Enzymen vorgeschlagene

Mutanbindedomäne, wie z.B. in Penicillium, ist in MutA nicht vorhanden.

Expressionsanalyse mittels einer Fusion des mutA Promotors mit sgfp zeigte eine

spezifische Induktion des Gens während der sexuellen Entwicklung in Hülle-Zellen. Um

die Rolle von mutA während der sexuellen Differenzierung zu untersuchen, wurde ein

mutA-Deletionsstamm hergestellt. Obwohl der Abbau von Mutan in diesem Stamm

beeinträchtigt war, war der Stamm noch in der Lage, Cleistothecien mit einer ähnlichen

Häufigkeit wie Wildtypstämme zu bilden. Das bedeutet, dass wahrscheinlich zusätzliche

Kohlenstoffquellen während der sexuellen Entwicklung zur Verfügung stehen. In

Minimalmedium, das mit isoliertem Mutan als einziger Kohlenstoffquelle supplementiert

wurde, zeigten mutA-Wildtyp und Überexpressionsstämme dichteres Wachstum als die

mutA Mutante. Progressive 5'-Deletionen des mutA-Promotors fusioniert mit sgfp führten

zur Identifikation von drei möglichen DNA-Bindungsregionen für regulatorische Faktoren.

3

Zusammenfassung

4

Einer dieser potenziellen Faktoren wurde als ein 40 kDa Protein charakterisiert. Eine

massenspektroskopische Analyse zur Identifikation dieses Proteins wurde noch nicht

durchgeführt.

Ein zweites Gen, das in der SSH-Genbank gefunden wurde, zeigte Homologie zu hoch-

affinen Hexosetransportergenen. Das Gen, hgtA, kodiert einen offenen Leserahmen von 2

kb, der von 6 Introns mit 44-67 bp Länge, unterbrochen wird. Die translatierte Sequenz

von 531 Aminosäuren besitzt eine molekulare Masse von 59 kDa.

Hydrophobizitätsanalysen des Proteins ergaben das Vorhandensein von 12 möglichen

Transmembrandomänen, was eine charakteristische Eigenschaft der "major facilitator

family" ist. Das HgtA Protein wies 32-42 % identische Aminosäuren zu anderen

Glucosetransportern anderer Pilze auf. �hgtA-Stämme zeigten keinen evidenten Phänotyp

im Vergleich zu Wildtypstämmen. Durch Expression von sgfp unter der Kontrolle des

hgtA-Promotors wurde eine spezifische Induktion von hgtA in ascogenen Hyphen

innerhalb der Cleistothecien festgestellt.

Zusätzlich zur Untersuchung der beiden differenziell exprimierten Gene, wurde ein

regulatorischer Faktor untersucht. Extrazelluläre Signale können durch MAP kinase

Kaskaden in intrazelluläre Antworten übersetzt werden. Eine sequenzielle

Phosphorylierung ergibt eine transiente Aktivierung einer MAP Kinase, die schliesslich

bestimmte Transkriptionsfaktoren und damit stadienspezifische Gene in ihrer Aktivität

reguliert. Die Signalkaskade ist evolutionär konserviert, so dass homologe Komponenten

von der Hefe bis zum Menschen beschrieben wurden. In der vorliegenden Arbeit wurde die

MAPKK Kinase, SteC, ein Homologes von Ste11 aus Saccharomyces cerevisiae, in A.

nidulans untersucht. Das 886 Aminosäuren lange Protein zeigte die höchste Ähnlichkeit zu

Neruospora crassa Nrc-1. Deletion des Gens in A. nidulans führte zu einer verringerten

Wachstumsrate, der Bildung von stärker verzweigten Hyphen, einer veränderten

Conidiophormorphologie, einer Hemmung der Heterokaryon- und der

Cleistothecienbildung. Das Gen wird transkriptionell während der asexuellen Entwicklung

induziert und reguliert die Phosphorylierung von mindestens zwei Kinasen.

摘要

摘要

钩巢曲霉 (Aspergillus nidulans) 属于同宗配合的丝状真菌。它可有丝分裂产生无性

的分生孢子 (condiospore, conidia) 和减数分裂产生有性的子囊孢子 (ascospore)。两种孢

子分别由分生孢子梗 (condiophore) 和子囊壳 (cleistothecium) 产生。无性孢子的形成过

程已有清晰的研究，但有性产孢的分子基础有较少的研究。由于钩巢曲霉为同宗配合的

真菌，因而作为一个有性产孢子机制研究的模式种。该工作为项目“钩巢曲霉有性产孢

的分子机制的研究”的一部分。

基于钩巢曲霉有性产孢的消减杂交文库 (SSH) 的建立。两个基因被进一步研究。

第一个基因显示和其它真菌�-1,3 葡聚糖裂解酶 (�-1,3 glucanase; 木糖酶, mutanase) 高

度的同源性。由于传统认为在钩巢曲霉无性产孢过程中菌丝累积大量 �-1,3 葡聚糖，而

在后期 �-1,3 葡聚糖被降解重新利用以提供该菌有性产孢中子囊壳形成的碳源和能源。

因而该基因被进一步研究。该基因转录为拥有 3 个内含子 1.5 kb 片段，编码一个 48 kDa

的酶，其 N-端信号肽序列表明该酶被分泌。该酶显示和其它真菌 �-1,3 葡聚糖裂解酶

24-42% 相同的氨基酸序列，但不同于青霉菌 (Penicillium) 和木霉菌 (Trichodema) 的

�-1,3 葡聚糖裂解酶，该酶缺乏一个预测的 C-端葡聚糖结合域。绿色荧光蛋白 (sgfp) 与

�-1,3 葡聚糖裂解酶启动子的融合揭示该酶特异性的在有性组织护卫细胞 (Hülle cells)

中的高效表达。为明确该酶的功能，一个 �-1,3 葡聚糖裂解酶缺失突变的菌系被转化获

得，尽管在该突变菌系中�-1,3 葡聚糖降解被大大的影响，但仍形成和野生型菌系相似

数量的子囊壳。因而表明在子囊壳发育中其它碳源被利用。仅用来源于 12 天生长的

�-1,3 葡聚糖裂解酶缺失突变菌系葡聚糖组分作为唯一碳源的培养基上，野生型和

�-1,3 葡聚糖裂解酶超表达菌系显示浓密的生长，而 �-1,3 葡聚糖裂解酶突变菌系显示

非常稀疏的生长。用绿色荧光蛋白作为一个报告基因，�-1,3 葡聚糖裂解酶启动子连续

的 5’-端删除显示 3 个预测的调控蛋白结合区域。调控蛋白的分离显示一个 40 kDa 调

控蛋白结合在启动子 -1.7 kb 的位置。另一个来源于消减杂交文库的基因显示和己糖转

运蛋白 (hexose transporter) 高度的同源性。该基因 (hgtA) 为 2 kb 有 6 个内含子的开

放阅读框，编码一个由 531 氨基酸组成的 59 kDa 的蛋白质。疏水值预测该蛋白拥有

12 个垮膜区。该蛋白显示和其它真菌葡萄糖转运蛋白 32-42 % 相同的氨基酸序列。比

较野生型菌系，该基因缺失突变菌系未显示明显的表型差异。绿色荧光蛋白与启动子

的融合揭示该蛋白特异性的在子囊壳中子囊产生菌丝中 (ascogenous hyphae) 的高度表

达。

5

摘要

6

除了与钩巢曲霉有性产孢的相关的目标基因的鉴定，上游与有性产孢信号转导相关

的基因也被研究。细胞外信号可被丝裂原活化蛋白激酶 (mitigen-activated protein kinase,

MAPK) 信号转导通路传递为胞内信号。依次的磷酸化导致一个 MAP 激酶的短暂激活，

磷酸化 MAP 激酶激活调控蛋白，因而导致特定的基因的表达。在本工作中，一个与

S. cerevisiae Ste11 同源的来源于钩巢曲霉激酶, SteC, 被进一步研究。该激酶由 886 氨基

酸组成，显示和脉孢菌 (N. crassa) 激酶高度的同源性。该基因缺失突变菌系显示较

慢的菌落生长速率，多分枝的菌丝，改变的分生孢子梗形态，正常异核形成的抑制以及

子囊壳发育的封闭。该基因在无性产孢的早期被转录激活，调控至少两个激酶的磷酸化。

Introduction

7

2 Introduction

Aspergillus nidulans is a filamentous, homothallic fungus belonging to the family of

ascomycetes which is ubiquitously distributed worldwide. The scientific name is derived from

two Latin words: aspergillum which is a device used to sprinkle holy water (as this resembles the

asexual reproductive structure called a conidiophore) and nidulans which means nest-like (which

refers to the sexual structure called a cleistothecium). Although its correct taxonomic name is

Emericella nidulans, it is more widely known as Aspergillus nidulans. Other members of the

ascomycete class include Neuraspora crassa (orange bread mould) and Saccharomyces

cerevisiae (baker’s yeast).

A. nidulans was established as genetic model organism in the 1950s (Pontecorvo et al., 1953).

It has a relatively small, haploid genome of 28.5 Mb with about 8000 genes, spread over eight

chromosomes with sizes ranging from 4.3-2.7 Mb. It is able to reproduce with mitotically derived

conidiospores (conidia) and meiotic ascospores (Adams et al., 1998). Both spore forms are

generated at or in morphologically differentiated structures called conidiophores and cleistothecia

(fruiting bodies), respectively (Fischer, 2002)(Fig. 2.1). Whereas the developmental program of

conidiophore formation is well studied, cleistothecium differentiation is only poorly understood.

A. nidulans is especially attractive to analyze fruiting body formation because it is a homothallic

fungus and does not require a mating partner to initiate the developmental program.

2.1 Sexual development and fruiting body formation in A. nidulans A. nidulans is able to form fruiting bodies in the absence of a partner in a process called

selfing, which is the development of cleistothecia in homokaryons, with two identical parent

nuclei fusing and subsequently undergoing meiosis. This process results in meiospores with

genotypes identical to the single parent nucleus. The A. nidulans mycelium can exist as homo- as

well as a heterokaryon, the latter containing two genetically different sorts of nuclei after fusion

of vegetative hyphae (Käfer, 1977; Upshall, 1981). In fungi with two different mating types, an

antheridium cell (“male”) fuses with an ascogonium (“famale”) to give a dikaryotic hyphae. In

the homothallic fungus A. nidulans, wild type sexual development is initiated either by mating of

two strains or by selfing. Mating types are not known for this fungus, and no antheridium or

Introduction

8

ascogonium structure can be observed. In analogy to other filamentous ascomycetes, it is

assumed that an A. nidulans cell functionally equivalent to an ascogonium fuses to a second cell

equivalent to an antheridium (Champe et al., 1994). Initiation of the sexual reproductive cycle

and differentiation of three sexual tissue types take place shortly after conidiation begins

(Champe, et al., 1994). The fused hyphae are surrounded by growing, unordered mycelium,

which forms an increasingly packed “nest¨ and differentiates to form large thick-walled,

multinucleated, globose Hülle cells (nurse cells), which develop by budding at the tips of

specialized hyphae and form a tissue that envelops the young cleistothecium (Hermann et al.,

1983).

The surrounding mycelium which forms the nest is subject to the formation of the

cleistothecial primordium, a red-pigmented protective shell at maturity. In these nests, dikaryotic

hyphae are formed by the fertilization events and subsequently undergo an extended series of

coordinated cell and nuclear divisions. The developing cleistothecium grows out of the

surrounding nest hyphae and Hülle cells, while the dikaryotic mycelium undergoes a switch from

the coordinated nuclear and cellular division of ascogeneous hyphae to the formation of the so-

called croziers (Fig. 2.2). Two nuclei are trapped in the topmost crozier cell by a series of

divisions which require exact nuclear positioning and cell wall insertion. In every single crozier,

a nuclear fusion event (karyogamy) forms a diploid nucleus �70/80 h after spore germination

(Pontecorvo, et al., 1953). This short zygote stage is immediately followed by meiosis, which

results in four nuclei. After meiosis, one round of mitosis produces eight nuclei which are

separated from each other by membranes. Another round of mitosis yields the eight binucleate

ascospores organized in an octad of an A. nidulans ascus. Mature cleistothecia of wild type

strains can reach a size of 200 µm and usually contain �80,000 viable ascospores. The ascospores

are red owing to the accumulation of a characteristic red pigment called asperthecin. Under

laboratory conditions, cleistothecia and ascospores reach maturity �100 h after the initial spore

germination.

Introduction

9

Fig. 2.1 Life cycle of Aspergillus nidulans. The fungal mycelium of A. nidulans is a web of branched filaments (hyphae) of connected compartments or cells, which each contain several nuclei (see centre figure). This mycelium, or homokaryon, which develops from a single haploid spore, differentiates many identical asexual spores known as conidia or conidiospores (see the asexual cycle in the figure). A. nidulans is homothallic, which means that it is self-fertile, but crosses can be initiated by hyphal fusions between homokaryons with genetically different nuclei (shown by white and dark green nuclei). The resulting heterokaryons are not stable, but can be forced to maintain a balanced ratio of the component nuclei by including complementing auxotrophic mutations in the parental nuclei and forcing growth without the corresponding supplements. A. nidulans can also reproduce sexually. In the fruiting body, which produces the sexual spores, a pair of nuclei that is destined for meiosis divides in synchrony to form a mass of cells known as the ascogenous hyphae. These hyphae are highly branched and each tip cell becomes an ascus (a specialized cell) in which the two haploid nuclei fuse. The diploid nucleus undergoes meiosis followed by a post-meiotic mitosis, which results in the formation of eight haploid ascospores. The fruiting body, called the cleistothecium, can hold tens of thousands of ascospores, which are released

Introduction

10

into the environment when the cleistothecium bursts open. In addition to an asexual cycle and sexual cycle, a parasexual cycle offers the genetic benefits of meiosis achieved through a mitotic route (Pontecorvo & Kafer, 1958). The parasexual cycle is initiated when haploid nuclei fuse in the vegetative cells of a heterokaryon and continue to divide mitotically. Crossing over might occur between homologues and random chromosome loss restores the haploid chromosome number, which is eight in the case of A. nidulans. These events can be used to map gene orders and assign new genes to the eight linkage groups. Many closely related fungi of economic or medical importance, such as A. niger, A. fumigatus, Fusarium oxysporum and Penicillium chrysogenum, have no sexual cycle but are exploited experimentally or genetically using technologies developed for A. nidulans (Clutterbuck, 1992) (Taken from Casselton & Zolan (2002)).

e

a

b

cd

ascogenous hyphae

Fig. 2.2 iIlustration of crozier formation. In the ascogeneous, heterokaryotic hyphae (a) containing two haploid nuclei of opposite mating type, a hook-shaped structure is formed in which nuclei divide synchronously. (b) The penultimate, dikaryotic cell of the ascogonium forms the top crozier cell (c) in which, after fusion of the end cell and basal cell (d) karogamy (e) and further ascus development take place (Revised from Braus et al., 2002).

2.2 Determinants influencing fruiting body formation in A. nidulans 2.2.1. Environmental factors affecting sexual development

Environment factors such as light, CO2, surface exposure, nutritional status, amino acids and

hormones influence sexual development. Incubation of A. nidulans wild type in the dark for 24 h

after inoculation leads to higher densities of fruiting bodies and less conidiation than during

incubation in the light. In the dark, cleistothecia formation can also be initiated at an earlier time

point than light-grown colonies (Zonneveld, 1977). A. nidulans wild type strains produce �2000

fruiting bodies per cm2 on petri dishes while developing approximately double the amount of

cleistothecia and fewer conidiospores when air exchange is limited by taping the plates, which is

Introduction

11

attributed to an increase of the CO2 content. Usually, a mycelium does not differentiate in

submerged culture but can be induced by transfer to solid medium. Sexual development requires

a constant surface on which develop. A surface is indispensable for cleistothecia formation

(Galbriath & Smith, 1968). Major nutritional compounds in the medium play a crucial role in

sexual development. With reduced carbon source (0.8% glucose), fruiting body development was

reduced or blocked compared to growth on 3% glucose (Han et al., 1994). Low nitrogen levels

inhibit cleistothecia development (Zonneveld, 1975). A hormone was reported to influence sexual

development, the so-called psi factor (precocious sexual inducer), an endogenous mixture of

hydroxylinoleic acid moieties (Champe & El-Zayat, 1989; Champe et al., 1987). The psi factor

extracted from growth medium of A. nidulans to a confluent plate culture strongly inhibits

asexual sporulation and induces premature sexual sporulation.

2.2.2 Genetic determinants regulating fruiting body development

Perception of the environmental status as well as signal transduction relies on specific systems

encoded by genetic determinants. From the �8000 genes encoded by the A. nidulans genome, an

estimated 6000 are required for “housekeeping” biochemical functions. A large proportion of the

remainder of the genes is expected to be required for development and differentiation processes,

e.g., the detection of environmental signals, signal transduction processes within developmental

programs, or altered gene expression for coordinating the action of general and specialized

biosynthetic enzymes.

The veA gene was identified as a gene responsible for a velvet-like phenotype (Käfer, 1965).

The veA1 mutation delays and reduces the development of sexual organs, resulting in the

preferential development of asexual sporulation (Champe et al., 1981). Compared with the wild

type (e.g. FGSC4), asexual development of the veA1 mutant is much less affected by various

environmental factors such as nutrients (Han et al., 1994a), light (Mooney & Yager, 1990) and

temperature (Champe, et al., 1981). Therefore, it was proposed that veA acted as a negative

regulator of asexual development and an activator of sexual development in response to the

various environmental factors (Han, et al., 1994a; Timberlake, 1990). Protection of the culture

plate from aeration and light inhibits asexual sporulation of the veA+ strains almost completely

(Han et al., 1990; Mooney & Yager, 1990). The veA gene was recently cloned and its

overexpression induced a larger number of sexual structure (Kim et al., 2002).

Introduction

12

Although numerous signal transduction pathways are described and conserved among

eukaryotes, the signalling compounds necessary for sexual development in A. nidulans are hardly

known. This might be partally due to the fact that there is a spatiotemporal order of the two

programs of asexual and sexual development subsequent to hyphal growth. However, both

reproduction pathways seem to share signal transduction compounds. In addition, conidiation

starts significantly earlier than sexual development, so intrinsic signals play an important role for

the decision of the fungus to start cleistothecia formation. Accordingly, there has to be crosstalk

between regulatory proteins specific for the asexual cycle and the sexual cycle of development.

Some environmental parameters, which have to be perceived and translated into internal signals,

are essential for both differentiation pathways. Numerous mutant strains altered in genes essential

for both spore-producing developmental programs which have been described as defective in

sexual sporulation, had originally been isolated for other characteristic phenotypes.

These genes include e.g. some of the flu (= fluffy) genes or their suppressors which influence

the development of the vegetative mycelium as well as sporulation program (Wieser et al., 1997).

Flu mutations generate colonies with profuse aerial hyphae, giving them the appearance of cotton

wool (Wieser et al., 1994). Genetic analysis of flu genes and their suppressors revealed several

elements of signalling pathways. Although initially described as defective in asexual sporulation,

the flu phenotype is typically correlated with the inability to perform the sexual cycle, indicating

that the gene products exert a connecting role between the two developmental programs.

In addition, elements of heterotrimeric G-proteins have been identified which are involved in

development. The sfaD gene encodes the �–subunit (Rosen et al., 1999), fadA the �–subunit of a

heterotrimeric G-protein (Adams et al., 1992). The flbA gene, a homologue of yeast SST2,

encodes a RGS protein (� regulator of G-protein signalling) and seems to antagonize the action of

the heterotrimeric G-protein (Yu et al., 1996). The major role of this G-protein might be to decide

between growth as vegetative mycelium and the initiation of a developmental program like

sporulation. It is unclear in A. nidulans whether the isolated heterotrimeric G-protein is connected

to the cAMP-dependent PKA (protein kinase A) pathway. This connection exists in budding

yeast between response to the nutritional situation in the environment and initiation of a

development program, the filamentous pseudohyphal growth (Mösch & Fink, 1997; Kübler et al.,

1997). The gene product of flbE is another protein which is presumably involved in signal

transduction and required for developmental processes, but its exact molecular function is

unknown.

Introduction

13

Transcription factors

medA, stuA steA, dopA

rosA

DNA repair

nuv�-1, 3-glucan

dcl

Hormones

psi FluGp

Pigment

yB

cytoskeleton

tub

light

veA

Time

Space(surface)

Air exchange

Fluffy genesSignal transduction

G protein sfaD, fadA

flbA, rasA

Carbon source

Nitrate source

Amino acids

cpcA, cpcB

Nsd, Bsd, asd

Revised from Braus et al., 2002

C

H

Fig. 2.3 Determinants influencing fruiting body formation in A. nidulans. Environmental factors are shown in boxes, whereas relevant genetic determinants are presented in the inner boxes (see text for details). The electron micrograph shows a mature A. nidulans cleistothecium (C) surrounded by Hülle cells (H) was taken from Scherer & Fischer (1998).

The RasA, a small G-protein encoded by a homologue of the ras genes, has been shown to be

essential for regulating an ordered developmental program. The active GTP-bound and the

inactive GDP-bound form of the protein have been mimicked by the construction of dominant

alleles with the appropriate mutations. The overexpression of constitutive inactive rasA alleles

resulted in an acleistothecial phenotype (Hoffmann et al., 2000).

Several transcription factors have been identified which are involved in asexual or/and sexual

development of A. nidulans. For the asexual pathway, the most prominent genes are brlA and

abaA. brlA encodes a C2/H2 Zinc finger protein and overexpression of either flbC or flbD in

submerged hyphae activates its expression (Adams, et al., 1998; Wieser & Adams, 1995). abaA

encodes a protein with an ATTS/TEA domain which is conserved among other members of the

family. The abaA gene product acts downstream of brlA. These two key regulatory proteins seem

to be specific for conidiation without obvious influence on cleistothecia formation. In contrast to

this, medA and stuA are two modifier genes of development where mutant alleles exist which

exhibit clear effects on asexual as well as on sexual differentiation with the medA gene product

Introduction

14

having a more general role influencing both sporulation pathways. The corresponding wild type

protein is responsible for the correct temporal expression of both transcripts of the asexual

regulator brlA and also functions as coactivator required for normal levels of abaA expression

(Busby et al., 1996). Mutations in the medA result in aberrant conidiophores with branching

chains of metulae, delayed conidiophores (Clutterbuck, 1969). The medA mutant strains produce

only Hülle cells during the sexual cycle. The stuA mutant strains are completely acleistothecial

and exhibit spatially abnormal conidiophores with spore production from the vesicles (Miller et

al., 1991). Another transcription factor DopA is also involved in asexual and sexual sporulation.

The deletion of dopA in A. nidulans results in several morphologically distinguishable defects:

vegetative hyphae and conidiophores show an abnormal morphology, the sexual cycle is

abolished, suggesting a very early block in sexual development (Pascon & Miller, 2000).

Deletion of the transcription factor nsdD prevents fruiting body development and Hülle cell

formation. In contrast, when the nsdD gene was overexpressed, sexual-specific organ (Hülle

cells) was formed even in submerged culture, which normally completely blocked sexual

development, and the number of cleistothecia was also dramatically increased on solid medium.

These results lead to propose that the nsdD gene functions in activating sexual development of A.

nidulans. A. nidulans steA encodes a protein with a homeodomain 63-75% identical to those of

other Ste12 proteins, with greatest similarity to Ste12� of F. neoformans. SteA and Ste12� lack

the pheromone induction domain found in budding yeast Ste12, but have C-terminal C2/H2-Zn+2

finger domains not present in the other Ste12 proteins. A �steA strain is sterile and differentiates

neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual

cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation

of PH-like (pseudohaphae-like) asexual reproductive cells (metulae and phialides) are normal in

the deletion strain. Northern analysis of key regulators of asexual and sexual reproduction cycles

support the observation that although SteA function is restricted to the sexual cycle, cross

regulation between the two developmental pathways exists (Vallim et al., 2000). RosA (repressor

of sexual development) is a Zn(II)2Cys6 transcription factor characterized recently in our group.

(Vienken, 2003). Overexpression of rosA in A. nidulans led to a reduction of the hyphal growth

rate, a complete block of development and the proliferation of aerial mycelium giving the

colonies a fluffy, cotton-like appearance. Deletion of the gene induced the production of masses

of Hülle cells in submerged culture, which has never been observed in the wild type. Because of

the observation that overexpression of the transcription factors NsdD or VeA induce Hülle cell

Introduction

15

formation in liquid culture comparable to the deletion of rosA, it was proposed that a

coordianated action of activators such as NsdD and VeA and the repressor RosA mediate the

transition from vegetative growth and asexual development to sexual development.

The A. nidulans sexual cycle consists of several developmental programs which are

interconnected: the formation of ascogenous hyphae, asci, and ascospores; the formation of Hülle

cells; and the formation of the fruiting body envelope surrounding the asci. Since several A.

nidulans mutant strains exhibit only Hülle cells, the formation of this specific cell type can be

uncoupled from the other processes. Furthermore, sexual and asexual development seem to be

interconnected as the two programs presumably share regulatory elements necessary for both

sporulation programs. Accordingly, cleistothecia formation depends on the regulation of a large

number of genes. In addition, the molecular analysis of a number of mutant alleles of genes

which might play a role in cleistothecia formation will increase within the years.

2.3 Objective of this study As described above, although a few genes involved in cleistothecium differentiation were

identified, the process of sexual development is still poorly understood. In this study, the

functions of three genes were mainly analysesd using a reverse genetic approach. Two of them,

�-1,3-glucanase (mutanase, mutA) and a high-affinity hexose transporter (hgtA) which were

discovered in a differential cDNA library (Scherer, 2001), were further analysed. In the search for

factors of upstream signalling pathways triggering sexual development of A. nidulans, a MAP

kinase kinase kinase (steC) was also studied.

2.3.1 Carbon cycle related to �-1,3-glucanase (mutanase) and high-affinity hexose

transporters in sexual development of A. nidulans

The cell wall plays an important role in the growth and development of fungi. In addition to its

function as the primary osmotic barrier of the cell, the temporal and spatial regulation of wall

polymer synthesis is critical to the morphogenesis of the cell types characteristic of many fungi.

During vegetative growth of A. nidulans, the mycelium stores large amounts of �-1,3-glucan

(mutan), which occupies most of the alkali-soluble fraction (about 22% dry weight of complete

cell wall) in the cell wall of A. nidulans (Zonneveld, 1971; 1972a). Later when the external

Introduction

16

glucose supply is depleted, the glucan is broken down as glucose by an exo-splitting �-1,3-

glucanase (mutanase) and reutilised as a carbon and energy source for cleistothecium formation

(Zonneveld, 1972b; 1974)(Fig. 2.4). A mutant that lacks �-1,3-glucan is acleistothecial

(Martinelli & Bainbridge, 1974; Polacheck & Rosenberger, 1977). It is assumed that the

requirement of �-1,3-glucan for the formation of cell walls of the cleistothecium depends on

increased �-1,3-glucanase activity, �-1,3-glucanase activity correlates with the density of

cleitothecia. An A. nidulans mutant strain exhibiting an increased density of cleistothecia has

been isolated which showed increased �-1,3-glucanase activity (Zonneveld, 1974). In this study,

I characterized the �-1,3-glucanase gene (mutA) for �-1,3-glucan degradation, which is found in

substractive hybridization library (SSH)(Scherer, 2001). It was shown that the gene is specifically

expressed in Hülle cells, and is dispensable for sexual development.

Glucose

�-1,3-glucansynthesis

Glucose

Starvation

�-1,3-glucandegradation

Fig. 2.4 Carbon cycle including the cell wall as storage compartment. A. nidulans synthesizes α-1,3-glucan (mutan) using the carbon source such as glucose during vegetative growth and deposits it as cell wall material. After depletion of external glucose, α-1,3-glucan can be degraded by α -1,3-glucanase and reutilised as a carbon and energy source during the formation of cleistothecia (Zonneveld, 1972b; 1974).

The initial step in glucose metabolism is the uptake of glucose, which is carried out by hexose

transporter proteins localized in the plasma membrane (Bisson et al., 1993; Boles & Hollenberg,

1997; Kruckeberg, 1996; Ozcan & Johnston, 1999). Eukaryotic and bacterial sugar transporter

proteins are related in protein sequence and cellular topology, forming the sugar permease

superfamily (Bisson et al., 1993). In the yeast Saccharomyces cerevisiae, hexose transporter

(HXT) proteins transport glucose across the plama membrane. The HXT proteins are encoded by

a multigene family with 20 members, of which HXT1-4p and HXT6-7p are the major hexose

transporters. The remaining HXT proteins have other or unknown function (Ozcan & Johnston,

1999; Reifenberger et al., 1995; Wieczorke et al., 1999). The existence of such a multigene

Introduction

17

family of glucose transporters in this yeast resembles the situation in the mammalian systems

(Gould & Bell, 1990; Weierstall et al., 1999), in which six glucose carriers have already been

identified. In A. nidulans, it is anticiptated that the extracellularly monosaccharide released by the

action of mutanase can be taken up by the cells and transferred to the metabolism by hexose

transporters. Indeed in the subtractive hybridisation library (Scherer, 2001), in which also the

mutanase gene was discovered, a fragment, whose translation sequence exhibited similarity to a

high-affinity hexose transporter gene of the yeast, was found. This enable us to examine glucose

utilization during sexual development was examined. Here a new A. nidulans gene, hgtA (high-

affinity glucose transporter), which may code for the major high-affinity glucose carrier in this

fungus was analysed.

2.3.2 MAP kinase cascade

Mitogen-activated protein kinases (MAP kinases) are ubiquitous among eukaryotes. MAP

kinases are components of MAP kinase cascades, which are major signalling modules by which

cells transduce extracellular cues into intracellular responses (Gustin et al., 1998; Stork &

Schmitt, 2002). Originally they were described as protein kinases, which were transiently

activated by a variety of mitogens, including insulin or growth factors and are thus implicated in

cell proliferation and regulation of the cell cycle. Misregulation in animal cells leads to

inappropriate activation of cell division and might result in the development of cancer

(Schramek, 2002). The basic mechanism of signal transduction appears to be very similar in

different MAP kinase cascades, namely a sequential activation of protein kinases upon the

external stimulation with a signal. One early kinase after signal recognition, is a MAP kinase

kinase kinase, which phosphorylates a MAP kinase kinase at two amino acid residues. The latter

kinase in turn activates a MAP kinase, again by dual phosphorylation. The phosphorylated MAP

kinase triggers the activity of transcription factors, and thereby the external signal is transmitted

from the surface of the cell into the nucleus.

The evolutionary conservation of MAP kinase signalling pathways allows to use lower

eukaryotes such as Saccharomyces cerevisiae or Schizosaccharomyces pombe as models to

unravel the molecular and biochemical functions of the components (Herskowitz, 1995). In S.

cerevisiae at least 6 different MAP kinase cascades exist which differ in the signals, which are

perceived and transmitted, in the activation of the specific MAP kinase and ultimately a specific

Introduction

18

transcription factor (Fig. 2.5)(Gustin, et al., 1998; Hohmann, 2002). The cascades are involved in

mating, nutrient sensing and pseudohyphal growth, osmoregulation and stress adaptation, cell

integrity, and ascospore formation. Thus the cellular responses are as diverse as the induction of

mating upon pheromone perception and the synthesis of compatible solutes upon osmotic stress.

Nevertheless, some signalling molecules are used in different cascades. Likewise the MAPKK

kinase Ste11 is involved in mating, pseudohyphal growth and osmoregulation. The regulation of

the specificity of each cascade and the prevention of cross talk between them is one important

and largely unsolved question (Sabbagh et al., 2001). In plant or human pathogenic fungi MAP

kinase cascades are involved in triggering the pathogenic program and the adaptation to the host-

specific environmental conditions (Lengeler et al., 2000; Mayorga & Gold, 1999; Müller et al.,

1999; Xu, 2000).

Ste2Ste3

Sho1?Sin1 Mid2 Wsc1

? ?

Ste20

Ste50 Ste50

Ste20 Ste20 Ste20

Ste50 Ste50

Pkc1 Sps1Ssk1

Gpa1 Ste4 Ste18

Cdc24

Cdc42

Ras2

Cdc24

Cdc42

Cdc24

Cdc42 Ypd1 Rho1

Rom2

Ste11 Ste11 Ste11 Ste11 Ssk2Ssk22

Bck1 ?

Ste7 Ste7 Ste7 Pbs2 Mkk1Mkk2 ?

Fus3 Kss1 Kss1 Hog1 Slt2 Smk1

Ste12 Ste12 Ste12 Rim1 ?

Ste5

?

MATING RESPONSE FILAMENTOUS GROWTH

CELL WALL INTEGRITY

Several factors

OSMOREGULATION CELL WALL CONSTRUCTION ASCOSPORE

FORMATION

Receptor/Sensor

Upstream control

MAPKKK

MAPKK

MAPK

Fig. 2.5 Six S. cerevisiae MAP kinase pathways. The MAPKKK, MAPKK, and MAP kinase cascades regulating mating responses, filamentous growth, cell integrity, and osmoregulation in vegetative cells grown under different conditions. The SMK1 pathway is expressed only during ascospore formation (Revised from Hohmann, 2002)

Introduction

19

MAP kinase cascades are involved in major developmental transitions in the life cycle of the

unicellular fungus S. cerevisiae (Fig. 2.5). In comparison to S. cerevisiae, the life style of the

filamentous fungus A. nidulans is much more complex (Adams, et al., 1998; Braus, et al., 2002;

Fischer, 2002). Unicellular spores initiate polarized growth and emerge germtubes at one pole of

the spore. This hyphae extends by continuous elongation of the hyphal tip. The mycelium is

metabolically very versatile and can adapt to many different environmental conditions, such as

high osmolarity, different temperatures or desiccation. Besides hyphal growth A. nidulans can

undergo different developmental programs. After about 20 h of vegetative growth, asexual

reproductive structures, called conidiophores emerge into the air. From a vesicle on top of a stalk

two layers of unicellular, uninucleate cells, the metulae and phialides are generated in a budding-

like process. The phialides are the spore-producing cells, which continuously protrude

conidiospores. The growth pattern in the conidiophore resembles the pseudohyphal growth type

of S. cerevisiae (Gimeno et al., 1992). After about 5-6 days and after depletion of the carbon

source, the mycelium of the fungus enters the sexual cycle. A. nidulans is a homothallic fungus

and does not require a mating partner although a cross to other strains is favoured. The sexual

developmental pathway leads to the formation of highly differentiated structures, the

cleistothecia, in which karyogamy and subsequent meiosis occurs to generate haploid ascospores.

The developmental decisions are partly triggered by a pheromone system consisting of

interconvertible C-18 fatty acids (Champe & El-Zayat, 1989). However, the exact function of the

pheromones as well as their perception and cellular responses are largely unknown. To get

insights into the involvement of MAP kinase cascades during the life cycle of A. nidulans the

MAPKK kinase SteC, a homologue of S. cerevisiae Ste11, was studied.

Materials

20

3 Materials

3.1 Equipment and Chemicals

Table 3.1 Main equipment used in this study

Equipment Type Manufacturer

SORVALL RC 5B plus (HB-6)

SORVALL ultra pro80

SORVALL RC 28S

SORVALL, Bad Homburg, Centrifuge

with rotors

Centrifuge 5403 Eppendorf, Hamburg

Electroporation apparatus Gene Pulser II, Pulse Controller Bio-Rad, Munich

Electrotransfer apparatus Mini Trans-blot Electrophoretic

Transfer Cell

Bio-Rad, Munich

Hybridization oven Personal HybTM Stratagene, Heidelberg

Rapid Cycler Idaho Technology PCR machines

Personnal Cycler Biometra, Goettingen

SDS-PAGE apparatus Mini Protein II Bio-Rad, Munich

UV-cross Linker UV Stratalinker 2400 Stratagene, Heidelberg

UV/Visible spectrophotometer Ultrospec 3100 pro Amersham Pharmacia

Biotech, Freiburg

Table 3.2 Kits used in this study

Kit Manufacturer

BM Chemimminescence Blotting Substrate (POD) Roche, Mannheim

DNeasy Plant Kit Qiagen, Hilden

Nucleobond� AX Macherey-Nagel, Düren

RNeasy Mini Kit Qiagen, Hilden

QIAEX� II Gel Extraction Kit (150) Qiagen, Hilden

QIAquick� PCR Purification Kit Qiagen, Hilden

Materials

21

Chemicals were purchased from Boehringer (Mannheim), Merck (Darmstadt) Sigma

(Diesenhofen), Roth (Karlsruhe), Biomol (Hamburg) and Difco Laboratories (Detroit, MI, USA).

Restriction enzymes and other DNA-modifying enzymes from Amersham (Braunschweig), New

England Biolabs (NEB) (Frankfurt) or Invitrogen (NV Leek, The Netherlands). The enzymes for

PCR from Qiagen (Hilden) or Promaga (Mannheim). Radionucleotide [�-32P]-dATP from

Hartmann Analytics (Braunschweig). The autoradiographic film from Kodak (Rochester, NY,

USA) or Fuji (New RX, Fuji, Japan). Filter (Miracloth) from Calbiochem-Novabiochem (Bad

Soden / Ts.). Anti-phospho MAPK antibodies from New England Biolabs.

3.2 Media

Ingredients were added to ddH2O, poured into bottles with loosen caps and autoclaved 20 min

at 15 lb/in2, For solid media, 1.5% (w/v) agar was added in media. Glassware and porcelain was

sterilized for 3 h at 180�C. Heat-sensitive solutions such as antibiotics, amino acids and vitamins

were filter-sterilized with 0.22 �m pore filter membranes (Millipore, France)

Standard media for Escherichia coli according to Sambrook & Russel (1999) were shown in

Table 3.3, and supplements in Table 3.4.

Table 3.3 Media for E. coli

Medium Ingredients (1 liter)

LB 10 g Bacto-Trypton; 5 g Bacto-Yeast Extract; 10 g NaCl

SOC 20 g Bacto-Trypton; 1 g Bacto-Yeast Extract; 5 g NaCl; 0.185 g KCl; 2.03 g

MgCl2 x 7H2O; 2.46 g MgSO4 x 7H2O; 3.6 g Glucose

Table 3.4 Antibiotics and supplements for E. coli media

Substance End concentration

Ampicillin ( Ap) 100 µg/ml

Kanamycin (Km) 50 µg/ml

X-Gal 40 µg/ml

IPTG 8 µg/ml

Materials

22

Minimal and complete media for A. nidulans growth were prepared according to the protocols

(Pontecorvo et al., 1953). For protoplast transformation of A. nidulans, 0.6 M KCl as

osmoprotection substance was added into minimal media (Table 3.5). The supplemented

vitamins, amino acids and ncleotides for auxotrophic A. nidulans strains were listed in Table 3.6.

Table 3.5 Media and stock solutions for A. nidulans

Media or Stock Preparation (per liter)

20 x Salt stock solution 120 g NaNO3; 10.4 g KCl; 10.4 g MgSO4 x 7H2O; 30.4 g KH2PO4

1000 x Microelement stock

solution

22 g ZnSO4 x 7H2O; 11 g H3BO3; 5 g MnCl2 x 4H2O; 5 g FeSO4 x 7H2O;

1.6 g CoCl2 x 5H2O; 1.6 g CuSO4 x 5H2O; 1.1 g (NH4)6Mo7O24 x 4H2O;

50 g Na4 EDTA; adjust to pH 6.5-6.8 using KOH

Minimal medium (MM) 50 ml Salt stock solution; 1 ml Microelement stock solution; 20 g

Glucose; adjust to pH 6.5 using 10 N NaOH

Complete medium (CM) Minimal medium with 2 g Peptone; 1 g Yeast extract; 1 g Casamino-

acids; 1 ml Vitamin stock solution; 1 ml Microelement stock solution;

adjust to pH 6.5 using 10 N NaOH

Table 3.6 Vitamins, amino acids and medium components

Component Stock Concentration Volume per liter

Biotin 0.05 % 1ml

PABA 0.1% 1ml

Pyridoxin-hydrochloride 0.1 % 1 ml

Arginine 500 mM 10 ml

Uracil - 1 g

Uridine 500 mM 10 g

3.3 A. nidulans and E. coli strains

Table 3.7 A. nidulans and E. coli strains used in this study

Strain Genotype Source

FGSC26 biA1; veA1 FGSC, Kansas, USA

FGSCA4 wild type FGSC, Kansas, USA

Materials

23

SRF200 pyrG89; �argB::trpC�B; pyroA4; veA1 (Karos and Fischer,

1999)

GR5 pyrG89; wA3; pyroA4; veA1 G. May, Houston, TX,

USA

RMSO11 pabaA1, yA2; �argB::trpC�B; veA1 (Stringer et al., 1991)

AJC48 biA1; medA26; veA1 J. Clutterbuck,

Glasgow, UK

SHW1 and 44 RMSO11 transformed with mutA::argB (pMut-argB);

homologous integration

This study

SHW26 and 29 RMSO11 transformed with mutA::argB (pMut-argB);

no replacement of mutA; considered wild type

This study

SHW44-13 pabaA1, yA2; veA1; mutA::argB

SWH44 crossed to SRF200; mutA replacement selected

This study

SHW-p-sgfp17

and 19

RMSO11 transformed with mutA(p)::sgfp (pMut-p-

sgfp) and pDC1

This study

SHW-gpd3, 4, 5 RMSO11 transformed with gpd(p)::mutA (pMut-gpd)

and pDC1

This study

SWHH11 SRF200 transformed with hgtA::argB (pHHRarg11),


This study

SWHgfp1, 7 SRF200 transformed with hgtA::sgfp (pHHgfp4) This study

SWTB SRF200 transformed with pDC1, wild type phenotype (Schier, 2001)

SWH33 and 35 RMSO11 transformed with steC::argB (pHSAB3);


This study

SWH51, 57

SRF200 transformed with steC::argB (pHSAB3);


This study

SWHSR3 �steC strain SWH51 retransformed with pHSKS2 This study

SWHSGP4, 6, 8 �steC strain SWH51 retransformed with pHSKP3 This study

SWHSGR3, 5, 8 �steC strain SWH51 retransformed with pHSKR4 This study

SWHSGSa3, 4 �steC strain SWH51 retransformed with pHSKSa4 This study

SWHSGSt2, 4, 8 �steC strain SWH51 retransformed with pHSKSt4 This study

SWHSgfp3, 21 RMSO11 transformed by pHSRB-gfp1 and pDC1, This study

Materials

24

Escherichia coli

XL1-Blue recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac

[F’proABlacIQZ.M15::Tn10 (TetR)]

Stratagene,

Heidelberg

Top10F’ F’[lacIQ, Tn10 (TetR)] mcrA .(mrr-hsdRMS-mcrBC ), O80

lacZ .M15.lacX74, deoR, recA1, araD139.(ara-leu)7679,

galU, galK, rpsL, (StrR) endA1, nupG

Invitrogen, Leek,

Netherlands

GM2159 thr-1, araC14, leuB6, DE(gpt-proA)62, lacY1, tsx-33,

glnV44(AS), galK2(Oc), LAM-, hisG4(Oc), rpsL31(strR),

dam-13::Tn9, xylA5, mtl-1, recF143, argE3(Oc), thi-1

E. coli Genetic

Stock Center, New

Haven, CT, USA

3.4 Plasmids and Cosmids

Table 3.8 Plasmids and cosmids used in this study

Cosmids/

Plasmids

Construction Source

pUC18 Cloning vector MBI Fermentas, St. Leon-

Rot

pBluescript

KS-

Cloning vector Invitrogen (NV Leek, The

Netherlands)

pCR®-Blunt Cloning vector Invitrogen (NV Leek, The

Netherlands)

pCR2.1TOPO Cloning vector Invitrogen (NV Leek, The

Netherlands)

pDC1 A. nidulans argB gene in pIC20R (Aramayo et al., 1989)

pRG1 N. crassa pyr-4 gene as selection marker (Waring et al., 1989)

pHG2 mutanase containing cosmid This study

pHGE5 7.3 kb EcoRV mutanase-containing fragment

cloned into pCR®-Blunt

This study

pHGE5-Sac pHGE5 digested with SacI and religated This study

pHW-arg argB released with KpnI-XhoI from pDC1 and

cloned into corresponding sites in pBluescript

This study

Materials

25

pMut-arg argB released with BamHI from pHW-arg and

cloned into BamHI digested pHGE5-Sac

This study

pMS-gpd gpd promoter inserted into BamHI and argB into

NotI of pBluescript KS-

(Scherer, 2001)

pMut-gpd mutA open reading frame cloned behind the gpd

promoter

This study

pRF917 alcA(p)::kinA::sgfp, sgfp cloned as a NotI fragment (Requena et al., 2001)

pMut-p-sgfp 1.8 kb mutA promoter fused with sgfp from

pRF917 into EcoRV and NotI of pBluescript KS-

This study

pH38E7 a hgtA-containing cosmid from A.nidulans PUI

cosmid library

This study

pHHH1 4 kb HindIII hgtA-containing fragment was

subcloned into PCR2.1TOPO

This study

pHHPS4 3 kb PstI-SalI fragment containing the promoter

and partial N-terminal was subcloned into

pBluescript KS-

This study

pHHRarg11 hgtA deletion construct (stratagy see result 5.1.5) This study

pHHgfp4 KS-Rev/Hex-Not1 amplyfied fagement (2.5 kb)

from pHHPS4 were inserted into EcoRV of

pBluescript KS-, sgfp from pMut-p-sgfp was

inserted into NotI

This study

pAG1 steC-containing cosmid (Geißenhöner et al., 2001)

pHSKS2 8 kb KpnI-SalI steC-containing fragment from

pAG1 cloned into pCR2.1TOPO

This study

pHSAB3 BamHI-released argB from pHW-arg inserted into

pHSKS2, steC deletion construct

This study

pHSRB-gfp1 4 kb EcoRV-BamHI fragment containing steC

promoter and N-terminal from pHSKS2 inserted

into EcoRV-BamHI and sgfp into NotI of

pBluescript KS-

This study

pHSKGP3 gpd::steC / PvuII-KpnI fragment in pMS-gpd This study

pHSKGR2 gpd::steC / EcoRI-KpnI fragment in pMS-gpd This study

Materials

26

pHSKGSa4 gpd::steC / SacI-KpnI fragment in pMS-gpd This study

pHSKGSt4 gpd::steC / StuI-KpnI fragment in pMS-gpd This study

3.5 Oligonucleotides

All primers used in this study were synthesized by MWG Biotech (Ebersberg)

Table 3.9 Primers used for PCR

Glucan1 5'-CAGTAGACCAGCTGGTCAG-3'

Glucan2 5'-GGAGGTTCAGGAATAATCTC-3'

Glucan-ex1 5'-CGCATCCCAGGCAACACA-3'

Glucan-ex2 5'-CTGCACGCACCCTCCCTA-3'

glu-P1 5'-CTCGTCGTGGCTGTGTGGAT-3'

glu-P2 5'-GCGGCCGCCATTGCGGCGTCAGTTGCT-3'

glu-P3 5'-GCGTAAGGGTATGAGATGGT-3'

glu-P4 5'-AGCCACGCCGTATGAGGAAT-3'

glu-P5 5'-TTCGCGCGATACCCATCAGT-3'

glu-P6 5'-GCGCAAGGTCTTAACATTGCCT-3'

glu-P7 5'-TTCGAGATTTTCCAGCGAGCATC-3'

glu-P8 5'-GTTAAATAGATGCCCGTGTCGCT -3'

glu-P9 5'-CCAGTCAGTATCTCTCAAAGCCT-3'

KS-gfp 5'-CGACTCACTATAGGGCGAATT-3'

GluP1B Biotin-5'-CTCGTCGTGGCTGTGTGGAT-3'

GluP6F 5'-CAATGTTAAGACCTTGCGCT-3'

GluP4B Biotin-5'-AGCCACGCCGTATGAGGAAT-3'

GluP4F 5'-ACCTGCCACCGGAAGACACT-3'

HexA5’ 5’-GTTTCGACATCTCGTCGATG-3’

HexA3’ 5’-CCAGCGGTCCTTGCTCG-3’

HexB5’ 5’-CAATTGTCGGGAATGAACGTC-3’

HexB3’ 5’-GTTCAAGGCGGCGACAGTG-3’

HexR1 5'-CCTTGGTGTTTCTCTTCTTCCTG-3'

HexR2 5'-TGAAGATGGCTTTCAAGAAGTCCT-3'

HexR3 5'-CATGACCCTTGGTATGAAACCGT-3'

Materials

27

HexF 5'-GACCAGCAAGGGGATACCGT-3'

Hex-Not1 5'-CGCGGCCGCCATCTTCAAAGGCGGTGCT-3'

KS-Rev1 5'-GCCAAGCGCGCAATTAACCCTCACT-3'

Hex-ex2 5'-AATGGACTCGAGATTACACCGCCTTCTCCGGT-3'

SteC-R1 5'-ATGTCCCGTTCCCTTAGATTCA -3'

SteC-R8 5'-GTTCTGCGACATGGCTAAATGATCGTGTCC -3'

SteC-R9 5'-TCCTCTACGAATCCGGATCAATATCTCTCG -3'

SteC-F4 5'-TCCTTTCGCCCTGAATCCAT-3'

SteC-F6 5'-CTGGTCGCATTCGATAAGGT-3'

Ste11A 5'-CGAGAGCCAACAGGCGC-3'

Ste11B 5'-TCATCGGCGGTAGTTCC-3'

Ste11C 5'-GTCTTGATGCTGTGAGGTG-3'

Ste11D 5'-GGGTCAGGGTCCAAGCC-3'

Ste11-ex3 5'-CGTCCTCATCGTTACGTTGCT-3'

Ste11-ex2 5'-TTGGTTGTTTTGTCGCGTGAGT-3'

Methods

4 Methods

4.1 Growth conditions and storage of transformed E. coli and A. nidulans strains Transformed E. coli was overnight cultivated on LB plates with appropriate antibiotics at

37�C. Liquid culture was inoculated from a single colony and incubated in LB medium

containing appropriate antibiotics at 37�C with 250 rpm overnight shaking. Freshly grown

bacterial suspension was adjusted to 15% end concentration of sterile glycerol and stored at –

80�C.

The A. nidulans strains were grown on minimal or complete medium plates. Pieces of a colony

were cut from an agar plate and suspended in 15-20% sterile glycerol and stored at –80˚C.

4.2 Transformation of A. nidulans

Standard procedures of Aspergillius protoplast transformation were used (Yelton et al., 1984).

4.2.1 Preparation of protoplast

A 500 ml volume of spore culture in minimal medium with appropriate components was

shaked at 30ºC in water bath for 12-16 h until spores germinated. The culture was filtered

through sterile miracloth followed by washing using Wash solution. The washed mycelium was

collected into a sterile flask that was set on ice. Then 5 ml of Osmotic medium, 200 mg of

GlucanX (Novozyme) in 1 ml sterile water and 6 mg BSA in 0.5 ml water was successively

added into the flask. The digestion mixture in the flask was incubated at 30ºC in water bath for 1-

3 h until enough protoplasts became free. The digestion mixture was transferred into a 30 ml

corex tube and 10 ml of Trapping buffer was slowly added on top of the mixture, followed by a

centrifugation at 5000 rpm for 15 min using a HB-6 rotor. Then a protoplast band was transferred

into a new sterile tube, followed by washing two times using STC with centrifugation at 7000

rpm for 8 min. The protoplast pellet was gently resuspended in 200-1000 �l STC for

transformation.

28

Methods

4.2.2 Protoplast transformation

100 �l of protoplasts in STC and 100 �l DNA (10 �g DNA filled with 100 �l STC) were

mixed and incubated 25 min at room temperature in a falcon tube. Then, 2 ml PEG was added

and the tube was rolled until the mixture was homogeneous, followed by 20 min incubation in

room temperature. Finally, 8 ml STC was added and the entire mixture spread onto osmatically

stabilized medium (MM + 0.6 M KCl) with appropriate selection markers. The plates were

incubated at 37ºC until clear colonies were formed after 3-4 days.

Mycelium wash solution 0.6 M MgSO4

Osmotic medium 1.2 M MgSO4; 10 mM Na3PO4 buffer (pH 5.8)

Trapping buffer 0.6 M Sorbitol; 0.1 M Tris-HCl (pH7.0)

STC 1.2 M Sorbitol; 10 mM CaCl2; 10 mM Tris-HCl (pH 7.5)

PEG 60% PEG 4000; 10 mM CaCl2; 10 mM Tris-HCl (pH 7.5)

4.3 DNA and RNA manipulations 4.3.1 Plasmid DNA preparation from E. coli cells

An alkali-lysis method was used for the isolation of plasmid or cosmid DNA (Sambrook &

Russel, 1999). For a small volume of liquid culture (Miniprep), 1.5 ml overnight culture was

centrifuged 1 min at 13000 rpm, and the pellet resuspended in 200 �l Tris-EDTA buffer. Then

200 �l Alkali-lysis buffer was added, gently mixed, with the cell suspension, followed by the

addition of 200 �l Neutralization buffer. After a 10 min centrifugation, plasmid DNA-containing

supernatant was precipitated with 0.7 vol. isopropanol followed by 70% EtOH washing. After

drying, the pellet was resuspended in TE buffer. For a large volume of liquid culture (50-100 ml;

Midipreps), plasmid DNA was prepared using the Qiagen Midi Plasmid Purification Kit.

Plasmid DNA concentration was determined via absorption measurement with 260 and 280

nm in a spectrophotometer (Pharmacia LKB-UltrospecIII) in a quartz cuvette or compared the

intensity of Ethidium bromide stained DNA bands on an agarose gel with the intensity of defined

standards.

29

Methods

Tris-EDTA buffer 5 ml 1 M Tris-HCl (pH7.5); 2 ml 0.5 M EDTA (pH8.0); 10 mg RNAse in 100

ml

Alkali-lysis buffer 0.2 M NaOH; 1% SDS

Neutralization buffer 1.5 M K-Acetate (pH4.8)

TE buffer 10 mM Tris-HCl; 1 mM EDTA; pH8.0

4.3.2 Genomic DNA preparation from A. nidulans

For the preparation of A. nidulans genomic DNA, around 25 ml fresh liquid minimal media in

a 9 cm plastic plate was inoculated using a spore suspension prepared the colony grown on an

agar plate. The culture was incubated for 12-15 h at 37�C. Then, the mycelium was taken off with

a spatula and pressed briefly until dry between paper towels, and put into liquid N2. The frozen

mycelium was grinded in liquid N2 or kept at –80˚C until isolation. A. nidulans genomic DNA

was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden). Finally, 5 �l DNA extraction

was used to check quality and yield via running a 1% agarose gel.

4.3.3 Precipitation of DNA

Contamination by small nucleic acid fragments, protein and salt can be reduced to acceptable

levels by precipitating the DNA. 2.5 volume of ethanol and 1/10 3.0 M NaAc (pH 5.2) were

added into the DNA solution. The sample was mixed, kept at –80ºC for more than 10 min and

centrifuged for 10 min at 10000 rpm. The supernatant was discarded and the pellet was washed

with 70% EtOH, followed by centrifugation at 10000 rpm for 5-10 min. The purified DNA pellet

was completely dried in a speed vacuum or at 50ºC for 10-20 min, and then dissolved in sterile

water or TE buffer.

4.3.4 DNA electrophoresis through agarose gel

DNA electrophoresis through agarose gel is a standard method to separate, identify and purify

DNA fragments. An agarose gel of 0.8-1.2% was prepared by boiling agarose in 0.5 or 1 x TAE

buffer and pouring it into gel-making models. DNA samples were mixed with 1/10 10 x DNA

loading buffer. DNA samples together with a DNA marker (Eco130I-cut � DNA, MBI 30

Methods

Fermentas, St. Leon-Rot) were separated at 10 V/cm for 30 min-4 h depending the length of the

gel in 0.5 or 1 x TAE buffer. Then, the gel was stained in 0.5 x TAE buffer with Ethidium

bromide for 15-30 min. The DNA bands in the gel were visualizd by 302 nm UV light and

pictures were taken with a camera (INTAS, Goettingen) connected to a video printer.

50 x TAE buffer (pH 8.0) 40 mM Tris-Acetate; 1 mM EDTA; pH 8.0

10 x Loading buffer 20% Ficoll 400; 0.1 M Na2EDTA (pH 8.0); 1% SDS; 0.25%

Bromphenol blue; 0.25% Xylene cyanol

4.3.5 Digestion of DNA by restriction endonucleases

DNA samples (200 ng – 1 �g) were digested by restriction endonucleases using

corresponding reaction buffers. Enzyme, DNA, buffer and reaction time varied depending on the

specific requirements (generally, 37˚C from 1 h to overnight). A. nidulans genomic DNA was

generally digested for more than 2 h. For cloning, it was enough for 1-2 h. In the case where it

was necessary to treat the same DNA sample with different enzymes, the digestion was carried

out first in the buffer with low salt concentration or the buffer compatible to different enzymes.

4.3.6 PCR

Polymerase chain reaction (PCR) was accomplished with Taq (Gibco or Invitrogen), Expand

(Boehringer) or Pfu (Promega) according to manufacturer protocols. The synthesis of oligo

nucleotides was made by MWG Biotech (Ebersberg). As beginning oligonucleotide

concentrations, 5-20 �M were used in a reaction volume of 10-100 �l. The PCR reaction took

place in a Personal Cycler (Biometra), or a capillary Rapid Cycler (Idaho Technology, Idaho

Falls, ID, USA). RT-PCR was carried out using SUPERSCRIPTTM II RNase H- Reverse

Transcriptase (Invitrogen) according to the manufacturer protocol.

31

Methods

32

A Standard PCR reaction in Rapid Cycler

1 �l 2.5 mM dNTP

1 �l templete DNA

1 �l 10 x buffer

1 �l 50 mM MgCl2

1 �l 10 x BSA

1 �l 10 x Ficcol

1 �l each 5 �M Primer A and B

0.2 �l Taq DNA polymerase

1.8 �l Autocloved ddH2O

A Standard PCR reaction in Personal Cycler

5 �l 2.5 mM dNTP

5 �l templete DNA

5 �l 10 x buffer

5 �l 50 mM MgCl2


1 �l Taq DNA polymerase

24 �l Autocloved ddH2O

4.3.7 DNA isolation from agarose gel

For DNA fragment isolation, 0.8% - 1% “low melting” gel was often used. The low melting

gel separated by gel electrophoresis was stained in 0.5 x TAE with ethidium bromide. The

appropriate DNA bands were cut out under UV light. The DNA purification was carried out

according to the protocol of WizardTM PCR Preps DNA Purification System (Promega, Madison,

WI, USA). Alternatively, the DNA in normal agarose gels was isolated with the QIAEX II Gel

Extraction System (Qiagen, Hilden).

4.3.8 Dephosphorylation of digested DNA

After the digestion with restriction enzymes, the vector was dephosphorylated by Shrimp

alkaline phosphatase (SAP) to remove the phosphate group at the 5’-end which prevent religation

of the vector. 0.1 unit / �M 5’-end with buffer was added into one sample. The mix was

incubated around 30 min at 37ºC. Less SAP and shorter incubation time were used for the

protruding 5’ termini than for recessed 5’ termini. If two enzymes with incompatible termini were

used, the dephosphorylation process was omitted.

Methods

33

4.3.9 DNA ligation

DNA ligation was carried out using T4 ligase (Amersham-Pharmacia Biotech, Freiburg) at

16ºC or Fast LinkTM System (Biozym, Hessisch Oldendorf) in a volume of 10-20 �l. The

concentration of vector and insert DNA was measured on the basis of DNA marker. Around 50

ng vector was used in one ligation. The ratio of vector to insert was 1: 2-3 and 1:5-10 respectively

for sticky and blunt end ligation. For the cloning of PCR products, it was often done to add

restriction enzyme sites in both primers. In addition, PCR fragments were cloned blunt end into

EcoRV or SmaI of pBluescript or into PCR-Blunt (Invitrogen, NV Leek, The Netherlands). For

TA cloning, the PCR products amplified by Taq or Taq-containing polymerases were cloned into

PCR2.1TOPO (Invitrogen).

4.3.10 DNA sequencing

DNA sequencing was done by commercial sequencing (MWG Biotech, Ebersberg).

4.3.11 Transformation of E. coli

The transformation of electrocompetent E. coli cells was done as described (Ausubel et al.,

1995). A fresh single E. coli colony was cultured overnight in 37ºC. The culture was centrifuged,

followed by repeatly washing using cool sterile water at 2ºC. The cells were resuspended in 10%

glycerol and aliquot frozen at –80ºC for use. After desalting of ligation reaction solution, 2 �l

ligation solution and 50 �l E. coli cells were mixed on ice and filled into transformation cuvette

(PEQLAB, Erlangen). The plasmids were transformed by electroporation (Gene-Pulser, Bio-Rad)

into electrocompetent E. coli cells XL1-Blue (Stratagene, La Jolla, USA). Alternatively,

electrocompetent E. coli strain TOP10F’ (Invitrogen, Leek, Nethelands ) was used.

4.3.12 DNA-DNA hybridization (Southern blot analysis)

DNA-DNA hybridization (Southern blot analysis) according to Sambrook & Russel (1999)

was accomplished using radioactive �-32P-dATP or �-32P-dCTP. The production of probes was

made by means of random priming (usb, Freiburg) or in a PCR reaction with specific primers.

Methods

34

The DNA samples isolated in agarose gel were capillary transferred to the positively charged

nylon filter (Biodyne A, Pall, Ann Arbor, MI, USA). The filter was cross-linked under UV

radiation with a dose of 1.2 x 105 �J (UV Stratalinker 2400, Stratagene, Heidelberg). The probe

was purified through prespin Mobispin S-300 Column (Mo Bi Tec GmbH, Goettingen). The filter

was prehybridized in Hybridization solution supplemented with 100 �g/ml Salmon sperm DNA

more than 1 h at 68ºC and afterwards hybridized overnight with the probe at 68ºC, followed by

stringent washing at 68ºC, first, 1 time in 2 x SSC / 0.1% SDS for 10 min, and then 2 times for

each 10 min in 0.1-0.2 x SSC / 0.1% SDS. The detection was carried out by means of

autoradiography using the films from Kodak (Rochester, NY, USA) or Fuji (New RX, Fuji,

Japan). If the filter was reused, a process of stripping was carried out in 0.5% SDS at 95ºC for 2-4

times. The stripping result was radioactively checked.

Hybridization solution 5 x SSC; 1% skim milk; 0.1% Lauroylsarcocine sodium salt; 0.02% SDS

Acidic solution 0.25 M HCl

Denaturation solution 100 g NaOH; 438.3 g NaCl in 5 liter

Netralization solution 242 g Tris; 347 g NaCl in 4 l; pH 7.2

20 x SSC 441.3 g Na3Citrate; 876.3 g NaCl in 5 l; pH 7.0

2 x Wash 100 ml 20 x SSC; 10 ml 10% SDS in 1 liter

0.2 x Wash 10 ml 20 x SSC; 10 ml 10% SDS in 1 liter

4.3.13 Isolation of total RNA from A. nidulans

For isolation of RNA in development stages, approximately 103 spores per 9 cm plate was

inoculated onto complete medium plates covered with sterile preserving membrane (Ostmann,

Bielefeld). Alternatively, a 500 ml CM liquid culture inoculated by spore suspension from one

plate was shaken at 200 rpm for 14 h at 37ºC, 50 ml of liquid culture was vacuum filtered through

miracloth, the filtered mycelium was put on CM agar plates, incubated at 37ºC. Then, the

mycelium together with membrane or miracloth was harvested at defined times, removed extra

water between paper towels, frozen in liquid nitrogen and grinded in a mortar. RNA isolation

from grinded mycelium powder was carried out with TRIZOL (Gibco or Invitrogen) according to

manufacturer protocol. The RNA was finally dissolved in 40-50 �l sterile DEPC H2O with 0.5

U/�l RNase inhibitor (Promega, Mannheim). The RNA concentration was measured in a

Methods

35

spectrophotometer (Pharmacia LKB, UltrospecIII). The RNA samples were diluted to 2 �g/�l

with DEPC H2O containing RNase inhibitor and kept at –80�C.

4.3.14 DNA-RNA hybridization (Northern blot analysis)

DNA-RNA hybridization (Northern blot) was accomplished as described (Sambrook &

Russel, 1999). The RNA was denatured with formamide and separated in denaturing

formaldehyde agarose gel, followed by capillary transfer to a positively charged nylon membrane

(Biodyne Plus, Pall, Ann Arbor, MI, USA). For size estimation, RNA marker of Promega

company (Mannheim) was used. The filter was cross-linked as for Southern blots. Then, the

membrane was stained by Methylene blue and washed with H2O. The two clear rRNA bands

should appear. The picture was taken via a camera (INTAS, Goettingen) and video printer (Ann

Arbor, MI, USA). The filter was destained in Destaining solution. The filter was prehybridized in

Northern hybridization solution with 100 �g/ml Salmon sperm DNA more than 1 h at 42�C and

afterwards hybridized overnight with the probe at 42�C, followed by stringent washing at 65-

68�C, 1 time in 2 x SSC / 0.1% SDS for 10 min, and then 2 times for each 10 min in 0.5 x SSC /

0.1% SDS. The detection was carried out as in Southern blots.

DEPC water 0.1% DEPC, stir overnight, autoclave

10 x MOPS 0.4 M MOPS (pH7.0); 0.1 M Sodium acetate; 0.01 M EDTA;

autoclave

RNA sample buffer 100 �l Formamide; 38 �l 37% Formaldehyde; 20 �l 10 x

MOPS; 42 �l DEPC water; 20 �l RNA loading buffer

RNA loading buffer 80% formamide; 1 mM EDTA; 0.1% Bromphenol blue; 0.1%

xylene cyanol

Northern staining solution 0.03% Methylene blue in 0.3 M Na-Acetate

Northern destaining solution 1% SDS; 1 x SSC

100 x Denhardt’s solution 10 g Ficoll 400; 10 g polyvinylpyrrolidone; 10 g BSA (Pentax

fraction V); H2O to 500 ml, filter and store at –20˚C in 25 ml

aliquots

Prehybridization / Hybridization

solution

5 x SSC; 1% SDS; 5 x Denhardts; 50% formamide

Methods

4 Methods

4.1 Growth conditions and storage of transformed E. coli and A. nidulans strains Transformed E. coli was overnight cultivated on LB plates with appropriate antibiotics at

37�C. Liquid culture was inoculated from a single colony and incubated in LB medium

containing appropriate antibiotics at 37�C with 250 rpm overnight shaking. Freshly grown

bacterial suspension was adjusted to 15% end concentration of sterile glycerol and stored at –

80�C.

The A. nidulans strains were grown on minimal or complete medium plates. Pieces of a colony

were cut from an agar plate and suspended in 15-20% sterile glycerol and stored at –80˚C.

4.2 Transformation of A. nidulans

Standard procedures of Aspergillius protoplast transformation were used (Yelton et al., 1984).

4.2.1 Preparation of protoplast

A 500 ml volume of spore culture in minimal medium with appropriate components was

shaked at 30ºC in water bath for 12-16 h until spores germinated. The culture was filtered

through sterile miracloth followed by washing using Wash solution. The washed mycelium was

collected into a sterile flask that was set on ice. Then 5 ml of Osmotic medium, 200 mg of

GlucanX (Novozyme) in 1 ml sterile water and 6 mg BSA in 0.5 ml water was successively

added into the flask. The digestion mixture in the flask was incubated at 30ºC in water bath for 1-

3 h until enough protoplasts became free. The digestion mixture was transferred into a 30 ml

corex tube and 10 ml of Trapping buffer was slowly added on top of the mixture, followed by a

centrifugation at 5000 rpm for 15 min using a HB-6 rotor. Then a protoplast band was transferred

into a new sterile tube, followed by washing two times using STC with centrifugation at 7000

rpm for 8 min. The protoplast pellet was gently resuspended in 200-1000 �l STC for

transformation.

28

Methods

4.2.2 Protoplast transformation

100 �l of protoplasts in STC and 100 �l DNA (10 �g DNA filled with 100 �l STC) were

mixed and incubated 25 min at room temperature in a falcon tube. Then, 2 ml PEG was added

and the tube was rolled until the mixture was homogeneous, followed by 20 min incubation in

room temperature. Finally, 8 ml STC was added and the entire mixture spread onto osmatically

stabilized medium (MM + 0.6 M KCl) with appropriate selection markers. The plates were

incubated at 37ºC until clear colonies were formed after 3-4 days.

Mycelium wash solution 0.6 M MgSO4

Osmotic medium 1.2 M MgSO4; 10 mM Na3PO4 buffer (pH 5.8)

Trapping buffer 0.6 M Sorbitol; 0.1 M Tris-HCl (pH7.0)

STC 1.2 M Sorbitol; 10 mM CaCl2; 10 mM Tris-HCl (pH 7.5)

PEG 60% PEG 4000; 10 mM CaCl2; 10 mM Tris-HCl (pH 7.5)

4.3 DNA and RNA manipulations 4.3.1 Plasmid DNA preparation from E. coli cells

An alkali-lysis method was used for the isolation of plasmid or cosmid DNA (Sambrook &

Russel, 1999). For a small volume of liquid culture (Miniprep), 1.5 ml overnight culture was

centrifuged 1 min at 13000 rpm, and the pellet resuspended in 200 �l Tris-EDTA buffer. Then

200 �l Alkali-lysis buffer was added, gently mixed, with the cell suspension, followed by the

addition of 200 �l Neutralization buffer. After a 10 min centrifugation, plasmid DNA-containing

supernatant was precipitated with 0.7 vol. isopropanol followed by 70% EtOH washing. After

drying, the pellet was resuspended in TE buffer. For a large volume of liquid culture (50-100 ml;

Midipreps), plasmid DNA was prepared using the Qiagen Midi Plasmid Purification Kit.

Plasmid DNA concentration was determined via absorption measurement with 260 and 280

nm in a spectrophotometer (Pharmacia LKB-UltrospecIII) in a quartz cuvette or compared the

intensity of Ethidium bromide stained DNA bands on an agarose gel with the intensity of defined

standards.

29

Methods

Tris-EDTA buffer 5 ml 1 M Tris-HCl (pH7.5); 2 ml 0.5 M EDTA (pH8.0); 10 mg RNAse in 100

ml

Alkali-lysis buffer 0.2 M NaOH; 1% SDS

Neutralization buffer 1.5 M K-Acetate (pH4.8)

TE buffer 10 mM Tris-HCl; 1 mM EDTA; pH8.0

4.3.2 Genomic DNA preparation from A. nidulans

For the preparation of A. nidulans genomic DNA, around 25 ml fresh liquid minimal media in

a 9 cm plastic plate was inoculated using a spore suspension prepared the colony grown on an

agar plate. The culture was incubated for 12-15 h at 37�C. Then, the mycelium was taken off with

a spatula and pressed briefly until dry between paper towels, and put into liquid N2. The frozen

mycelium was grinded in liquid N2 or kept at –80˚C until isolation. A. nidulans genomic DNA

was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden). Finally, 5 �l DNA extraction

was used to check quality and yield via running a 1% agarose gel.

4.3.3 Precipitation of DNA

Contamination by small nucleic acid fragments, protein and salt can be reduced to acceptable

levels by precipitating the DNA. 2.5 volume of ethanol and 1/10 3.0 M NaAc (pH 5.2) were

added into the DNA solution. The sample was mixed, kept at –80ºC for more than 10 min and

centrifuged for 10 min at 10000 rpm. The supernatant was discarded and the pellet was washed

with 70% EtOH, followed by centrifugation at 10000 rpm for 5-10 min. The purified DNA pellet

was completely dried in a speed vacuum or at 50ºC for 10-20 min, and then dissolved in sterile

water or TE buffer.

4.3.4 DNA electrophoresis through agarose gel

DNA electrophoresis through agarose gel is a standard method to separate, identify and purify

DNA fragments. An agarose gel of 0.8-1.2% was prepared by boiling agarose in 0.5 or 1 x TAE

buffer and pouring it into gel-making models. DNA samples were mixed with 1/10 10 x DNA

loading buffer. DNA samples together with a DNA marker (Eco130I-cut � DNA, MBI 30

Methods

Fermentas, St. Leon-Rot) were separated at 10 V/cm for 30 min-4 h depending the length of the

gel in 0.5 or 1 x TAE buffer. Then, the gel was stained in 0.5 x TAE buffer with Ethidium

bromide for 15-30 min. The DNA bands in the gel were visualizd by 302 nm UV light and

pictures were taken with a camera (INTAS, Goettingen) connected to a video printer.

50 x TAE buffer (pH 8.0) 40 mM Tris-Acetate; 1 mM EDTA; pH 8.0

10 x Loading buffer 20% Ficoll 400; 0.1 M Na2EDTA (pH 8.0); 1% SDS; 0.25%

Bromphenol blue; 0.25% Xylene cyanol

4.3.5 Digestion of DNA by restriction endonucleases

DNA samples (200 ng – 1 �g) were digested by restriction endonucleases using

corresponding reaction buffers. Enzyme, DNA, buffer and reaction time varied depending on the

specific requirements (generally, 37˚C from 1 h to overnight). A. nidulans genomic DNA was

generally digested for more than 2 h. For cloning, it was enough for 1-2 h. In the case where it

was necessary to treat the same DNA sample with different enzymes, the digestion was carried

out first in the buffer with low salt concentration or the buffer compatible to different enzymes.

4.3.6 PCR

Polymerase chain reaction (PCR) was accomplished with Taq (Gibco or Invitrogen), Expand

(Boehringer) or Pfu (Promega) according to manufacturer protocols. The synthesis of oligo

nucleotides was made by MWG Biotech (Ebersberg). As beginning oligonucleotide

concentrations, 5-20 �M were used in a reaction volume of 10-100 �l. The PCR reaction took

place in a Personal Cycler (Biometra), or a capillary Rapid Cycler (Idaho Technology, Idaho

Falls, ID, USA). RT-PCR was carried out using SUPERSCRIPTTM II RNase H- Reverse

Transcriptase (Invitrogen) according to the manufacturer protocol.

31

Methods

32

A Standard PCR reaction in Rapid Cycler

1 �l 2.5 mM dNTP

1 �l templete DNA

1 �l 10 x buffer

1 �l 50 mM MgCl2

1 �l 10 x BSA

1 �l 10 x Ficcol


0.2 �l Taq DNA polymerase

1.8 �l Autocloved ddH2O

A Standard PCR reaction in Personal Cycler

5 �l 2.5 mM dNTP

5 �l templete DNA

5 �l 10 x buffer

5 �l 50 mM MgCl2


1 �l Taq DNA polymerase

24 �l Autocloved ddH2O

4.3.7 DNA isolation from agarose gel

For DNA fragment isolation, 0.8% - 1% “low melting” gel was often used. The low melting

gel separated by gel electrophoresis was stained in 0.5 x TAE with ethidium bromide. The

appropriate DNA bands were cut out under UV light. The DNA purification was carried out

according to the protocol of WizardTM PCR Preps DNA Purification System (Promega, Madison,

WI, USA). Alternatively, the DNA in normal agarose gels was isolated with the QIAEX II Gel

Extraction System (Qiagen, Hilden).

4.3.8 Dephosphorylation of digested DNA

After the digestion with restriction enzymes, the vector was dephosphorylated by Shrimp

alkaline phosphatase (SAP) to remove the phosphate group at the 5’-end which prevent religation

of the vector. 0.1 unit / �M 5’-end with buffer was added into one sample. The mix was

incubated around 30 min at 37ºC. Less SAP and shorter incubation time were used for the

protruding 5’ termini than for recessed 5’ termini. If two enzymes with incompatible termini were

used, the dephosphorylation process was omitted.

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33

4.3.9 DNA ligation

DNA ligation was carried out using T4 ligase (Amersham-Pharmacia Biotech, Freiburg) at

16ºC or Fast LinkTM System (Biozym, Hessisch Oldendorf) in a volume of 10-20 �l. The

concentration of vector and insert DNA was measured on the basis of DNA marker. Around 50

ng vector was used in one ligation. The ratio of vector to insert was 1: 2-3 and 1:5-10 respectively

for sticky and blunt end ligation. For the cloning of PCR products, it was often done to add

restriction enzyme sites in both primers. In addition, PCR fragments were cloned blunt end into

EcoRV or SmaI of pBluescript or into PCR-Blunt (Invitrogen, NV Leek, The Netherlands). For

TA cloning, the PCR products amplified by Taq or Taq-containing polymerases were cloned into

PCR2.1TOPO (Invitrogen).

4.3.10 DNA sequencing

DNA sequencing was done by commercial sequencing (MWG Biotech, Ebersberg).

4.3.11 Transformation of E. coli

The transformation of electrocompetent E. coli cells was done as described (Ausubel et al.,

1995). A fresh single E. coli colony was cultured overnight in 37ºC. The culture was centrifuged,

followed by repeatly washing using cool sterile water at 2ºC. The cells were resuspended in 10%

glycerol and aliquot frozen at –80ºC for use. After desalting of ligation reaction solution, 2 �l

ligation solution and 50 �l E. coli cells were mixed on ice and filled into transformation cuvette

(PEQLAB, Erlangen). The plasmids were transformed by electroporation (Gene-Pulser, Bio-Rad)

into electrocompetent E. coli cells XL1-Blue (Stratagene, La Jolla, USA). Alternatively,

electrocompetent E. coli strain TOP10F’ (Invitrogen, Leek, Nethelands ) was used.

4.3.12 DNA-DNA hybridization (Southern blot analysis)

DNA-DNA hybridization (Southern blot analysis) according to Sambrook & Russel (1999)

was accomplished using radioactive �-32P-dATP or �-32P-dCTP. The production of probes was

made by means of random priming (usb, Freiburg) or in a PCR reaction with specific primers.

Methods

34

The DNA samples isolated in agarose gel were capillary transferred to the positively charged

nylon filter (Biodyne A, Pall, Ann Arbor, MI, USA). The filter was cross-linked under UV

radiation with a dose of 1.2 x 105 �J (UV Stratalinker 2400, Stratagene, Heidelberg). The probe

was purified through prespin Mobispin S-300 Column (Mo Bi Tec GmbH, Goettingen). The filter

was prehybridized in Hybridization solution supplemented with 100 �g/ml Salmon sperm DNA

more than 1 h at 68ºC and afterwards hybridized overnight with the probe at 68ºC, followed by

stringent washing at 68ºC, first, 1 time in 2 x SSC / 0.1% SDS for 10 min, and then 2 times for

each 10 min in 0.1-0.2 x SSC / 0.1% SDS. The detection was carried out by means of

autoradiography using the films from Kodak (Rochester, NY, USA) or Fuji (New RX, Fuji,

Japan). If the filter was reused, a process of stripping was carried out in 0.5% SDS at 95ºC for 2-4

times. The stripping result was radioactively checked.

Hybridization solution 5 x SSC; 1% skim milk; 0.1% Lauroylsarcocine sodium salt; 0.02% SDS

Acidic solution 0.25 M HCl

Denaturation solution 100 g NaOH; 438.3 g NaCl in 5 liter

Netralization solution 242 g Tris; 347 g NaCl in 4 l; pH 7.2

20 x SSC 441.3 g Na3Citrate; 876.3 g NaCl in 5 l; pH 7.0

2 x Wash 100 ml 20 x SSC; 10 ml 10% SDS in 1 liter

0.2 x Wash 10 ml 20 x SSC; 10 ml 10% SDS in 1 liter

4.3.13 Isolation of total RNA from A. nidulans

For isolation of RNA in development stages, approximately 103 spores per 9 cm plate was

inoculated onto complete medium plates covered with sterile preserving membrane (Ostmann,

Bielefeld). Alternatively, a 500 ml CM liquid culture inoculated by spore suspension from one

plate was shaken at 200 rpm for 14 h at 37ºC, 50 ml of liquid culture was vacuum filtered through

miracloth, the filtered mycelium was put on CM agar plates, incubated at 37ºC. Then, the

mycelium together with membrane or miracloth was harvested at defined times, removed extra

water between paper towels, frozen in liquid nitrogen and grinded in a mortar. RNA isolation

from grinded mycelium powder was carried out with TRIZOL (Gibco or Invitrogen) according to

manufacturer protocol. The RNA was finally dissolved in 40-50 �l sterile DEPC H2O with 0.5

U/�l RNase inhibitor (Promega, Mannheim). The RNA concentration was measured in a

Methods

35

spectrophotometer (Pharmacia LKB, UltrospecIII). The RNA samples were diluted to 2 �g/�l

with DEPC H2O containing RNase inhibitor and kept at –80�C.

4.3.14 DNA-RNA hybridization (Northern blot analysis)

DNA-RNA hybridization (Northern blot) was accomplished as described (Sambrook &

Russel, 1999). The RNA was denatured with formamide and separated in denaturing

formaldehyde agarose gel, followed by capillary transfer to a positively charged nylon membrane

(Biodyne Plus, Pall, Ann Arbor, MI, USA). For size estimation, RNA marker of Promega

company (Mannheim) was used. The filter was cross-linked as for Southern blots. Then, the

membrane was stained by Methylene blue and washed with H2O. The two clear rRNA bands

should appear. The picture was taken via a camera (INTAS, Goettingen) and video printer (Ann

Arbor, MI, USA). The filter was destained in Destaining solution. The filter was prehybridized in

Northern hybridization solution with 100 �g/ml Salmon sperm DNA more than 1 h at 42�C and

afterwards hybridized overnight with the probe at 42�C, followed by stringent washing at 65-

68�C, 1 time in 2 x SSC / 0.1% SDS for 10 min, and then 2 times for each 10 min in 0.5 x SSC /

0.1% SDS. The detection was carried out as in Southern blots.

DEPC water 0.1% DEPC, stir overnight, autoclave

10 x MOPS 0.4 M MOPS (pH7.0); 0.1 M Sodium acetate; 0.01 M EDTA;

autoclave

RNA sample buffer 100 �l Formamide; 38 �l 37% Formaldehyde; 20 �l 10 x

MOPS; 42 �l DEPC water; 20 �l RNA loading buffer

RNA loading buffer 80% formamide; 1 mM EDTA; 0.1% Bromphenol blue; 0.1%

xylene cyanol

Northern staining solution 0.03% Methylene blue in 0.3 M Na-Acetate

Northern destaining solution 1% SDS; 1 x SSC

100 x Denhardt’s solution 10 g Ficoll 400; 10 g polyvinylpyrrolidone; 10 g BSA (Pentax

fraction V); H2O to 500 ml, filter and store at –20˚C in 25 ml

aliquots

Prehybridization / Hybridization

solution

5 x SSC; 1% SDS; 5 x Denhardts; 50% formamide

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36

Northern running buffer 100 ml 10 x MOPS, 20 ml 37% Formaldehyde, 880 ml DEPC

water

Northern mini gel 0.36 g Agarose, 21 ml DEPC water; boiling, after reaching

70�C; add 6 ml 37% formaldehyde, 3 ml 10 x MOPS

4.3.15 Construction of DNA plasmids

Cloning of the mutA gene

The partial sequence obtained from the SSH library (Scherer, 2001) was used to design

primers for amplification of a specific mutanase fragment (Glucan1, Glucan2). This fragment

was used to identify two cosmids carrying the entire gene from a cosmid library (PUI). From one

cosmid (pHG2), a 7.3-kb mutanase-containing EcoRV restriction fragment was subcloned into

the pCR®-Blunt vector (Invitrogen) (pHGE5).

Cloning of the mutA disruption construct (pMut-arg)

Plasmid pHGE5 was cut with SacI and religated in order to remove a BamHI restriction site in

the polylinker of the plasmid and one close to the 5'-end of the EcoRV fragment (for scheme see

result Fig. 5.3). Within this plasmid (pHGE5-Sac) a 0.6-kb BamHI fragment was replaced by

argB from plasmid pHW-arg, which was constructed by cloning KpnI-XhoI released argB from

pDC1 into pBluescript. The final plasmid was cut with KpnI and XbaI, which releases the entire

construct plus some vector border sequences, and transformed into A. nidulans RMSO11.

Cloning of the mutA overexpression construct (pMut-gpd)

The mutA open reading frame was amplified with Glucan-ex1 and Glucan-ex2 using the proof

reading polymerase Pfu (Promega, Mannheim) and pHGE5 as a template. The PCR product was

cloned blunt-end into pBluescirpt KS- EcoRV. The insert was released with EcoRI and XhoI and

inserted behind the gpd promoter in pMS-gpd.

Construction of the mutA::sgfp transcriptional fusion

The putative mutA promoter region (1.8-kb) was amplified by PCR with glu-P1 and glu-P2

using Pfu polymerase. The PCR fragment was cloned blunt end into pBluescript EcoRV. The

glu-P2 primer introduced a NotI restriction site at the start codon of the mutanase. This NotI site

Methods

37

and the one in the pBluescript polylinker were used to insert sGFP as a NotI fragment obtained

from pRF917. Translation of the construct will be terminated by a stop codon within the

polylinker after the sgfp.

Cloning of the hgtA gene

The hgtA sequence obtained from A. nidulans genomic database (Cereon, USA) was used to

design primers for amplification of a specific hgtA fragment (HexA5’/3’ and HexB5’/3’). This

fragment was used to identify a cosmid carrying the entire gene from a cosmid library (PUI) as

described for the mutA gene. From this cosmid (pH38E7), a 4-kb hgtA-containing HindIII and a

3-kb SalI-PstI restriction fragments were respectively subcloned into the pCR2.1TOPO vector

(Invitrogen) (pHHH1) and pBluescript KS (pHHPS4).

Cloning of the hgtA deletion

The fragment containing partial hgtA from pHHPS4 was cut by SmaI-SalI and inserted

EcoRV-XhoI of pCR2.1TOPO vector. The fragment was released by EcoRI-XhaI and inserted

into EcoRI-XhaI of pHHH1, in which the argB released from pDC1 by EcoRI was inserted

EcoRI site, leading to the hgtA deletion construct (pHHRarg11). The final plasmid was linearized

by XhaI, and transformed into A. nidulans wild type strain SRF200 (also see Fig. 5.7 for

procedure).

Construction of the hgtA::sgfp transcriptional fusion

The putative hgtA promoter region (2.5-kb) from the plasmid pHHPS4 was amplified by PCR

with KS-Rev1 and Hex-Not1 using Pfu polymerase. The PCR fragment was cloned blunt end into

pBluescript EcoRV. The Hex-Not1 primer introduced a NotI restriction site at the start codon of

the hgtA. This NotI site and the one in the pBluescript polylinker were used to insert sgfp as a

NotI fragment obtained from pMut-p-gfp. Translation of the construct will be terminated by a

stop codon within the polylinker after the sgfp.

Construction of 5’ deletion of mutA promoter

The construct pMut-p-sgfp was used as the template for a PCR to amplify mutA promoter

fragments together with the sgfp reporter gene. Here used the different 5’ primers corresponding

-1.6, -1.45, -1.38, -1.0, -0.9 and -0.75 kb positions in mutA promoter and a 3’ primer KS-gfp

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38

under the downstream of sgfp gene in the polylinker of pBluescript KS- (see Table 3.9 for

primers). The amplified fragments were inserted into the EcoRV site of pBluescript KS-. The

constructs with argB-carrying plasmid pDC1 was used for co-transformation with 5’ progressive

deletion construct into A. nidulans wild type strain RMSO11.

Cloning of the steC gene

The partial steC sequence upstream of digA was used to probe different restriction enzyme-cut

cosmid (pAG1) by Southern blotting. A 8 kb SalI-KpnI restriction fragment containing the entire

steC gene and 4-5 kb upstream and 1 kb downstream of steC was subcloned into XhoI-KpnI of

PCR2.1-TOPO vector (Invitrogen; pHSKS2).

Cloning of the steC disruption construct (pHSAB3, 5)

In plasmid pHSKS2, a 0.6 kb BamHI fragment in steC was replaced by argB from plasmid

pHW-arg. The final plasmid was linearized with KpnI, and transformed into A. nidulans wild

type strains RMSO11 and SRF200.

Cloning of the steC overexpression constructs

PvuII-KpnI, EcoRI-KpnI, SacI-KpnI and StuI-KpnI fragments from pHSKS2, which contain

whole or partial sequence of the steC open reading frame, were respectively inserted behind the

gpd promoter in pMS-gpd.

Construction of the steC::sgfp transcriptional fusion

A 4 kb EcoRV-BamHI fragment containing the putative steC promoter region and 2/3 of steC

ORF was inserted into pBluescript. The NotI site in the pBluescript polylinker was used to insert

sgfp as a NotI fragment obtained from pRF917. Translation of the construct will be terminated by

a stop codon within the polylinker after the sgfp.

4.4 Biochemical methods 4.4.1 Isolation of protein from A. nidulans

Mycelium was harvested, dried and powered in a mortar in liquid nitrogen. 2 x Laemmli

sample buffer was added into the grinded powder of samples and completely mixed. The reaction

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39

tubes containing samples were put into boiling water or heated at 95˚C for 5-10 min and then

cooled on ice, followed by strong vortex and centrifugation at 10000 rpm for 5-10 min. The

supernatants were transferred into new tubes and kept at –20˚C.

2 x Laemmli sample buffer 100 mM Tris-HCl (pH 6.8)� 4% SDS� 20% Glycerol� 100 mM DTT

4.4.2 Determination of protein concentration (Bradford Assay)

Protein concentration was determined according to Bradford (Bradford, 1976) using the Bio-

Rad protein assay (Bio-Rad, Munich). The measurement is based upon Coomassie� Brilliant

Blue G-250 dye-binding assay. Acryl-cuvettes (Sarstedt, Nümbrecht) were used for the

determination of protein concentration. 200 �l Bio-Rad Protein Dye (Bio-Rad) were added into

samples and standard (BSA) (0-50 �g / 0.8 ml H2O with diluted sample isolation buffer), and

well but gently mixed to avoid bubbles. After 5 min, the measurement was carried out in the

spectrophotometer (Pharmacia LKB, UltrospecIII) at 595 nm.

4.4.3 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

The SDS-PAGE gel consisted of a resolving gel topped by a stacking gel. The seperating gel

was casted between the glass plates using Bio-Rad Mini Protean II equipment and overlayed by

isopropanol. After gel polymerisation, the isopropnol was removed and the gel chamber was

filled up with stacking gel and a comb was inserted. The protein samples were diluted to

appropriate concentrations using 2 x Laemmli sample buffer, heated at 95ºC for 5-10 min and the

samples were loaded onto the gel. Electrophoresis was accomplished at room temperature first

with 50 V until the samples moved out of the sample wells and then 100-120 V until the tracking

dye reached the bottom of the separating gel.

Stacking gel Separating gel

4% 6% 10% 12%

Acrylamid / Bisacrylamid 40% 0.36 ml 1.1 ml 1.9 ml 2.25 ml

1 M Tris-HCl pH 6.8 0.45 ml

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1 M Tris-HCl pH 8.8 1.88 ml 1.88 ml 1.88 ml

H2O 2.8 ml 4.4 ml 3.6 ml 3.25 ml

10% SDS 36 �l 125 �l 125 �l 125 �l

10% APS 15 �l 40 �l 40 �l 40 �l

TEMED 3 �l 5 �l 5 �l 5 �l

10 x Electrophoresis running buffer 30.3 g Tris� 144 g Glycine� 2 g SDS in 1 liter of ddH2O.

4.4.4 Western blotting

After electrophoresis, the proteins in the gel were transferred to Hybond ECL nitrocellulose

membrane (Arersham-Pharmacia Biotech, Freiburg). Electroblotting was performed in a

“sandwich” assemble in Transfer buffer for 2 h to overnight at 60 V at 4�C using Mini Trans-Blot

Apparatus (Bio-Rad, Munich). After transfer, the membrane was stained for 5 min in Ponceau S

solution, then washed using water until the protein bands appeared in the desired way. The

membrane was washed in TBST solution 5 x 5 min, blocked in Blocking solution for 1 h, washed

again 3 x 5 min in TBST, hybridized overnight at 4�C with the primary antibody in Blocking

solution, washed 3 x 5 min in TBST, then, incubated with the second antibody for 1 h at room

temperature, followed by 3 x 5 min washing in TBST. The detection was done with the BM

chemiluminescence kit from Roche (Mannheim).

10 x Transfer buffer 30.3 g Tris� 144 g Glycine in 1 liter of ddH2O.

Transfer buffer 800 ml H2O; 100 ml 10 x Transfer buffer; 200 ml methanol

Ponceau S 0.1% Poncean S in 1% Acetic acid, reusable

10 x TBS 24.2 g Tris; 80 g NaCl in 1 liter of ddH2O pH 7.6

TBST 1 x TBS; 0.1% Tween 20 (100%)

Blocking solution TBST with 3% BSA

4.4.5 Preparation of A. nidulans nuclear extracts

The mycelium after overnight growth at 37˚C was grinded to a very fine powder. Then 20 ml

NIB-A was added. Once a slurry was homogenous, 40 ml NIB-B buffer was added, and then

Methods

41

transferred to a 30 ml Corex tube and centrifuged at 1480 g for 7 min at 4˚C. The supernatant was

transferred into a new Corex tube, 5 ml NIB-C buffer was layered, followed by centrifuge at

16500 g for 25 min. The pellet was resuspended in NS buffer or processed as below. The crude

nuclear pellet was resuspended in 4 ml of 1 x NEB buffer, and incubated on ice for 30 min with

shaking, and then pelleted at 35000 rpm for 30 min. The supernatant was dialysed against 2 x 1

litre of BIND buffer for 4 h at 4˚C. The sample was concentrated using vacuum dialysis or

ultrafiltration through a Centriprep 10 unit (Amicon). The protein concentration was determined

using Bio-Rad assay.

NIB-A buffer 50 mM Tris-HCl (pH7.5); 5 mM Mg(C2H3O2)2.4H2O; 20% glycerol; 5 mM EGTA;

3 mM CaCl2; 1M sorbitol; 7% Ficoll

NIB-B buffer 25 mM Tris-HCl (pH7.5); 5 mM Mg(C2H3O2)2.4H2O; 10% glycerol; 5 mM EGTA;

NIB-C buffer 25 mM Tris-HCl (pH7.5); 5 mM Mg(C2H3O2)2.4H2O; 10% glycerol; 1 M sucrose

5 x NEB buffer 75 mM HEPES-KOH (pH7.6); 0.5 mM EGTA; 25 mM MgCl2; 25% glycerol; 2 M

KCl

NS buffer 25 mM Tris-HCl (pH7.5); 5 mM Mg(C2H3O2)2.4H2O; 25% glycerol; 0.1 mM

Na2EDTA

5 x BIND buffer 125 mM HEPES-KOH (pH7.6); 200 mM KCl; 5 mM Na2EDTA; 50% glycerol

All the buffers were sterilized by autoclaving and stored at 4˚C, immediately prior to use, 5

mM DTT and proteinase inhibitor cocktail (Sigma) were added

4.4.6 Purification of DNA-binding proteins

The method was adapted from Joern Kalinowski (University of Bielefeld). Standard PCR

amplified 150-200 bp DNA fragments of the promoter with 20-25 bp primers and one of the

primers was modified with biotin at the 5’-end. 200-400 �l of Streptavidin agarose was washed

3-5 times with 1 x DNA binding buffer, resuspended in 400 �l 1 x DNA binding buffer. The

purified PCR product (about 2 µg / 200 �l) was mixed with 2 x DNA binding buffer (1:1) and

400 �l Streptavidin agarose was added, and then incubated several hours or overnight at room

temperature (PCR product binds with biotin to Streptavidin), followed by washing 2-3 times with

1 x DNA binding buffer and then equilibrated with 1 ml protein binding buffer for 30 min. PCR

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42

fragment bound to Streptavidin was incubated with nuclear extract for 10-15 min, followed by

washing with 1 ml protein binding buffer. 10-20 �l protein elution buffer was added. The pellet

was vortexed carefully and placed on ice for 5 min. After centrifugation, the supernatant was

transferred into a new tube and SDS-loading buffer was added to run a SDS-PAGE gel. The gel

was stained by silver staining. The separated bands were eluted and digested for MALDI-TOF

Mass Spectrum analysis. A PCR fragment of nonspecific binding of transcription factors was

used as a control.

2 x DNA binding buffer 10 mM Tris-HCl; 1 mM EDTA; 2 M NaCl; pH 7.5

Protein binding buffer 20 mM Tris-HCl; 1 mM EDTA; 1 mM DTT; 100 mM NaCl; 10%

Glycerine; 0.05% Triton X 100; pH 8.0

Protein elution buffer 20 mM Tris-HCl; 1 mM EDTA; 1 mM DTT; 1 M NaCl; 10% Glycerine;

0.05% Triton X 100; pH 8.0

4.5 Fluorescence microscopy

sGFP-expressing strains were observed with a Zeiss Axiophot microscope and appropriate

filter combinations. For the documentation a Seascan CCD camera (INTAS, Goettingen) was

used.

4.6 Other methods 4.6.1 Quantification of mutan (alkali-soluble fraction)

For each time point 6 agar plates (9 cm diameter) were inoculated and incubated at 37°C.

Development was followed microscopically. Mycelium was harvested and the alkali-soluble

fraction isolated as described (Zonneveld, 1973). At different time points, the mycelium from 6

plates (9 cm diameter) on minimal solid medium was harvested and then poured into 2 liters of

boiling water. After the agar was solubilized, the mycelium mats were washed in fresh boiling

water and dried at 80˚C. The dried material was weighed and inoculated with 5% KOH (100 ml/g

dry mycelium) for 18 h with shaking at 37˚C, after which it was centrifuged and washed again

with 5% KOH. The remaining pellet after centrifugation was called the alkali-insoluble fraction.

Methods

43

The alkaline extracts were neutralized with glacial acetic acid; the resulting flocculent precipitate

was centrifuged, washed three times with water, and dried. This alkali-soluble fraction contained

the �-1,3-glucan (mutan).

4.6.2 Growth of �mutA, wild type and mutA overexpression strains in mutan medium

The alkali-soluble fraction (mutan) was used as sole carbon source. Approximately 1% alkali-

soluble fraction from 12 d growth of �mutA strain (SHW1) mixed with other minimal medium

components except glucose was used to pour agar plates. Spore suspensions of �mutA, wild type

and mutA overexpression strains were inoculated at the center of the agar plate. The plates were

incubated at 37ºC and the phenotypes were investigated microscopically.

Results

44

5 Results

5.1 Analysis of the carbon cycle during sexual development in A. nidulans 5.1.1 Molecular cloning of the mutA gene

A partial sequence (350 bp) obtained in a substractive hybridization (SSH) library specific for

sexual development (Scherer, 2001) encoded a peptide with high homology to fungal �-1,3

glucanases (= mutanase). The partial cDNA sequence was used as a probe to isolate two

corresponding cosmids from the PUI library (kindly provided by B. Miller, Idaho, USA). From

one of the cosmids (pHG2), a 7.3 kb EcoRV restriction fragment was identified by Southern blot

containing the mutanase gene (Fig.5.1). It was subcloned (pHGE5) and sequenced (Fig.5.1). The

genomic sequence revealed that the mutanase gene was located in a region close to the 3' end of

the EcoRV restriction fragment. This area, spanning from the BglII restriction site at about 4000

bp to the EcoRV site at the 3'-end, was sequenced on both strands. In order to deduce a putative

mutanase protein sequence, a corresponding cDNA was sequenced, which was identified in the

EST database (clone j9c02) at the Fungal Genetics Stock Centre (FGSC, http://www.fgsc.net).

Comparison of the genomic DNA with the cDNA sequence revealed the presence of three short

introns, 48, 52 and 45 bp in length. One is located at the 5'-end of the gene and two in the middle

of the coding region. Interestingly, the positions of the first and the second introns are conserved

in comparison to the mutanase from Penicillium purpurogenum (Fuglsang et al., 2000). The

polyadenylation site was derived from the cDNA sequence 50 bp downstream of the stop codon

and is also indicated in Fig. 5.1. A putative promoter was identified using a promoter prediction

program (http://www.fruitfly.org/cgi-bin/seq:tools/promoter.pl), which determined the start of

transcription 54 bp upstream of the start of translation. A putative TATA box motif is located 32

bp upstream of the transcriptional start site. After removal of the intron sequences, an open

reading frame of 431 amino acids could be deduced, which encodes a putative protein with a

calculated molecular mass of 48 kDa. The protein is hydrophilic with a calculated isoelectric

point of 4.6. However, at the N-terminus a 22 amino acid long hydrophobic signal peptide was

detected (http://www.cbs.dtu.dk). This suggests that the mutanase protein is secreted. Several

short (10 amino acids) stretches of hydrophobic residues are found throughout the sequence and a

50 amino acid long area at the C-terminus of the protein. Pairwise comparison with other fungal

http://www.fgsc.net/

http://www.fruitfly.org/cgi-bin/seq:tools/promotor.pl)

http://www.cbs.dtu.dk/

Results

45

mutanases revealed identities between 24 and 42 % to the Neurospora crassa, P. pupurogenum,

T. harzianum, and Schizosaccharomyces pombe proteins (Fig.5.2). The sequence of A. nidulans

mutA was deposited in the EMBL database and is available under the accession number

AJ310441.

AG AT CTT CC GGC AG T T CGAGT TGG T T GCGAG TCT T A T CA CG CA CCA CAGT C AT ATT CAGCC GT CTC GT GCC CA TC AT GCC CAT GA GAACGCT C TC CGC AGAC ATG CT AAT GG AGA CT T AT 120

AC CA T AC T C AGG CT AC GT AGG GGT GAT AGGC GT G GT AGG T G TG CAT CT AAT TT CAT CAT T T AG GAG AT GCGT A AG GGGT A TGA GA T GGT ACAG GC CAC CGGC AAC GT T T GCG CT T AT AAG 240

AC CG T CT T G AAC AT GACGC T G GT G GT GAAT C TCA GC AAC CC TA ACT CGGGA AG GAG AACGGT C CTT GGAT AAT TG AAAT A TAA CT CT C AC AGT GC AAG GAT A CTG AGCGGGC TGA T AGGA 360

CG AA CGA GT T T T AT AGCT T GT GAT AT AT T CC CAC T A GCA CT TG AGC CAGAA GA GAT AT AT AT G AAG AGT GT T G GA ACGT T CAT AA AAAAAAAA AA AGA T ACC CTG AGCGAT T CAT CACGA 480

GA AT T TC CA CAT AA T GGT GT T AAT CGGT GT A GAT AT T GC GG TG CCA GT CAA GA ACG AT ACT GA AAC T C GT GT C CT AT C T T CT T CG GAT AGAT G TA AGC T GGC GCC T C AAGAT ACG CGGGG 600

AC AG T AC CT CGC TC T T GCC T T GAA CT ACACC TGC AC CCC GC AA AGG GGCGT TG T CT CGCT C T A AAT T C T AC GG GG CGT GA TCC CA T AT GC T T G GT GTT CC T G CGC GGACGGA CAA AAGCC 720

GG GT CGT CA T GA GT AAT GGAT TGC CT AT C CA AGT GC AAC CC TA GAG AAGGT CA GGC GAT CC AT T CT T T GAGCG AC CAT T T CAC CG AAT T T AT A GA T GT CC AT GGT T T CT GCG CT C AAAGT 840

CT CT T TA CT CAG AG T AT AT AT AT G GGCCT GA AAT GT CGG CT TG GAA AT T GC AA GTT CGCAT CC CAG GC AAC AC AG CCT CA AGC AA GCAAC T GA CG CCG CAAT GAA GAT CT T C CAC CGCT G 960M K I F H R C

CT GC GCA GC CCT AA CT CT T CT AGC CAAT GCT CT C CC GGC T C TA T CG CT ACC GG GAG CGAAC AG T CT CACT AT T CG CAAAG ACT CG AAC AAAT A TG T CA CGGC GCA T T T CAT G GT A T T CCT 108 0C A A L T L L A N A L P A L S L P G A N S L T I R K D S N K Y V T A H F M

CC TC T CC AA CAT TG AACGC T G CCG GT GT T GA CAT AG CAA GG TT GGC AT CGT CG AAA AC T AT AC CGT CGACGAC TG GAAGC ACG AT AT GGAGCT AG CCA AGGA AAC GGGAAT C GAC GC AT T 120 0V G I V E N Y T V D D W K H D M E L A K E T G I D A F

CG CT CTT AA CT G CG CC AGC AT CGA CT CCT AC ACA GA CAA GC AG CTG GC AT A TG CGT AC GAGGC AGC T GAAGAG GT CGACT TT A AG GT GT T T AT CT CGT T C GA CTT CGCAT AT TGG T C GAA 132 0A L N C A S I D S Y T D K Q L A Y A Y E A A E E V D F K V F I S F D F A Y W S N

CG GC GAC AC T GC CA GAAT T AC CT C CAT T AT G CAG AC CT A T G CC GAC CAT CC AG GCC AGT T T CA ATA T AAT GGT GC T GC GT TAG TG AGC AC GT T CG T CG GGGA CAG T T T CGAC TGG GGT CC 144 0G D T A R I T S I M Q T Y A D H P G Q F Q Y N G A A L V S T F V G D S F D W G P

AG TC AAG AG GGC AG T AGAT CA TCC AAT CT T T GCG GT T CC GA AT CTG CAGGA TC CGA AC T GGGC CGG CC ACGCG AC GAC AT CGA TT GAT GGAGC GT T TT CGT G GTA T GCGT GG CCG AC GGA 156 0V K R A V D H P I F A V P N L Q D P N W A G H A T T S I D G A F S W Y A W P T D

T G GA GGA AA T AG TA T T AT C AA GGG CC CCAT G ACG AC AAT T T GG GAT GAT AG GT T CA GGAAT AA T CT CAAGGAC AA GGT T T AT A TG GCT CGT AG GT T GT CC AT T CT T T T GC T C CT G AGACG 168 0G G N S I I K G P M T T I W D D R F R N N L K D K V Y M A

AC GC T GA T G ACG AT GC T AT AG CT G T C T CGCC GT G GT T T T CA AC CCA CT T CA AC ACC AAGAACT GGG T AT T T AT CT GT GAA GAC CT T CC AC AT C TC CGC T GGC AGC AGAT GCT GGA AAT GC 180 0V S P W F S T H F N T K N W V F I C E D L P H L R W Q Q M L E M

AG CC AGA AT T GA TC GAGAT CA TT T CC T GGAA TGG T G AGT CT CT CCT T T GAC AT T AG CAGGC AT GAA T AACC GT GA AAGAC TAC GG CGAGT CCC AC T AC AT CG GCC CT T AC T C AGA AGCCC 192 0Q P E L I E I I S W N Y G E S H Y I G P Y S E A

AC TC T GA CG ACG GC T C T GC GC AAT GGACGAA AGA CT T T C CC CA CGA CGCAT GG CGC AT CAT CG CCA AGCCC T A CA T CGCC GCA TA CAAGGCGG GA GAG AGAG AGC CC ACT GT GGA GT CT G 204 0H S D D G S A Q W T K D F P H D A W R I I A K P Y I A A Y K A G E R E P T V E S

AC CA GCT GG T CT AC T GGT ACA GAC CGACACC GAA AG CCG T T AC CTG CT CGA AG GAT CC GCT T G GTC CGCCAAA TG GAAT C AAC CT GCT T GAGG AT AGT GT CT T TG T T ACGAC GCT GC T CA 216 0D Q L V Y W Y R P T P K A V T C S K D P L G P P N G I N L L E D S V F V T T L L

CC GA GCC GG CCA CA T T GAC T G TCG GC AGC GG CT C CC T GG AG TT T TC GGT T G AC GTA GAT GC T G GCA T T GT GAC AA ACAGC TT C CC CAT GGGT G TC GGC T C GC AGG CC T T T T C TGT CACCC 228 0T E P A T L T V G S G S L E F S V D V D A G I V T N S F P M G V G S Q A F S V T

GC GA CGG CG AGG AG AT CCT CG GCG GC GAT GG AGG GC T GG AT GT T CA GGAT A GA T GT GACT AT T ATA AC T T C AA TG T T T AT GT T GG CT C T T T T A GC GCC T AGT GCT AGAAGGA TCG AGCCA 240 0R D G E E I L G G D G G L D V Q D R C D Y Y N F N V Y V G S F S A .

GA GG CCA AT AAG AT T T GT T GA TT C AAACC T G AAC GA AGG CC AA GCG AT T AT GT CAA CT AGACT ATT GAAT AT T AT GCC GA GGG TT T CT T AAT C TT T TT T T CC T TG T C GCGT T GAC AGAGT 252 0

T A AT AAG GC GCT CT GAAT T T C AGT AGT T T T G TCG GC T CA CA GG CTT CT T GA AC GCC AAAAAGA GAT CAGCC AT GG T CT GT CAG AA GAAT T T CG TC CAA CC AC CGC CACGGCC AAT CT AT T 264 0

T C AA ATT CT CCC GG GGGCAGC AGT AAT CC GG ACC AT T CT CC GA T GG GC AGA GC T GG GAAAACT AAC AAT GT GA CG AAT AT CAA GG CAAT ACT A GG GAG GGT G CGT GC AGGT A GCA T ACT C 276 0

T G CC AAA GG CT A GT T T AT T CA AT G GT GCT CA TCA T A GGA GC TT T GA T GCAA GA GAT GC ACT T T GGA GGAAGGC CG CAAAC GCG AA T T T CC CAG GG ACC T GGT CTT T C CGC T T TCA CT CGG 288 0

T T GA GGC T A CCA TG AC GGT AT CGT T GGGAGG CT T T T AGA AC CC GAT AT AGA TC CCG AT ACAGC ATA AAACT GA AA T GAT C GCC GC T GC AGGT T GC T CG GGCC AGG GGCT GT G AAG GAACT 300 0

GT TC GTC T T T T G TT AGCAAAC GAT GC GAGAG AT A AC CT G AA GA T AC T C T AC GG ATC AAGCC T C T CC AT CT AGC CA T CGGT CAG GC AAGCC GGT GT AAT CAAA ATC CGACC AA GGC GAAAA 312 0

AG GA CTG GA T GT GA GAT C 3 138

In tron 1

In tron 2

P

In tron 3 D

*

*

A

B

EcoR

VB

amH

I

SacI

Bgl

II

Bgl

II�

Bgl

II

Bam

HI

Bam

HI

EcoR

V

G enom ic 7 .3 kb

T ra nscr ip t 1 .4 kb

P ro te in 48kD a

1kb

Fig. 5.1 Sequence of A. nidulans mutA. (A) Scheme of the mutA gene locus. (B) The BglII (with asterisk)-EcoRV restriction fragment covering the mutA gene was sequenced on both strands. The predicted start of transcription is indicated in the promoter region and the polyadenylation site found in one cDNA is indicated both with an asterisk above the sequence. The predicted N-terminal secretion signal is shadowed. The BamHI sites used for the construction of the disruption allele are boxed (see below). DNA and protein analysis was performed with the DNAStar program. The sequence of A. nidulans mutA is available under the accession number AJ291452.

Results

46

M K I F H R C C A A L T L L A N A L P A L S L P G A N S L T I R K - - - - - - D S N K Y V T A H F M V G I V E N Y T - V D D W K H D M E L A1M W L F - - - - - - - - - - - - T L L A V L L A C A Q V Q A - - - - - - - - - - - - K A V F A H F M - - - - - - - - - - D Q W K V D M R L A1M - - - - - - - K V S S A F A A T L S A I I A A C S A - L P S D S M V S R R S T S D R L V F A H F M V G I V S D R T S A S D Y D A D M Q G A1M L G V V R R L G L G A L A A A A L S S L G S A A P A N V A I R S L E E R A S S A D R L V F C H F M I G I V G D R G S S A D Y D D D M Q R A1M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V V A H F I V G N T Y P Y T - V S N W E E D I Q D A1M - - - - - - - - - - A S L T T A L P - - - - - - - - - - - - - - - - - - - - - - N K A V V A H F M M G L T Y N Y A - Q S D F Q N D I Q N A1

K E T G I D A F A L N C A S I D S Y T D K Q L A Y A Y E A A E E V D - - F K V F I S F D F A Y W S N G D T A R I T S I M Q T Y A D H P G Q F64Q A A H I D G F A L N I R A D D T T S G V S L S R A F D A A D Q L G - - F K V I L S F D Y A G G G P W G A N L V S S M I L L Y S G Y D S - Y37K A Y G I D A F A L N - I G T D T F S D Q Q L G Y A Y E S A A N N D - - M K V F I S F D F N W W S T S Q A T E I G Q K I A Q Y G S L P G Q L63K A A G I D A F A L N - I G V D G Y T D Q Q L G Y A Y D S A D R N G - - M K V F I S F D F N W W S P G N A V G V G Q K I A Q Y A S R P A Q L71I A V G I D G F A L N - M G S D A W Q V E R I E D A Y D A A A S V S S D F K L F I S F D M S I I S A - D A D F I E G V V R R F A D K P N Q L27I S L G L D G F V L N - F G N D S W M M S K L T L M Y N A A D A L N L Q F L L Y L N L D M S E M S T V P A S T L V T Y V Q T F A N R G H Q A38

Q Y N - - G A A L V S T F V G D S F D W G - - - - - - - - - - P V K R A - - - V D H P I F A V P N L Q D P N W A G H A T T S I - - - - - - -132Y I D K Q G R P V V S T F E G P A Q A - - - - - - - G T W Q Y I I K K T N A A - - - - - - F I P D - - - - - W S S L G A K A A L A A A P C V104M Y D - - D K I F V S S F A G D G V D V A - - - - - - - - - - A L K S A - - - A G G N V F F A P N F H P S Y - - G T D L S D V - - - - - - -130Y V D - - N R P F A S S F A G D G L D V N - - - - - - - - - - A L R S A - - - A G S N V Y F V P N F H P G Q - - S S P - S N I - - - - - - -138Y Y D - - G K V F V S T F A G E T D T F G Y S D V S T G W D S A V K E P L A S A G Y P I Y F V P S - - - - - W T S L G Q G A L E E S - - - V95R I N - - N N V V V G T F L G Q D I N F G Q S S V N Q G W Q V A F K N A L A S A G I N I F F M P T - - - - - W - P L D A S T I Y Q T Y P - V107

- D G A F S W Y A W P T D G G N S I I K - G P M T T I W D - - D R F R N N L K D K V Y M A P V S P W F S T H F N - - - - - T K N W V F I C E180P E G L F S W A P W P W - G N T D G N T Y V D A S Y I E F M E T A E K N C A M D M Q Y M M P A S P W F Y T N L P - - - G Y K K N W L W R G D156- D G L L N W M G W P S N G N N K A P T A G A N V T V E E G D E E Y I T A L D G K P Y I A P A S P W F S T H F G P E V T Y S K N W V F P S D176- D G A L N W M A W D N D G N N K A P K P G Q T V T V A D G D N A Y K N W L G G K P Y L A P V S P W F F T H F G P E V S Y S K N W V F P G G183A D G F L S W N A W P T - T D A D M N D N D D I G Y Q N L A N S L - - - - - - G K L Y V A P V S P W F Y T H L S - - - - - Y K N W A Y K S D155A D G F C K W N C W P Y Y T S S P T S D A E D L V Y I Q N S K A T - - - - - - N K K Y M A T V S P I F Y T H F T - - - - - S K N Y S F F S E168

D L P H L R W Q Q M L E M Q P E L I E I I S W N D Y G E S H Y I G P Y S E A H - - - - - S D D G S A - - Q W T K D F P H D A W R I I A K P Y241D L W F D R W E Q I R F M N P D Y V E I I S W N D F G E S H H I G P L Y V E G D S Y E A F T V G K A P F N Y A L D M P H D G W R E L L P F T222L L F Y Q R W N D L L N L G P Q F I E V V T W N D Y G E S Q Y V G P L N S P H - - - - - T D D G S S - - R W A N D M P H D G W L D L A K P Y245P L I Y N R W Q Q V L Q Q G F P M V E I V T W N D Y G E S H Y V G P L K S K H - - - - - F D D G N S - - K W V N D M P H D G F L D L S K P F252W L I I D R W N E M L S V Q P D M I E V L T W N D Y G E S H Y I G - - N I Q G A - L P - - - A G S E - - G Y V D G F D H T A W R Y L M S P Y213G L W F T R W M Q L I K D Q P N Y V Q V L T W N D Y G E S T Y I G P T N Y A A D - F P V I G S N S H - - E W V D S F T H A P L S Y S L P L F227

I A A Y K A G E R E P T - - - V E S D Q L V Y W Y R P T P K A V T C S K D P - - - - - - - - - - - - - - - L G P P N G I N L L E D S V F V T304I D L Y K T G V A T - - - - - I E Q E K L V A W Y R V T S K N A A C N D G W T T G N T A S Q L Q L E - - F T P Q - - - - D V V Q D K I F Y S292I A A F H D G A T S L S S S Y I T E D Q L I Y W Y R P Q P R L M D C D A T D T - C M V A A N N D T G N Y F E G R P N G W E S M E D A V F V V308I A A Y K N R D T D I S K - Y V Q N E Q L V Y W Y R R N L K A L D C D A T D T T S N R P A N N G S G N Y F M G R P D G W Q T M D D T V Y V A315I S A Y K L - - - G L S E P Y I N F E S L F Y W Y R P T P K S A T A T A D S - - - - - - - - - - - - - - - L S Y P S G G D Y M E D E I F V L275I Q M Y K Q N T T G L P S N F S G I S Q L Y V T Y R V H S K N A T A S S D S - - - - - - - - - - - - - - - I P R P D N Y Q N S S D V I S V I294

T L L T E P A T L T V G S G S L E F S V D - - - - V D A G I V T N S F P M G V G S Q - A F S V T R X G E E I L G G D G G L D V Q D - - R C D356A L L A S S Q P V T V T V G G V N V G A T W T K T P S G G A G I Y H G S V D F G S N T G A V V V T V G S M T V N K R P I A A N C D D T N - G351A L L Q S A G T V Q V T S G P N T E T F D - - - - A P A G A S A F Q V P M G F G P Q - S F S L S R D G E T V L S G T S L K D I I D G C L C G377A L L K T A G S V T V T S G G T T Q T F Q - - - - A N A G A N L F Q I P A S I G Q Q - K F A L T R N G Q T V F S G T S L M D I T N V C S C G384V Y L L Q S A E V T V T C G S T T Q T F S - - - - G V P G V N Q F T I P M E T N A S P S F T V A R Q G G T L A S G T G - - - - - - - - - - -327S F A K S S Y T L R V S V N G T V L G T T - - - - N V N A G V Q - - - - - - - S A N V S F I V N N T A A A - - - - - - - - - - - - - - - - -349

Y Y N F N V Y V G S F S - A .419Y T N W N A F V A S T T G A A V S A T V N S T Q W V C V E G T A A A G F D E L C A F T C K Y G Y C P V G A C L C T K M G P G N K L P G P E T420I Y N F N A Y V G S L P - A T F S D P L E P P S L N A F - - - - - - - S E G L K V S T C S A T - - - - - - - - - P S L G L T S T T P P E T I442I Y N F N P Y V G T I P - A G F D D P L Q A D G L F S L - - - - - - - T I G L H V T T C Q A K - - - - - - - - - P S L G - - - T N P P V T S449- - - - - - - - - - - - - - - - P E I V D S L S I Y N - - - - - - - - F N A Y T G V L - - - - - - - - - - - - - - - - - - - - - - - - - - -382- - - - - - - - - - - - - - - - - - - - - G L P L F Q I - - - - - - - L N G T T V I A - - - - - - - - - - - - - - - - - - - - - - - - - - -391

432P G F G T V G F P A K G R T A S Y G G L C S F A C N Y G Y G C P N A Y C D T V E H E L V I P S V S P F T P D A C T S G V G E G G - - - - L G490P T G T I T P G S A I T - - G A A T T T S T I S T T S T I S T T S T F I S T T T T T T S S A A T S T - T T G T C I A G T G P D - - - - N Y S495G P V S S L P A S S T T R A S S P P V S S T R V S S P P V S S P P V S R T S S P P P P P A S S T P P - S G Q V C V A G T V A D G E S G N Y I499- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -406

432G L C S F S C N F G F C P - - - - - I H S C T C T S T G A L H V P P G A S D T I T G K A D P S V D E R T Y G P L C E F A C S R G Y C P E G A556G L C S F C C N Y G Y C P G S D G S A G P C T C T A Y G D - P V P T P P V T G T V G V P L D G E G D - S Y L G L C S F A C N H G Y C P S T A558G L C Q F S C N Y G Y C P - - - - - P G P C K C T A F G A - P I S P P A S N G R N G C P L P G E G D - G Y L G L C S F S C N H N Y C P P T A568- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -406

432C V Q S D V D D G N P Y K N V N P A Y I G P Q V Y E T P T A A C E S P C V L V L P P S K L P A T T T F R L P P Y P T S F Q V G S T T T T V T621C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -626C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -631- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -406

432L Y P A D I V T D Q V S F S N I N I T G A V T D G A V F P M C T S L K P D A I P V T L S Y V S N A K T T V T V R Q V E L P P W P Q I T Q G P691- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -627- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -632- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -406

432P D Q W T S T C G G W V T G T I T N T H N D T D G P I G W I P P P T T T I T T P R P T Y T G I F P P A I V E P I V D P I D A Q C H N N R C V761- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -627- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -632- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -406

432P E A D D D N E H S N H V V I K V K C D E L W F F V F C I H T E H I Q V F G W K F T F P K A V I G P Y E L831- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q V E S627- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q Y - C632- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y F401- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q G406

A. nidulansN. crassaP. purpurogenumT. harzianumS. pompe 1S. pompe 2














*

*

Fig. 5.2 Alignment of A. nidulans MutA with homologous sequences of N. crassa (Q9P563), P. pupurogenum (Q9P8T5), T. harcianum (AF214480) and S. pombe (O13716 and O94510). If amino acids were identical in two or more sequences they were shaded. The alignment was done with DNAStar using Megalign (Clustal) with a gap penalty and a gap length penalty of 10. The putative mutan binding domains from P. purpurogenum and T. harcianum are indicated with two asterisks above the corresponding amino acids.

Results

47

5.1.2 mutA disruption and overexpression

To gain insights into the biochemical function of MutA in A. nidulans, a null mutant was

constructed, in which part of the open reading frame of mutA was substituted with the nutritional

marker gene argB (Fig. 5.3; see Methods 4.3.15). Colony-purified arginine-prototrophic

transformants were tested by Southern blot for homologous replacement of the mutA gene using

different restriction digests (Fig. 5.3). In 5 of 46 strains the observed bands indicated disruption

of the wild type copy. One strain contained additional integrations of the knock-out construct and

was therefore discarded. To show linkage between the disruption event and the nutritional marker

gene argB, two mutA disruption strains (SHW1 and SHW44) were crossed to SRF200 and

progeny strains analysed for the mutA deletion. Twenty argB+ strains were tested in a Southern

blot. They all displayed the banding pattern of the deletion. Two strains (SHW1 and SHW44-13)

were selected for phenotypic characterisation and comparison with mutA wild type strains

(SHW26 and SHW29) in minimal and complete medium. No difference with regards to

vegetative growth, asexual development or sexual fruiting body formation was found. The

number of cleistothecia per cm2, the number of ascospores per cleistothecium and the viability of

the ascospores were also similiar. To further characterise the effect of a loss-of-function mutation

of mutA, the amount of "alkali-soluble material" (this fraction contains mutan) accumulated

during growth was determined (see methods 4.6.1; Fig. 5.3). A wild type and a deletion strain

were inoculated on minimal media and grown for different times at 37 °C. Under these growth

conditions the strains initiated cleistothecia formation after 4 days. The alkali-soluble material

(mutan) was isolated as described (Methods 4.6.1). Whereas in wild type the amount of mutan

reached a maximum after 4 days, in the deletion strain the mass of mutan increased steadily. The

experiment was repeated three times where mycelium after 6 and 12 days was analysed.

Generally, the relative amount of mutan in the deletion strain was higher after 12 days than after

6 days. This might be due to degradation of other cell components and the remaining mutan. The

absolute values of the mutan fraction differed from experiment to experiment, but the amount of

mutan in the wild type after 12 days was always about 50 % of the mutan in the mutA mutant. In

our experiment, if estimating the absolute amount of mutan as the mutan fraction of mutA

deletion strain minused by that of mutA overexpression strain after longer growth such as 12

Results

48

days, the mutan occupied around 5-8% of the amount of supplemented glucose in minimal

medium.

In addition to the disruption of the gene, the mutA gene was overexpressed under the control

of the constitutive gpd promoter (pMut-gpd) in the wild type RMSO11 (see methods 4.3.15 for

cloning). Three strains (SHW-gpd3, 4 and 5), which showed high transcriptional expression

levels in liquid culture after 15 h growth at 37 ºC (Fig. 5.3), were analysed for development and

mutan degradation. No difference was found with respect to asexual development and the timing

of cleistothecium formation but the amount of mutan was slightly lower than in wild type (Fig.

5.3).

The evident difference in mutan depletion among wild type, �mutA and mutA overexpression

strains suggested mutan could be degraded by MutA and reultilized as carbon source. To further

visualize the function of MutA, two wild type (SHW26, 29), and two mutA-deletion (SHW1, 44-

13) and two mutA overexpression strains (SHW-gpd4, 5) were inoculated at the center of minimal

medium plates where only 1% alkali-soluble material (mutan), harvested from a mutA deletion

strain (SHW1) after 12 d growth, was used as a sole carbon source instead of 2% glucose

(method 4.6.2). The result showed the number of conidiophores in wild type and mutA

overexpression strains were much higher than in mutA deletion strains. During the early days,

overexpression strains grew much denser than wild type strains (result not shown). After

prolonged growth, the density of conidiophores looked similiar between wild type and

overexpression strains, however, more cleistothecia appeared in overexpression strains. This

result suggested mutan could be degraded by MutA and contributed to sexual development under

the normal growth conditions. A sparse growth of the mutA deletion strain was probably due to

other carbon sources in the alkali-soluble fraction (Fig. 5.3).

Results

49

1.4kb

SHW-g

pd3

SHW-g

pd4

SHW-gp

d5

gpd mutAATG

Bam

HI

EcoRIEcoRV

EcoRV

Bam

HI

probe

EcoR

V

Bam

HI

Bam

HI

1 kb

EcoR

V

mutA

EcoR

I

EcoR

I

EcoR

I

SacI

Bam

HI

argB

A

WT �mutA

Time (d)2 3 4 5 86

Alka

li-so

lubl

e fra

ctio

n m

g/g

dry

wei

ght

0

100

200

300

400

500 WT�mutAgpd::mutA

Wild type �mutA gpd::mutA

CoCo

CoCl

C

E

WT �mutAG H

B

D

F

Fig. 5.3 Disruption of the mutA gene and overexpression. (A) Scheme of the disruption construct (for cloning see Methods 4.3.15). (B) Southern blot analysis of mutA disruption strains. Genomic DNA of a wild type (RMSO11) and two mutA disruption strains (SHW1 and SHW44) was isolated, restricted with EcoRI (left panel) and EcoRV (right panel), separated on a 1% agarose gel, blotted and hybridised with the probe indicated in (A). (C) Scheme of constitutive overexpression (see Methods 4.3.15). (D) Northern blot analysis of different mutA overexpression strains (SHW-gpd3, 4, 5) after overnight growth in liquid culture. (E) Conidia-derived colonies 3 days after inoculation onto the

Results

50

agar plates with complete midium. No difference in vegetative growth, asexual development or later sexual fruiting body formation between a wild type strain (SHW26) and a �mutA strain (SHW1) was observed. (F) Analysis of the accumulation of "alkali-soluble material" (contains mutan) during growth and development in mycelia of a wild type strain (SHW26) (solid bar), a mutA mutant (SHW1) (striped bar) and a strain which overexpresses MutA (SHW-gpd4) (stippled bar). For each time point 6 agar plates were inoculated, grown and processed for mutan isolation as described in methods 4.6.1. At time point 4 d the amount was not measured for SHW-gpd4. (G) Alkali-soluble material isolated from wild type (SHW26) and SHW1 after 12 days from 6 agar plates. The material was isolated and finally collected in test tubes but not dried as in (C). (H) Wild type (SHW26), �mutA (SHW1) and MutA overexpression strain (SHW-gpd5) were inoculated in minimal agar plates using 1% alkali-soluble fraction (mutan fraction) as sole carbon source instead of glucose, where the mutan fraction was harvested from SHW1 after 12 d growth as in (F) and (G). Microscopic observation of colonies was carried out after 7 d growth at 37ºC.

5.1.3 MutA is expressed in Hülle cells

To analyse the spatial expression pattern within the different tissues, the sgfp gene was fused

to the promoter of the mutA gene (pMut-p-sgfp). This construct was co-transformed together with

the argB-carrying plasmid pDC1 into the arginine-auxotrophic strain RMSO11 and arginine-

prototrophic strains were analysed for GFP fluorescence. Two strains (SHW-p-sgfp17 and 19)

were further characterised. Bright fluorescence signals were visible on agar plates at hyphal

aggregations surrounding cleistothecia. Higher magnification of the areas revealed that Hülle

cells and the connecting hyphae showed high expression levels, whereas conidiophores,

cleistothecial primordia or ascospores showed no fluoresence (Fig. 5.4). Under growth conditions

where no Hülle cells and fruiting bodies were formed, no GFP-fluorescence was observed. The

expression pattern resembled the localisation of a catalase peroxidase, CpeA (Scherer et al.,

2002).

The obtained results suggested that Hülle cells could act as nurse cells and provide a carbon

source to the developing cleistothecium. Hülle cells appear to secrete the a-1,3 glucanase which

most likely releases glucose from neighboring hyphae. The question was whether Hülle cells

would be also involved in the uptake of glucose. To address this question, a high-affinity hexose

transporter was studied.

Results

51

A B

DC D

Asco.

H

E FH

Conid.

GFP-H

C

Fig. 5.4. Cellular localisation of mutA expression. The mutA promoter was fused to sgfp and transformed into RMSO11. The strain (SHW-p-sgfp17) was grown on agar plates and sexual development induced. (A) Young cleistothecium with Hülle cells (H). (B) Mature cleistothecium. (C) View onto a developing culture. Dark areas are conidiophores, green shining areas are young fruiting bodies. (D) One isolated cleistothecium with attached Hülle cells and interconnecting hyphae. Ascospores (Asco.) are not stained. (E) Isolated Hülle cells and conidiospores. Some Hülle cells are brightly fluorescent (GFP-H.) whereas others (H) are not. (F) Brightly stained Hülle cells and non-fluorescent conidiophore (Conid.). The SEM pictures (A, B) were taken from Scherer & Fischer (1998). The pictures in (C, D, E, F) were taken in dimmed bright field light plus fluorescence light to excite GFP. The scale bar represents 100 µm in (A, B, D), 200 µm in (C) and 20 µm in (E, F).

Results

52

5.1.4 Molecular cloning of the hgtA gene

A partial sequence obtained in the substractive hybridization (SSH) library (Scherer, 2001)

encoded a peptide with high homology to a high-affinity hexose transporter gene. The partial

sequence information was used to search for corresponding sequences in the Cereon Genomics

LLC (Cambridge, MA, USA; http://www.cereon.com) database and a 2.7 kb region was

assembled, which covers the entire ORF (Weber, 2002). This gene was named hgtA. However,

the DNA sequence deposited in the Cereon database could be incorrect in some places.

Therefore, it was considered as an appropriate to isolate a genomic DNA clone and resequence

the hgtA locus. PCR was used to screen the A. nidulans PUI cosmid library (see method 4.3.15).

A hgtA-containing cosmid was identified (pH38E7). From this cosmid, a 3 kb SalI-PstI fragment

containing a hgtA upstream and partial hgtA 5’ region and a 4 kb HindIII containing full hgtA

sequence were respectively subcloned (methods 4.3.15). The hgtA locus was sequenced and some

nucleotide errors removed by comparison with the assembled sequence. The cDNA was

generated by reverse transcription-PCR with total RNA from the stage of sexual development as

a template for the reverse transcriptase reaction (Weber, 2002). However, the cDNA was

incomplete and thus the predicted protein was wrong. Therefore, the cDNA was amplified again

using oligonucleotides from further upstream sequences. Sequence comparison revealed an extra

intron in the 5’ region and revealed an open reading frame of 2 kb interrupted by 6 introns with

44-67 bp in length in the coding region. The translated sequence was predicted to encode a

protein of 531 amino acids in length with a molecular weight of 59 kDa. Hydrophobicity values

determined at each residue by the Program TMHMM 2.0 (http://www.cbs.dtu.dk) showed the

presence of 12 putative transmembrane (TM) domains, a larger cytoplasmatic loop between the

sixth and seventh transmembrane helix, a characteristic feature of the major facilitator

superfamily, which includes a variety of transport systems in eukaryotes and in prokaryotes

(Fig.5.5)(Marger & Saier, 1993).

The HgtA protein shows the high homology to other glucose transporter genes such as

glucose transporter (42% identity) in T. harzianum (Delgado-Jarana and Benitez, 2000), Hgt1

(37% identity) in K. lactis (Billard P. et al., 1996), CaHgt1 (35%) in C. albicans (Varma A. et al.,

2000), Ght6 (32% identity) in S. pombe (Wood V, 2002) and Snf3 (32% identity) in S. cerevisiae

(Goffeau A., 1996). The alignment revealed a conserved signature (CDRFGRRPAILIG) typical

http://www.cereon.com/


Results

53

of sugar facilitators (Marger & Saier, 1993). The sequence of A. nidulans hgtA was deposited in

the EMBL database and is available under the accession number AJ535663.

SalI

XhoI

XhoI

Hin

dIII

PstI

SalI

EcoR

IPs

tISm

aI

Hin

dIII

1 kbG en o m ic D N A 6 .5 kb

T ran sc ip t 2 kb

Pro te in 59 kD

A

BGAT T CGATGGGCAGAT T CT CGAGAACCGGT GT ACAAAAAACCT AAAAACT GAAT AAAAAAGT GCAAAGAAT GT AGTAAT GT ACT GAAT GCAGACTCGAGAAT AT T ACAGCAT CTCCGT CTAT GT A 12 5

T GT AT GCTGAT T GAT T CAGAT GT T GCCCAGT CTGAAACTGCT GGCCGCT GGT CAT T CTCGT CT CT CGCGTCT T T ACAGCAT CTCGCT AAT GT CT CCAGT GGCT AT ACCT T GCT TGT CACGCGT GC 25 0

T TGGAT GTCT AT ACCCCCGGT CT GGGCGCGGGGAGGT T TAT GCGGT T CT GAT T GGGAGCCT CCACAGCAGT CCAAGAAAACCCCGGGCGAGT GGCGCCCACT AAAAT GT CT T GTGAGCT TT CAAA 37 5

GGGGGCT AAT ACGGGAT ACTCCCAACCCAAT GGACACGGCT GGATGT T ACGT T GCACTGCGGCCT GT T T TGGGAGCGAAACT TAACAT CGCT GGTAT GGGGGAGGT T GCCCT GCT AT GT GGT T CG 50 0

GGT GCGCAGCAT GGCCT GGTCT T CCCCAGCT GAGCT CGACAAGGGT GCAT GT ACGGGGAAAT ATAAAGCTCCAT T AACCGCCTCCT T GGT GT T T CT CT T CTT CCT GATGCT T T TCT T CATT GAT T 62 5

AGT GT GCTGT T GGCGCCAGCACCGCCTT T GAAGAT GGCTT T CAAGAAGT CCT ACAAT GT T T ACTT CCT T TGCGGGTT CAT GACCCT T GGT AT GAAACCGT GAT T CT ACT GT GGGGAT GATACT GA 75 0M A F K K S Y N V Y F L C G F M T L

CTGGCCAGGT GGT GGT CT GTT CGGT T TCGACATCT CGT CGAT GAGT GGT AAGCT GAATGCT CT GCCT GCAGCGGCAAACAT CTCACCATCGCAGGT GT CT TGGGT ACCGCGGCCT ACAACAACT A 87 5G G G L F G F D I S S M S G V L G T A A Y N N Y

CTT T CAGGT CGGCGGCGGCAAT T ACAAACAGGTACT T T GGT GT T CT CAAT TCAAT T GCAGAT CTAT ACGCT AACGTT T T AGGGT GGAATCACT T GT GCT ATGCCAT T TGGAT CGCT CGT CGGT GC 10 0 0F Q V G G G N Y K Q G G I T C A M P F G S L V G A

CCT AGCCTCCAGT TT CCT T GCCGACAAAT ACT CGCGT GTCACT GCAAT T CAGT T CT CGT CT ACTT T GT GGAT T AT TGGAT CAGT GT ACGT AT AGTAT AT CTACT CT T TGACCT GAT CT GACT T T T 11 2 5L A S S F L A D K Y S R V T A I Q F S S T L W I I G S V

T AGCT T T CAAT GT GCCGCCAACGGT ATCCCCT TGCT GGTCGT CGGT CGT GTCAT T GCAGGCCT CT GCGT TGGCAT CGCCT CAGCT AT GGT T CCGGT CT ACAT CGCCGAAGT GAGCCCAAAGCAT A 12 5 0F Q C A A N G I P L L V V G R V I A G L C V G I A S A M V P V Y I A E V S P K H

T CCGAGGGCGAAT GAT AT CGCT ACAGCAAT GGGCAAT CACCT GGGGT AT CCT GAT CCAGT ACT TCAT T CAAT ACGGCGCT AGCAACGT CGACGGCGGGCCTGACAAT GAAACCCAGAGCACAGCG 13 7 5I R G R M I S L Q Q W A I T W G I L I Q Y F I Q Y G A S N V D G G P D N E T Q S T A

GCCT T CCGAAT T CCAT GGGGT AT CCAGAT T GT CCCCGGTGT AAT TT ACT T CGT T GGT TT GT T T CT AT ACCCT AAATCGCCCCGCT GGCTGGCGAGCAAGGACCGCT GGGACGAGT GCAT GCAT GT 15 0 0A F R I P W G I Q I V P G V I Y F V G L F L Y P K S P R W L A S K D R W D E C M H V

CCT CGCCCGCCT CCACGGGAACGGAGACAT GAACCACCCCCT T GTT CT GGCCCAGT ACAAGGAAAT T CAGGACGCGCT CGCCCT T GAACGT GAGCAGGCT AGCACCAGCT AT CAGGAGCTCAT CA 16 2 5L A R L H G N G D M N H P L V L A Q Y K E I Q D A L A L E R E Q A S T S Y Q E L I

AGCCACGCAT CGCAAAGCGTGT T T T CCT GGGCAT GAGT CT GCAGAT GT GGTCGCAAT TGT CGGGAAT GAACGT CATGAT GGT AAGAAT GT T CT T CGCCAT AAGAGCACAGT CAAAT T AACAAACG 17 5 0K P R I A K R V F L G M S L Q M W S Q L S G M N V M M

CAGT ACT ACAT CGTCT ACATCAT GCAGT CCACAGGAGCGGGCT CCCCT CT TCT AACAGCT T CCAT T CAATACAT CCT T AACACGGCCCTT ACT CTCCCAGCGAT T CT CT GGCT CGACCGCT T CGG 18 7 5Y Y I V Y I M Q S T G A G S P L L T A S I Q Y I L N T A L T L P A I L W L D R F G

CCGCCGCCCT GCGAT CCT CAT T GGCT TCACCATGCAAGCAAT CT TCCT T T ACAT T GAGGGT GGCCT GCAAGCCGGCT AT GGT CGCT CGACT CGACCGT CCGACGACCTAAACGCCAT CT CCT GGA 20 0 0R R P A I L I G F T M Q A I F L Y I E G G L Q A G Y G R S T R P S D D L N A I S W

CCGT GGCGGACCACCCGGGTGT GGGCAAGGCT AT CAT CGCCAT GTCGT ACCT CT T T GTCT GCT CCT T CGCAACGACCAT T GGTCCAACTT CCT GGACCT ACCCGGCT GAAAT CTACCCGGCCAAG 21 2 5T V A D H P G V G K A I I A M S Y L F V C S F A T T I G P T S W T Y P A E I Y P A K

GTCCGT GCCAAGGCT GT CT CACT GGCTACGGCGT CCAACT GGACCT GGAACT GT CT CCT T GCGCT CT T T GT T CCGCCACT T CTGT GGAAT AT CAACT GGAAGAT GT ACAT GAT TGT ACGTT T ACC 22 5 0V R A K A V S L A T A S N W T W N C L L A L F V P P L L W N I N W K M Y M I

CACCCACCAACCACT GCAT CCT T AT AAGCAAGAT ACT AACGGAGTT T T GT GT ACAGT TCGCAGCCT T CAACACT GTCGCCGCCT T GAACAT GT T CCT GACCGCCCCT GAAACAAAAGGATACACG 23 7 5F A A F N T V A A L N M F L T A P E T K G Y T

CTT GAGGAGAT GGACGAGGTCT T T GAGAGCGGCAT CGCGCCCT GGAAGAAAAGGAAGAT CAGCTCCCGT CT T GAGCAGAT CGAGAGGGAGAT T GCCGAGGGT AACCT CAAGGT TACGGACCGGAC 25 0 0L E E M D E V F E S G I A P W K K R K I S S R L E Q I E R E I A E G N L K V T D R T

T GGT CCCGT CGAT CCT GT GCAGGAGAACGT T GGT GAACCGGAGAAGGCGGTGT AAT CCCT T GT CCAT T T GGT AT GGT T ACT GAGCT T AAT GCGT GAGT AT GT AAT CAGGT AGGCGGCGT ACAACA 26 2 5G P V D P V Q E N V G E P E K A V .

GGGCT GT AGT ACCCAGGT AGGGT CT T CCCAAT CCGGT ATAGCT T TT GT T C 26 7 5

In tron 1

In tron 2

In tron 3

In tron 4

In tron 5

In tron 6

C

Prob

abili

ty

T ran sm em b ran e In s id e O u ts id e Fig. 5.5 Gene locus and hydrophobicity of A. nidulans hgtA. (A) Scheme of the hgtA gene locus. (B) Nucleotide and deduced amino acid sequence of the hgtA gene. The positions of six introns were denoted below the DNA sequence. The PstI and EcoRI sites used for the construction of the disruption allele are boxed (see below). DNA and protein analysis was performed with the DNAStar program. The sequence of A. nidulans hgtA is available under the accession number AJ535663. (C) Hydrophobicity plot of HgtA of A. nidulans. The plot was constructed from the hydrophobicity value with programm TMHMM 2.0 (http://www.cbs.dtu.dk)


Results

54

M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A F K K - - - - - S Y N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A I G N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S L K N W L L L R D I Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y E G - - - - - -M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S S K I - - - - - E R I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F S G P A L K I NM - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -M D P N S N S S S E T L R Q E K Q G F L D K A L Q R V K G I A L R R - - - - - N N S N K D H T T D D T T G S I R T P T S L Q R Q N S D R Q S N M T S - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - V Y F L C G F M T L G G G L F G F D I S S M S G V L G T A A Y N N Y F Q V G G G - - - - - - N Y - - - K -- - - - - - - - - - - - - - - - - - - - - - - - - - - I Y V I A G V S V V G G A L F G F D I S S V S A Q L A E Q S Y L C Y F N Q D E N - - - - - - P P T T A D GT F Y K K F P H V Y N - - - - - - - - - - - - - - - - I Y V I G F I A C I S G L M F G F D I A S M S S M I G T D V Y K D Y F S - - - - - - - - - - N P D S L T -T Y L D K L P K I Y N - - - - - - - - - - - - - - - - V F F I A S I S T I A G M M F G F D I S S M S A F I G A E H Y M R Y F N - - - - - - - - - - S P G S D I -- - - - - - - - - - - - - - - - - - - - - - - A K I L T I V M L V F V S M A G W M F G A D T G S I G G I T N M R D F Q S R Y A D R Y D P V T D T Y S Y S S A R -V F T D D I S T I D D N S I L F S E P P Q K Q S M M M S I C V G V F V A V G G F L F G Y D T G L I N S I T S M N Y V K S H V A P N H D - - - - - - S F T A Q Q -

- - - - - - - - - Q G G I T C A M P F G S L V G A L A S S F L A D K Y S R V T A I Q F S S T - L W I I G S V F Q C A A N G I - P L L V V G R V I A G L C V G I AK C G G P R S L V Q G G I T A S M A A G S W L G A L I S G P L S D R L G R K Y S I M V G C I - I W V I G S T L S C A S Q N I - G M L I V A R I I N G I S V G I E- - - - - - - - - Y G G I T A S M A G G S F L G S L I S P N F S D A F G R K V S L H I C A A - L W I I G A I L Q C A A Q D Q - A M L I V G R V I S G M G I G F G- - - - - - - - - Q G F I T S S M A L G S F F G S I A S S F V S E P F G R R L S L L T C A F - F W M V G A A I Q S S V Q N R - A Q L I I G R I I S G I G V G F G- - - - - - - - - Q G L L V G M V N T G T T V G C L L S S P L G D R F G K R K C I M G W T L - V Y I T G V I V Q L T T I P S W V Q M M V A K I W T G L G I G A L- - - - - - - - - M S I L V S F L S L G T F F G A L T A P F I S D S Y G R K P T I I F S T I F I F S I G N S L Q V G A G G I - T L L I V G R V I S G I G I G A I

S A M V P V Y I A E V S P K H I R G R M I S L Q Q W A I T W G I L I Q Y F I Q Y G A S N V D G G P D N - - E T Q S T A A F R I P W G I Q I V P G V I Y F V G L FS A Q V P V Y I A E I S P P S K R G R F I G M Q Q W A I T W G I L I M Y Y I S Y G C S F I G E D - N P - - V S Y N T A A W R I P W G L Q M I P A F F L F F M M MS S A A P V Y C S E I S P P K I R G T I S G L F Q F S V T V G I M V L F Y I G Y G C H F I D - - - - - - - - - - G A A A F R I T W G L Q M V P G L I L M V G V FS A V A P V Y G A E L A P R K I R G L I G G M F Q F F V T L G I M I M F Y L S F G L G H I N - - - - - - - - - - G V A S F R I A W G L Q I V P G L C L F L G C FS V I A P G Y Q S E S S P P H I R G A I V T T Y Q L F I T L G I F I - - - - - - - A A C I N M G - T H K Y T T H P E A Q W R V P I G I N L L W G I L M F F G M LS A V V P L Y Q A E A T H K S L R G A I I S T Y Q W A I T W G L L V - - - - - - - S S A V S Q G - T H - - A R N D A S S Y R I P I G L Q Y V W S S F L A I G M F

L Y P K S P R W L A S K D R W D E C M H V L A R L H G N G D M N H P L V L A Q Y K E I Q D A L A L E R E Q A S T S Y Q E L I K P R - - - - - I A K R V F L G M SP L P E S P R W L A R K D R W E D C R A V L T L V H G K G D P N H P F V A Y E L Q D I K D M C E F E R Q H A D V T Y L D L F K P R - - - - - M I N R T F I G L FF I P E S P R W L A N H D R W E E T S L I V A N I V A N G D V N N E Q V R F Q L E E I K E Q V I I D S A A K N F G Y K D L F R K K - - - - - T L P K T I V G V SF I P E S P R W L A K Q G Q W E A A E E I V A K I Q A H G D R E N P D V L I E I S E I K D Q L L L E E S S K Q I G Y A T L F T K K - - - - - Y I Q R T F T A I FF L P E S P R Y L A V K G R N E E C M K I L T R N A G - L P A D H P I M Q K E Y N A I Q A D V E A E L A G G P C S W P Q I F S N E - - - - - I R Y R T L L G M GF L P E S P R Y Y V L K D K L D E A A K S L S F L R G - V P V H D S G L L E E L V E I K A T Y D Y E A S F G S S N F I D C F I S S K S R P K Q T L R M F T G I A

L Q M W S Q L S G M N V M M Y Y I V Y I M Q S T G - - A G S P L L T A S I Q Y I L N T A L T L P A I L W L D R F G R R P A I L I G F T M Q A I F L Y I E G G - -T Q I W S Q L T G M N V M M Y Y I A N I F S M A G Y S G N A N L L A S S I Q Y I I N V L M T I P A L L W V D K W G R R P T L L I G S V L M A L W M Y A N A G I LA Q M W Q Q L C G M N V M M Y Y I V Y I F N M A G Y T G N T N L V A S S I Q Y V L N V V M T I P A L F L I D K F G R R P V L I I G G I F M F T W L F S V A G I LA Q I W Q Q L T G M N V M M Y Y I V Y I F Q M A G Y S G N S N L V A S S I Q Y V I N T C V T V P A L Y F I D K V G R R P L L I G G A T M M M A F Q F G L A G I LV M A F Q Q L T G N N Y F F Y Y G T Q V F R G T G - - L N S P F L A A L I L D A V N F G C T F G A I F V L E Y F G R R G P L I V G G V W Q S I C F F I Y A S - -L Q A F Q Q F S G I N F I F Y Y G V N F F N K T G - - V S N S Y L V S F I T Y A V N V V F N V P G L F F V E F F G R R K V L V V G G V I M T I A N F I V A I - -

- - - - - - L Q - - - - - - - A - - G - - - - - - - - - - Y G R S T R P S D D L N A I S W T V A D H P G V - - - - - - - - - G K A I I A M S Y L F V C S F A T TA T Y G E V V P - - - - - - - G - - G - - - - - - - - - - - - - - - - - - - - - - - - - - - - I D H V A A Q S M R V T G A P A K G L I A C T Y R F V A S F A P TA T Y S V P A P - - - - - - - G G V N G D D T V T I Q I P - - - - - - - - - - - - - - - - - - S E N T S A - - - - - - - - - A N G V I A S S Y L F V C F F A P TG Q Y S I P W P - - - - - - - D - - S G N D S V N I R I P - - - - - - - - - - - - - - - - - - E D N K S A - - - - - - - - - S K G A I A C C Y L F V A S F A F T- - - - - - V G D R A L T R P N - - G - - - - - - - - - - - - - - - - - - - - - - - - - - - - T S N H R A - - - - - - - - - G A V M I V F S C L F I F S F A Q T- - - - - - V G - - - - - - - C - - S - - - - - - - - - - - - - - - - - - - - - - - - - - - - L K T V A A - - - - - - - - - A K V M I A F I C L F I A A F S A T

I G P T S W T Y P A E I - Y P A K V R A K A V S L A T A S N W T W N C L L A L F V P P L - - - - - - L W N I N W K M Y M I F A A F N T V A A L H M F L T A P E TW G P V S W T Y P P E L - F P L R L R G K G V A M A T S G N W A F N T A L G L F T P V A - - - - - - F A N I K W K S Y L I F A V F N T V A F F H V F F V F P E TW G I G I W I Y C S E I - F N N M E R A K G S A L S A A T N W A F N F A L A M F V P S A - - - - - - F K N I S W K T Y I I F G V F S V A L T I Q T F F M F P E TW G V G I W V Y C A E I W G D N R V A Q R G N A I S T S A N W I L N F A I A M Y T P T G - - - - - - F K N I S W K T Y I I Y G V F C F A M A T H V Y F G F P E TW A P A A Y V I V G E S - Y P I R Y R S K C A A V A T A S N W F W N F M I S F F T P F I - - - - - - S N S I G F K Y G Y V F A A C N L C A A I I I F L F A K E TW G G V V W V I S A E L - Y P L G V R S K C T A I C A A A N W L V N F I C A L I T P Y I V D T G S H T S S L G A K I F F I W G S L N A M G V I V V Y L T V Y E T

K G Y T L E E M D E V F E S G I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A P W - - - - - - - - - - - - - - - - -A G K T L E E T E A M F E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -K G K T L E E I D Q M W V D N I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P A W R T - - - - - - - - - - - - - - -K G K R L E E I G Q M W E E R V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P A W R S - - - - - - - - - - - - - - -K G L T L E E I N Q L Y L S N I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K P W N T - - - - - - - - - - - - - - -K G L T L E E I D E L Y I K S S T G V V S P K F N K D I R E R A L K F Q Y D P L Q R L E D G K N T F V A K R N N F D D E T P R N D F R N T I S G E I D H S P N Q

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D P N G I P - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G A Y Q R D R E D I K Q S D S E -K E V H S I P E R V D I P T S T E I L E S P N K S S G M T V P V S P S L Q D V P I P Q T T E P A E I R T K Y V D L G N G L G L N T Y N R G P P S L S S D S S E D

- - - - - - - - - - - - - - - K K R K I S S R L E Q I E R - - - - - - - - - - - - - - - - - - - - - - - - - E I A E G N L K V T D - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - Y M G - - - - - - - - - - - - - - - - - - - - - - - - - - T P A W K T K V A - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A N Y I P Q L P - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R S W Q P T V P - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - K E R G P T S K L H E Y V E - - - - - - - - - - - - - - - - - - - - - - - - - H A P N S Y A S T - - - - - - - - - - - - - - - - -Y T E D E I G G P S S Q G D Q S N R S T M N D I N D Y M A R L I H S T S T A S N T T D K F S G N Q S T L R Y H T A S S H S D T T E E D S N L M D L G N G L A L N

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T S L T V R A E Q G D L E A K- - - - - - - - - - I V K D E E - - G N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K L G L L G N P Q H L E D V- - - - - - - - - - I A S D A E - L A R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K M E V E H E E D K L M N E- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A Y N R G P P S I L M N S S D E E - A N G G E T S D N L N T A Q D L A G M K E R M A Q F A Q S Y I D K R G G L E P E T Q S N I L S T S L S V M A D T N E H N N E

- - R T G P V D P V Q E N - - - - - - - V G E P E K A V .I A H D T E K P P I - - H - - - - - - - T H E E E - T T QH S - - - - - - - - - - N E K G L L D R S D S A S - N S ND S - - - - - - - - - - N - - - - - - - S E S R E - N Q A- - H S T E S E N Y P Q Q - - - - - - - V T N P V - G LI L H S S E E N A T N Q P - - - - - - - V N E N N - D L K

T. harzianumA. nidulans

K. lactis

S. pompeS. cerevisiae

C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans


K. lactis


C. albicans

Fig. 5.6 Alignment of A. nidulans HgtA with homologous sequences of T. harcianum (CAC81782), K. lactis (P49374), C. albicans (O74713), S. pombe (O74849) and S. cerevisiae (A31928). If amino acids were identical in two or more sequences they were shaded. A conserved signature (LDRFGRRPAILIG) typical of suger facilitors (Marger & Saier, 1993) was underlined. The alignment was done with DNAStar using Megalign (Clustal) with a gap penalty and a gap length penalty of 10.

Results

55

5.1.5 hgtA disruption

To gain insights into the biochemical function of hgtA in A. nidulans, a null mutant was

constructed. Part of the open reading frame of hgtA was substituted with the nutritional marker

gene argB (Fig. 5.7).

SalI

Xho

I

Xho

I

Hin

dIII

PstI

SalI

Eco

RI

PstI

SmaI

Hin

dIII

PstI

SmaI

SalI

Xho

I

pHH PS4

pCR2.1TO PO

Xho

I

Eco

RV

Eco

RI

Eco

RI

Xha

I

Eco

RI

Eco

RI

Eco

RV

Hin

dIII

pCR2.1TO PO

Eco

RI

Eco

RI

Xho

I

Hin

dIII

Hin

dIII

Eco

RI

Xha

I

pH HH 1

argB

Eco

RI

Eco

RI

Eco

RV

SalI

Xho

I

Xho

I

Hin

dIII

PstI

Eco

RI

PstI

SmaI

Hin

dIII

Eco

RI

argB

After Intergration

A

1 kb

CWT � hgtA

B

WT ��h

gtA

(SHW

H11)

19.3 kb

7.76.3

4.33.5

Probe

Fig. 5.7 Disruption of the hgtA gene. (A) Procedure for construction of the hgtA disruption. A 3 kb SalI-PstI fragment of the 5’ region of hgtA was inserted into pBluescript (pHHPS4), the SalI-SmaI released fragment from pHHPS4 was inserted into XhoI-EcoRV in pCR2.1-TOPO. From this construct, a XhaI-EcoRI released fragment was inserted into pHHH1, and then, argB from pDC1 was inserted into EcoRI, leading to the hgtA deletion construct, in which a 0.6 kb PstI-EcoRI fragment in hgtA was replaced by argB and some polylinker sequence of the vectors. (B) Southern blot analysis of a hgtA disruption strain. Genomic DNA of a wild type (SRF200) and a hgtA disruption strains (SWHH11) was isolated, restricted with BamHI, separated on a 1% agarose gel, blotted and hybridized with the probe indicated in (A). (C) Conidia-derived colonies 3 days after inoculation onto agar plates with complete medium. No difference in vegetative growth, asexual development or later sexual fruiting body formation between a wild type strain (SWTB) and a �hgtA strain (SWHH11) was observed.

Results

56

The deletion construct pHHRarg11 was linearized with XbaI and transformed into the A.

nidulans wild type strain SRF200. The purified arginine-prototrophic transformants were tested

by Southern blot for homologous replacement of the hgtA gene using different restriction digests

(Fig. 5.7). In 1 of 20 strains the bands suggested disruption of the wild type copy (SWHH11; Fig.

5.7). For phenotypic characterisation and comparison with hgtA wild type strains (SWTB) in

minimal medium and complete medium, no difference with regards to vegetative growth, asexual

development or sexual fruiting body formation was found.

5.1.6 hgtA is expressed in ascogenous hyphae within cleistothecia

To analyse the spatial expression pattern, the hgtA promoter was fused to sgfp and co-

transformed into A. nidulans wild type strain SRF200 with the plasmid pDC1. Green

fluorescence appeared on the transformation plate. After purifying, two representative strains

(SWHgfp1 and 7) were further characterised. Bright fluorescence signals were visible in swollen

hyphal structures, possibly the ascogenous hyphae after dissecting the cleistothecia. Higher

magnification of the areas revealed that ascogenous hyphae showed high expression levels. In

contrast, conidiophores, Hülle cells, cleistothecial primordia and ascospores showed no

fluorescence (Fig. 5.8). In addition, the GFP fluorescence showed changing intensity in different

parts of the cells.

Results

57

A B

C D

AH

As

AH

As

Fig. 5.8. Cellular localisation of hgtA expression. The hgtA promoter was fused to sgfp and transformed into SRF200. (A) Mature cleistothecium (Taken from Scherer & Fischer, 1998). (B) Green shining on the shell of a young cleistothecium. (C) One dissected cleistothecium, bright fluorescence appeared in the swollen hyphal structure, possibly ascogenous hyphae. Ascospores (As) are not stained. (D) The GFP fluorescence showed changing intensity in different parts of the cells (red arrows), possibly due to local aggregation of cytoplasm.

These results showed that the �-1,3 glucanase and the high-affinity hexose transporter are

expressed in different tissues. The next question was how the specificity of the expression is

achieved. To address this problem, the promoter region of mutA was characterized, and it was

tried to isolate regulatory proteins binding to this promoter.

5.1.7 Identification of regulatory regions in the upstream sequence of mutA

To further study the mechanisms underlying the induction of mutA expression in Hülle cells,

it was sought to locate the DNA-binding elements in the mutA promoter involved in the specific

induction. A progressive 5’-deletion analysis of the mutA promoter was performed to identify cis-

Results

58

acting elements involved in the Hülle cell specific expression. Serially deleted mutA promoter

fragments fused to sgfp reporter gene were produced by PCR (method 4.3.15). The constructs

were used to transform A. nidulans wild type strain RMSO11. At least 12 randomly picked

transformant strains for each promoter fragment were investigated microsopically for green

fluorescence intensity. A more quantitive assay using e.g. a spectrophotometer was difficult

because of the spatial restriction of the expression in the Hülle cells.

sgfp1.8 kb

1.6 kb

1.45 kb

1.38 kb

1.0 kb

0.9 kb

0.75 kb

++++Fluorenscence intensity

++

+++

+++++-

ATGActivator ？

Repressor ？

Activator ？

Fig. 5.9 Identification of regulatory regions in the upstream sequence of mutA. The progressive 5’ deletion fragments of the mutA promoter fused with sgfp were transformed into A. nidulans strain RMSO11. At least 12 transformant strains for each promoter fragment were investigated microscopically for green fluorescence and average values of fluorenscence intensity was estimated. – indicates that no fluorescence could be detected; + to ++++, increasing levels of green fluorescence. Putative repressor and activator sites are labeled.

Deletion of a 420 bp promoter fragment (from -1.38 to -1.8 kb) relative to the mutA start

codon, caused a decreased fluorescence, but, when the promoter fragment was reduced to the

position of -1.0 kb, GFP fluorescence increased again compared with the constructs at -1.38 kb

fusion. That suggested at least one putative repressor locates between -1.0 to -1.38 kb. Similarly,

at least one putative activator should locate respectively in the regions of -0.75 to -1.0 kb and -1.6

to -1.8 kb (Fig. 5.9). This result suggested that at least three putative binding motifs of

transcription factors exist in the mutA promoter.

Results

59

5.1.8 Binding protein isolation

Gel shift is a standard method used for identifying regulatory elements. But, in this study, a

new method for isolation of DNA-binding proteins was used (methods 4.4.5; 4.4.6). Wild type

strain RMSO11 was grown overnight at 37ºC in 2 liter minimal medium. The harvested

mycelium was used to isolate nuclei (methods 4.4.5). To identify potential transcription factors, a

150 bp DNA fragment ranged at –1.7 kb position in the promoter of the mutA gene was amplified

using primers (glu-P1B/glu-P6F) with one primer modified by biotin at the 5’ end, a 170 bp

fragment (glu-P4B/glu-P4F) at –1.3 kb in the promoter was amplified as a control. The purified

PCR fragments were individually mixed with the nuclear protein from A. nidulans (see methods

4.4.6). DNA-protein complexes were loaded onto a 10% SDS-PAGE gel. The gel staining was

carried out by silver staining. One protein band with a molecular mass of about 40 kDa was

found when the DNA fragment containing the putative binding sites was used, but not with the

control DNA (Fig.5.10).

agar

ose

Preparation of A. nidulansnuclear extracts (Adapted from Van Heeswijck et al)

Nuc lear proteins

+

+

DNA/RNA affinity beads

Add protein extracts

Separation

Elution

Biot

in Stre

ptav

idin

Identif ication

64 kDa

26 kDa

40 kDa

glu-

P1B/

glu-

P6F

glu -

P4B/

glu-

P4FA B

Fig. 5.10 Binding protein isolation in the promoter of mutA. (A) Procedure of binding protein isolation (see methods 4.4.5; 4.4.6) (B) Two fragment amplified by PCR using the primers glu-P4B/glu-P4F; glu-P1B/glu-P6F were used in DNA binding protein assay. A band around 40 kDa appeared to specifically bind to glu-P1B/glu-P6F fragment.

Results

60

Unfortunately, the identification using peptide mass fingerprinting is not yet established

because of the lack of the complete DNA sequence database at the time of investigation. This will

be available shortly and then it should be possible to get access to the regulatory proteins.

5.2 Signal transduction in sexual development of A. nidulans 5.2.1 Molecular cloning of the steC gene

After the characterization of two differentially induced target genes, the role of a MAP kinase

cascade as a potential signalling pathway which could be involved in the expression of sexual

cycle specific genes was investigated. During the cloning of digA (encoding a vesicle sorting

protein), a partial open reading frame was identified in the upstream region of digA (Geißenhöner

et al., 2001). The derived protein sequence (C-terminus) displayed homology to the MAPKK

kinases Ste11 from e.g. S. cerevisiae and Nrc-1 from N. crassa. Because of the similarity to

Ste11, the "sterile" phenotype of steC deletion mutants (see below), the previous characterization

of the homeodomain protein SteA of A. nidulans (Vallim et al., 2000), and the description of

another gene, steB, with similarity to Ste11 (Han & Prade, 2002), the gene was named as steC.

The partial sequence information was used to search for a corresponding sequences in the Cereon

Genomics LLC database and assembled a 4.8 kb region, which covers the entire open reading

frame. Restrction analysis and Southern blot analysis of digA-containing cosmid pAG1 using

steC partial open reading frame as a probe indicated that an 8 kb SalI-KpnI contains full steC

gene (result not shown), that was subcloned (pHSKS2) and sequenced in the steC region and

some errors in nucleotides were removed by comparing with the assembled sequence from

Cereon Genomics LLC. In Northern blot analyses of vegetative cells a transcript of 4 kb with

rather low abundance was detected (Fig. 5.18). The cDNA fragments were generated by RT-PCR

using different primer pairs and sequenced. Comparison of the cDNA with the genomic sequence

revealed the presence of three introns (51, 53 and 49 bp in length) in the 5'-region of the gene

(Fig. 5.11).

Results

61

CAAGTGCGAAATAAGAGCTGTGTCAATTTCATCGGTAGATCCGCTGGTTTAAAGTGTTGTTATCAGTGCTTCAGATCTCCTCTAAGCAAGACTGATGCGGTCTGCGGGTTATCAATACAGGCCGCAGTCTACCCTGCAAT 140

ACCACTTGAGAACGCGTGGTGCTTAGTTCTCGGGTTGTGGGTATGCAAAACTGGGAATCGGAGCGTGTTGACTTCCTTGGTCTGCATCATGTTCAGGTGTTGTCCACGCTTTCTGATCGGGCTGGATGTGCGGGCCTCCG280

GTGCCTCATCCTATATGTCATGGAGCCAGTCATCGCGCCCGCAATGGATATACCAATTGATGAGAGTCAAGACCAAATGTAGGAAACTAGTACCGAATATATACTGTGTAGAGTATATTGATTGGAAAGGGGAAGAACTA420

CCGGGATGCGTCCATGGTTCCAGGAAGCGGCGAAACCAAGAATAGATCATAGCAGAGGCGCGATAGTCTGACAGCCGCCACAGGGTTATTGTTTCAGGTTCCGGCCTTGACAAATCACTCCCCGGCCGTGCTGAACAAGG560

AGGAGTGGCTGCGATGTATATTTCTTTTAAAGACTAAACATTCTGTGAGGCACCTCCTGTGTTGCTCGAGCCAATCTTCATGGAACTCAGTTGCTGACGATTGCCAGTCCCATCCAACAGGAGCGCCCAGATTGAGACAG700

TCACATGATCTACCTAGCCCAAAGCTGAAACAAGCCCAGTGGAGGTGATCGGGCGGACAGACAGGAATCAGAAATAAGACGACGACGGCCTCCTTCCTTCCTCTATCTAATAATCACTCCGCCTGTTTAAAGGCGTCTTC840

AGCTGTTGATCGGCTGTCTTCTTCCCTTCGCTATCTTCTAGCTCTTTTGAGCTCTTTTGTCTTTCTTCGCGCCCCTGGATCTCCTGGATCGAAGGGCCTGTTCATTCAACGATAGAGCCGATCGTCAGCCTAGTCCCACG980

CCTATAGTCTAAACAACCGCAGAACATACAATTGAGGATACCGTGACCTCATTACCCCATAATCACTCTACTTTCTTTCTCTTTTACAGTACGCGTTCCCTTAGATTCAAGTTCGCTGGCTTGGGTGCGGGGATTGCTGG1120

CTTACATTTGTGATCCCAACCCTCTTTGTTCTGCGACATGGCTAAATGATCGTGTCCGCGGCTCTAGATCCCTTTATCATGCATTAGACGCCCTCAAACCTGCCGGGAACGCCTTCCTCTACGAATCCGGATCAATATCT 1260

CTCGCTAGGGGCACTCTAGAATTCTCGCTGTGAAGTCGTGGCCGGAAGAGACCGTTTGCGCTATACGCCCATCTACTAATCCCCTGCGATGCTCACCTCCAAAGCATACGCGGGCCCCCTGGGGCTGCCTTCGTCGCAAA1400M L T S K A Y A G P L G L P S S Q

CACCGACCGCATCTTATCATACCACGACGCAGAAGTCGTCAACATTTTCGGAATCACAAACGGGAACAGTGTATACGAGTCCAACCAAATCGGAGTTTTCTGAAGCTGACGACGGTCTTGATGCTGTGAGGTGCGCCGTT 1540T P T A S Y H T T T Q K S S T F S E S Q T G T V Y T S P T K S E F S E A D D G L D A V R

ATCGGAACACATAAGCGGGGTTTTCAAGCTGATTCCTCTAGGTCTTGGGATGAGAATCAAGTGATCTCATGGCTCCATAGTATCAACTGTCAACAATATGAGCCGTTATTCAGGGGTACGTGTATACCTGTCCTTCGGCG1680S W D E N Q V I S W L H S I N C Q Q Y E P L F R

GCGATGGACTATGTGTAACTGTTTCCAGCGAACAATTTTAACGGCAACAACCTTATCGAATGCGACCAGAAGATTCTGCAAGAGATGGGCATAAAGAAAATCGGTGATCGGGTGCGGATTTTCGTTGCTATCAAGCAGCT 1820A N N F N G N N L I E C D Q K I L Q E M G I K K I G D R V R I F V A I K Q L

TCGCAACAAGTCTGTGGTCAACAAAAAGCAAAAGAACCTGGTATTTCAGTAATCATCCCTTCTTAGTGACAGCTACTCACGTCTTACAGAGACAGTTGGCAGCTTTGGAAGCCGCTCACCAACAAGCTTCACCTGATTCC1960R N K S V V N K K Q K N L R Q L A A L E A A H Q Q A S P D S

GCCCGCTCGTACAGCGCACGTCAACAAACATCCAGTGCCGGTCATGCATCCAGGACCGGTGATTATAGCTATGGCAGACCTACGTCTCGTCCAGGGTCTCCGCTACGTCCTCATCGTTACGTTGCTAATAGCCCTATGGA2100A R S Y S A R Q Q T S S A G H A S R T G D Y S Y G R P T S R P G S P L R P H R Y V A N S P M D

TTCAGGGCGAAAGGATTATTTATCCGCGGGCTCAGGCGCCGGACGCAATCCCGGAACCCCGGTTGAGCGAATTGGAACCCATTCGAGGCAGAATCCTAGCCTTGATGGGATGACAATGGGCTCTTTGCTAAGCAATGCTC2240S G R K D Y L S A G S G A G R N P G T P V E R I G T H S R Q N P S L D G M T M G S L L S N A

CCGTGATCAGAGTGATTTACAGCGGTGGGCAAACGAAAGTTCTGGATATTAAGCACTGCAAAACGCCGGATGAGATCATCCTTTGTGTGTTGAAGAAATTACAACTTCCCGAGCACCAGTATCGGAATTATTGTTTCTAC2380P V I R V I Y S G G Q T K V L D I K H C K T P D E I I L C V L K K L Q L P E H Q Y R N Y C F Y

GTCCTTGATGGCTTGGACCCTGACCCATCAAATTGTCGGAGGCTCTCTGACCATGAGCTCATGGAGATATGCGAAGGTTTCCACAGGTCGGAACGCGGCCGGCTTATTCTTCGTAAAATCCATGCTGGGGAACCGGATGC2520V L D G L D P D P S N C R R L S D H E L M E I C E G F H R S E R G R L I L R K I H A G E P D A

TGAGGAGGTGCACCGTGCGGCCCAGCTCGCACTGGACGAGAGCCAACAGGCGCATATGAATGCTCTCAGCAGCTCGAATGCACGGAACCAAATGAAAATACAACAACTGACTGGTGAATCATGGCATAATATAAGGCAAC2660E E V H R A A Q L A L D E S Q Q A H M N A L S S S N A R N Q M K I Q Q L T G E S W H N I R Q

CCATGTCACCAGTTTCTTCTCGTCATAACACGACTCCTAGCGACCACGAAATAAGGCCTCCTCAAGTCAGCGAACGTGTCTCCAAGCTAAGATCCTTCTTTGGTGCCCGGCCACCCAGCGAGATGATTATTCATGAAATA2800P M S P V S S R H N T T P S D H E I R P P Q V S E R V S K L R S F F G A R P P S E M I I H E I

TCGTCGTACTTCCCTGGTCATCAGAGAGAGGACATTGAGAAAACGATGCGCATGTCTGTTCGCAGATCACAACGGTTGAGCAGAGCAGCCAGTCGATTAAGCGTCGTCAGTAATACAAGCTATGCTTCCAGCCTGCGGGA2940S S Y F P G H Q R E D I E K T M R M S V R R S Q R L S R A A S R L S V V S N T S Y A S S L R D

TGCACCACCCATCCCTAGTATCGCCGACACTTGGCTTAATAATGGCACACCGCCCACTCGGGCCGCACGGCCGCTCTCGGTTCTTTCGACGAGACCTGGTCTCCCTTCAACATCGTATCGGGATTCAATCGCATCCAGTT 3080A P P I P S I A D T W L N N G T P P T R A A R P L S V L S T R P G L P S T S Y R D S I A S S

CCCTCCATCCACTTCAGGAGGAATCACCTGTTGAGCCGAATCGGAAGTCCTACGTGTCGTTCGATAGTGGATCCGATGACCCTAACAATTCACGTCACAGCCTTCTCGATGAGAATGCAAGTGTTGCTGCAACAGATGGC3220S L H P L Q E E S P V E P N R K S Y V S F D S G S D D P N N S R H S L L D E N A S V A A T D G

GGCTCTTTCAATGAGCGTCTGAGCGTTCTCGTGGCTGAAGACGGGGAGGAAGAAGACGATGGATTGGCAGAATTTTTAGCTGGTAACAACTTCGTCAACTGGATGAAAGGCTCACTGATCGGCGAGG TTCCTTTGGCAG3360G S F N E R L S V L V A E D G E E E D D G L A E F L A G N N F V N W M K G S L I G E G S F G S

TGTGTTTTTGGCACTTCACTCGATTACTGGTGAGCTTATGGCTGTCAAACAAGTCGAGATCCCATCAGCAACCAAGGGCACCGAATTTGACAAGCGCAAGAACAGCATGGTGGAAGCGTTGAAACACGAGATAGATCTTC3500V F L A L H S I T G E L M A V K Q V E I P S A T K G T E F D K R K N S M V E A L K H E I D L

TACAGGGTCTTCATCATCCGAACATTGTCCAGTATCTGGGAACTACCGCCGATGATCAATATTTGAACATTTTCTTGGAGTACGTTCCTGGGGGCTCTATTGCTACAATGCTCAAGCAATACAACACCTTCCAGGAGCCA3640L Q G L H H P N I V Q Y L G T T A D D Q Y L N I F L E Y V P G G S I A T M L K Q Y N T F Q E P

TTGATAAAGAATTTCGTACGGCAAATCCTTGCGGGTCTGTCCTACCTCCACAGCAAGGATATTATACACCGTGATATTAAGGGGGCGAATGTTCTCGTTGACAACAAAGGTGGCATAAAAATCTCGGATTTTGGTATCTC3780L I K N F V R Q I L A G L S Y L H S K D I I H R D I K G A N V L V D N K G G I K I S D F G I S

CAAACGAGTTGAAGCATCTACTGTTCTTGGATCCCGAGCAAGCAATGGTGGGGGCCATATTCACCGGCCTTCGCTGCAGGGTAGCGTTTACTGGATGGCGCCCGAAGTCGTTCGTCAGACGGCGCATACAAAGAAGGCTG3920K R V E A S T V L G S R A S N G G G H I H R P S L Q G S V Y W M A P E V V R Q T A H T K K A

ACATTTGGAGTCTGGGATGTCTCGTCATTGAGATGTTCATCGGGTCTCACCCTTTCCCAGACTGTAGCCAGCTTCAAGCCATATTTGCGATTGGTAGCAACAAGGCTCGGCCTCCAGCCCCAGAACATGCTAGTAAGGAT 4060D I W S L G C L V I E M F I G S H P F P D C S Q L Q A I F A I G S N K A R P P A P E H A S K D

GCCGTTGCTTTCTTGGATATGACATTCCAGCTCGACCATGAGAAGCGACCTGACGCAGACGAGTTGCTCAAGTCGCCCTTCCTTGCTACAACACTTACCTGAAATCCTTTACGATGTCGGATAGACAATGGGCGTTTTTT 4200A V A F L D M T F Q L D H E K R P D A D E L L K S P F L A T T L T .

AGCATTGAGCTTGGAGATTTGGAGTTCGAATTATGTATCAGGGCAATAATACTTTTGGTCTTTTGCGTATTCTTTCCCCTTCGATATGATACCGTCCCTCTTGATTTTAATGCTGGCGTTGATTAAGGTAGATTTGGGAA4340

ATATATATATCCCCCAAGATATTGCGGGTCATCAAAACCCAATCGAAAGGTTCGTGGCGTCAGCAGGGGCTGGTTGCGGTTTTATGACCTTTTTCACCCGGGTCCACTGATTTTCATTTATAAAAATATAGCGTTTACCT 4480

TTTACCTGTTGTAATCTGAAGCTTCTCGTTGTAGGTCTCATTGACCTTATTTGATTCCTTGCTCGAAGTTAAGCATTTCTGGTTTATAACTCACTCACGCGACAAAACAACCAATCCAATATTGCGTACAGGTAAATTCT 4620

AGAGAACATGTGTAATGAAAATACTAAAACAGGAGTAGGTTACACGTATTCTAGCTGGTCTCTCAGTGCGGGTTTTACGCCAGTTCAAGATAGGGAGTGTTAATGGTTTTCTTTCCTGTTCTTAACCCTATCCCCTCCCA4760

TTTCCTATTCCTATTCCTACTCATCCTCTTCTCTTCTTTCTATCTTATACTTTCTGTACTTTCTACTTTCTCCTTCTTTTCTTATTTCCTTTC4853

Intron1 Intron 2

Intron 3

Fig. 5.11 Sequence of A. nidulans steC. A 4.8 kb steC-containing region assembled from Cereon Genomics LLC was sequenced again by using the plasmid pHSKS2 as a template. Three introns in the N-terminal were identified by RT-PCR. The deduced amino acid sequence is given below the DNA sequence in the one-letter code. The PvuII, EcoRI, SacI and StuI used for overexpression of steC or kinase catalytic domain as below were boxed, the corresponding start codon ATG are underlined. The two BamHI restriction sites used for deletion of steC were boxed in gray (see below). The concerved SAM domain and the kinase catalytic domain of SteC were shadowed in light yellow. The sequence is available in the EMBL database under the Accession No. AJ505944.

Results

62

Sal I

Bam

HI

Bam

HI

Kpn

I

Genomic locus 8 kb

Eco

RI

Transcript 4 kb

Protein 886 aa

A. nidulans (886)N. crassa (666)

P. carinii (823)U. maydis (1566)C. neoformans (1230)

S. pombe (659)S. cerevisiae (738)

Catalytic domain

SAM protein interaction domain

A

B

Fig. 5.12 Scheme of A. nidulans steC. (A) Scheme of the steC gene locus, the transcript with the three introns and the derived protein. (B) Scheme of the domain organisation of SteC and several homologous proteins from other fungi. The length of the proteins is given in brackets behind the species name. The NCBI accession numbers of the proteins are: A. nidulans AJ505944; N. crassa AF034090; P. carini AF312696; U. maydis AF542505.1 (Kpp4=Ubc4); C. neoformans AF294841; S. pombe M74293; S. cerevisiae X53431.

Results

63

70.7

010203040506070

P.cariniiS.cerevisiaeA.nidulansN.crassaS.pombeC.neoformansU.maydis

B

C

A. nidulansP. cariniiU. maydisC. neoformansS. pombeS. cerevisiae

A. nidulansP. cariniiU. maydisC. neoformansS. pombe

V R S W - D E N Q - - - V I S W L H S I N C Q Q Y E P L F R A - N N F N G N N L I E C D Q K I L Q E M G I K K I G D R VV R L W - S E E E - - - V G E W L E S N N F G D Y M D I F K E - N N I N G D I L L E C N A A V L K E L G V K K L G D R IW - - - - D D A D - - - V A S W L N A A R L G H Y A S I F A E - H D I R G S V I L D V D Q A A L K E M G I T M V K D R VN - - - W G S Q E - - - L I T F L N I H K C G Q Y L A I F Q K - N D I N G K I L L D L D M T A L K S M G I G K I S E R VM - - - - - E Y Y - - - T S K E V A E W L K S I G L E K Y I E - Q F S Q N N I E G R H L N H L T L P L L K D L G I E N TK - - - - - T N D L P F V Q L F L E E I G C T Q Y L D S F I Q C N L V T E E E I K Y L D K D I L I A L G V N K I G D R L

R I F V A I K Q L R NR L S V C I K G L R ER I S A A I K L LR L I G G I K D L RA K G K Q F L K Q R - D Y L R E FK I L R K S K S F Q S. cerevisiae

W M K G S L I G E G S F G S V F L A L H S I T G E L M A V K Q V E I P S A T K - G T - - - - - - - - - - - - - - - - - -W M K G S L I G Q G S F G S V Y L A L H A I T G E L L A V K Q V E T P A P G A - D S - - - - - - - - - - - - - - - - - -W I K G A L I G S G S F G S V F L G M N A L S G E L M A V K Q V E I - - - P S - I D - - - - - - - - - - - - - - - - - -W H K G A L I G A G S F G N V F L G M N A K T G L L M A V K Q V E L - - - P S G D S - - - - - - - - - - - - - - - - - -W I K G A L I G A G S F G S V Y L G M D A Q S G L L M A V K Q V E L - - - S A G S A - - - - - - - - - - - - - - - - - -W I R G A L I G S G S F G Q V Y L G M N A S S G E L M A V K Q V I L - - - D S - V S - - - - - - - - - - - - - - - - - -W L K G A C I G S G S F G S V Y L G M N A H T G E L M A V K Q V E I - - - K N - N N I G V P T D N N K Q A N S D E N N E

- - - - - - - - - - - - - - - - - - E F D - K R K N S M V E A L K H E I D L L Q G L H H P N I V Q Y L G T T A D D Q Y L- - - - - - - - - - - - - - - - - - K N D - A R K K S M I E A L K R E I T L L R D L Q H P N I V Q Y L G C S S S A E Y L- - - - - - - - - - - - - - - - - - I Q G C K R K R A M L D A L Q R E I S L L K E L H H E N I V Q Y L G S S M D E T H L- - - - - - - - - - - - - - - - - - H L D - Q R K K G M L E A L E R E I K L L K S L E H E N I V Q Y L D S F A D D S H L- - - - - - - - - - - - - - - - - - K N E - D R K R S M L S A L E R E I E L L K E L Q H E N I V Q Y L D S S V D A N H L- - - - - - - - - - - - - - - - - - E S K - D R H A K L L D A L A G E I A L L Q E L S H E H I V Q Y L G S N L N S D H LQ E E Q Q E K I E D V G A V S H P K T N Q - N I H R K M V D A L Q H E M N L L K E L H H E N I V T Y Y G A S Q E G G N L

N I F L E Y V P G G S I A T M L K Q Y N T F Q E P L I K N F V R Q I L A G L S Y L H S K D I I H R D I K G A N V L V D NN I F L E Y V P G G S V Q T M L D Q Y G A L P E S L V R S F V R Q I L Q G L S Y V H N R D I I H R D I K G A N I L V D NT F F L E Y V P G G S V T A L L N N Y G A F E E P L I R N F V R Q I L K G L N Y L H N K K I I H R D I K G A N I L V D NN I F L E Y V P G G S I V A L L R N Y G A F E E P L V R N F V R Q I L N G L S F L H N R G I M H R D I K G A N I L V D NN I F L E Y V P G G S V A A L L N N Y G A F E E A L V R N F V R Q I L T G L N Y L H M R G I V H R D I K G A N I L V D NN I F L E Y V P G G S V A G L L T M Y G S F E E T L V K N F I K Q T L K G L E Y L H S R G I V H R D I K G A N I L V D NN I F L E Y V P G G S V S S M L N N Y G P F E E S L I T N F T R Q I L I G V A Y L H K K N I I H R D I K G A N I L I D I

K G G I K I S D F G I S K R V E A S T V L - - - - - - - - - - - - G S R A S N G G G H I H R P S L Q G S V Y W M A P E VK G T I K I S D F G I S K K L E A T N I L - - - - - - - - - - - - - - - - N G A N N N K H R P S L Q G S V F W M A P E VK G G I K I S D F G I S K K V E A - N L L - - - - - - - - - - - - - - - - S M T R N Q - - R P S L Q G S V Y W M A P E VK G G I K I S D F G I S K K V E S - D L V L A T N K G G A G G G G - - - - A G G A A H - - R P S L Q G S V F W M A P E VK G G I K I S D F G I S K K V E N - S L I - - - - - - - - - - - - - - - - T G L R T N - - R P S L Q G S V F W M A P E VK G K I K I S D F G I S K K L E L N S T S - - - - - - - - - - - - - - - - T K T G G A - - R P S F Q G S S F W M A P E VK G C V K I T D F G I S K K L - - - S P L - - - - - - - - - - - - - - - - N K K Q N K - - R A S L Q G S V F W M S P E V

V R Q T A H T K K A D I W S L G C L V I E M F I G S H P F P D C S Q L Q A I F A I G S N K A R P P A P E H A S K D A V AV K Q T S Y T R K A D I W S L G C L V V E M M T G T H P F P D C T Q L Q A I F K I G G S K A S P T I P D N A S E E A K QV K Q T L Y T R K A D I W S L G C L I V E M F T G K H P F P K M N Q L Q A I F K I G Q Y - V S P D I P E H C T S E A R HV K Q T S Y T I K A D I W S L G C L V V E M I S G T H P W A E L N Q M Q A L F Q I G M G - R K P S L P D E I S N E C R DV K Q T S Y S P K A D I W S V G C L V V E M L T G T H P W A D L T Q M Q A I F R I G S L - A R P A P P S D I S V Q A D EV K Q T M H T E K T D I W S L G C L V I E M L T S K H P Y P N C D Q M Q A I F R I G E N - I L P E F P S N I S S S A I DV K Q T A T T A K A D I W S T G C V V I E M F T G K H P F P D F S Q M Q A I F K I G T N - T T P E I P S W A T S E G K N

F L D M T F Q L D H E K R P D A D E L L K S P F LF L A Q T F E I D H N K R P S A D E L M L S P F LF L E K I F E P D Y H A R P T A A D L L K Y S F LF L E K T F E L D Y N N R P S A D E L L N H A F MF L R K T F E I E H A K R P T A A Q L L K H P F IF L E K T F A I D C N L R P T A S E L L S H P F VF L R K A F E L D Y Q Y R P S A L E L L Q H P W L

A. nidulansN. crassaP. cariniiU. maydisC. neoformansS. pombeS. cerevisiae

A. nidulansN. crassaP. cariniiU. maydisC. neoformansS. pombeS. cerevisiaeA. nidulansN. crassaP. cariniiU. maydisC. neoformansS. pombeS. cerevisiae




A

Fig. 5.13 Alignment of A. nidulans SteC with homologues from different fungi. Phylogenetic tree of A. nidulans SteC showing evolutionary distances from the homologues of N. crassa (AF034090); P. carini (AF312696); U. maydis (AF542505.1); C. neoformans (AF294841); S. pombe (M74293); S. cerevisiae (X53431) (A). The SAM protein interaction domains (B) and the catalytic domains (C) of the SteC homologues of the species given in Fig. 5.12 were aligned. If amino acids were identical in two or more sequences they were shaded. The alignment was done with DNAStar using Megalign (Clustal) with a gap penalty and a gap length penalty of 10.

Results

64

After removal of these introns, the translated sequence was predicted to encode a protein of

886 amino acids in length with a molecular weight of 97.8 kDa (Fig. 5.11). The protein shares 50

% identical amino acids with Nrc-1 from N. crassa and 32 % (673 amino acids overlap) with

Ste11 from S. cerevisiae (Chaleff & Tatchell, 1985; Kothe & Free, 1998). The SteC protein

sequence contains a SAM protein interaction domain (sterile alpha domain) in the N-terminus

and the highly conserved catalytic domain typical of the serine-threonine protein kinase in the

very C-terminus (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi). The similarities of the

proteins are much higher if only the catalytic domains are compared (Fig. 5.13). The presence

and arrangement of the domains is similar to other MAPKK kinases, although the SAM domain

is missing in some species (e.g. N. crassa) (Fig. 5.12). Amnio acid identity over catalytic domain

is 67.5%, 61.1%, 58.2%, 58.5%, 54.3% and 53.5% for N. crassa, P. carinii, U. maydis, C.

neoformans, S. pombe and S. cerevisiae respectively. As a difference between S. cerevisiae Ste11

and the displayed six other fungal MAPKK kinases, Ste11 possesses an extra loop in the catalytic

domain. The percentage identity over SteC SAM domain is 48.5%, 41.0%, 39.7%, 24.2%, and

27.7% for P. carinii, U. maydis, C. neoformans, S. pombe and S. cerevisiae respectively.

5.2.2 Complementation analysis of steC mutant and steC overexpression

To overexpress steC and its kinase domain, and to address the question whether the SAM

domain is necessary for the signalling function of the protein, The steC and its N-terminally

truncated versions were overexpressed under the control of a constitutive promoter gpd and tested

them for complementation of the null phenotype, SWH51 (�steC, see next chapter) and a wild

type strain RMSO11 were respectively transformed with the plasmids pHSKP3 (full length, 886

aa), pHSKR2 (full length, 886 aa), pHSKSa4 (573 aa), pHSKSt4 (459 aa; Fig. 5.14).

Overexpression of different fragments was confirmed by Nothern blotting (Fig. 5.14). No evident

phenotypes were observed for the overexpression of any fragments. Wild type colonies were

obtained only in the case of the full length protein (pHSKP3; pHSKR2) after transformation of

steC mutant strain (SWH51). This demonstrates that the loss of 198 amino acids, including the

SAM domain, abolishes biological activity and thus suggests that this protein motif is essential

for full function.

Results

65

Pvu

IIE

co R

I

Sac

IS

tuI

SWHS

GP4

SWHS

GP6

SWHS

GP8

SWHS

GR3

SWHS

GR5

SWHS

GR8

SWHS

GSa3

SWHS

GSa4

SWHS

GSt2

SWHS

GSt4

SWHS

GSt8

steC

A B

Fig. 5.14 Complemention analysis of steC mutant and steC overexpression. (A) Different steC fragments, two full length clones (PvuII-KpnI fragment pHSKP3, EcoRI-KpnI fragment pHSKR2) and two N-terminal truncated fragments (SacI-KpnI fragment pHSKSa4, StuI-KpnI fragment pHSKSt4) were overexpressed under the control of the constitutive promoter gpd. The positions of the SAM and the catalytic domains were shaded with vertical lines. (B) Northern hybridization of the transformant strains after different steC overexpression versions transformed �steC strain SWH51. SWHSGP4,6,8 from pHSKP3-transformed SWH51; SWHSGR3,5,8 from pHSKR2; SWHSGSa3,4 from pHSKSa4; SWHSGSt2,4,8 from pHSKSt4. The transformant strains were grown overnight at 37˚C for RNA isolation. As a loading control, ribosomal RNA was stained with methylene blue after transfer to the nylon membrane (low panel).

5.2.3 steC deletion affects hyphal growth and conidiophore morphology

To gain insights into the function of the steC gene in A. nidulans, a deletion strain was

constructed. An internal 0.6 kb BamHI fragment was replaced with the nutritional marker argB in

the 8 kb SalI-KpnI subclone from the digA containing cosmid (pAG1; pHSAB3; Fig. 5.15).

Integration of the construct through a double crossing-over event leads to the loss of the catalytic

domain and disrupts the open reading frame. The construct pHSAB3 was linearized with KpnI

and used for transformation of two wild type strains, RMSO11 and SRF200. Transformation

yielded wild type-like colonies and about 15 % colonies with a distinct phenotype in orginal

transformation plate. Those strains showed a slower growth, the invading hyphae growth into the

agar and a darker color bottom of colonies. Southern blot analysis of 18 of those strains revealed

that the homologous integration event had taken place (result not shown). Two strains (SWH51

and SWH33) were chosen for further studies (Fig. 5. 15). To confirm that the observed phenotype

was due to the predicted integration event, the deletion strain SWH51 was transformed with the

Results

66

steC carrying plasmid pHSKS2. This plasmid rescued all mutant phenotypes, indicating that it

contained the entire steC gene and the observed phenotype was due to the disruption of steC (Fig.

5.15).

�ste

C(S

WH51

)

8 kb

A BWT

4.7 kb

2.5 kb

3.4 kb

Sal I

Bam

HI

Bam

HI

Kpn

I

Bam

HI

Bam

HI

Xho

I

Xho

IargB

probe

WT �steC�steC transformed

with steC

C

XhoI

Cl

Cl

Fig. 5.15 Disruption of the steC gene. (A) Scheme of the disruption construct (for cloning see text). (B) Southern blot analysis of a steC disruption strain. Genomic DNA of a wild type (SRF 200) and steC disruption strains (SWH51) were isolated, restricted with XhoI, separated on a 1 % agarose gel, blotted and hybridised with the probe indicated in (A). (C) Colonies of wild type (WT; SWHC18), the �steC mutant SWH51 and SWHSR3 (retransformed steC mutant by the plasmid pHSKS2) grown on an agar plate supplemented minimal medium and enlargements of the colonies (lower row of pictures) showing conidiophores and cleistothecia (boxed). Scale bar represents 0.5 cm (upper picture) and 1 mm (lower panels).

Results

67

The steC deletion strain was compared to wild type with respect to hyphal growth, hyphal

morphology, osmosensitivity, conidiophore development and sexual reproduction. Growth on

minimal media resulted in a reduced growth rate and hyphae appeared more curled and more

branched than the wild type. Conidiophore development was initiated like in wild type but the

height of the stalks was uniform in wild type and varied in the mutant (Fig. 5.16). Metulae and

phialides appeared to be normal. However, in a small percentage of conidiophores, the

morphology was drastically altered. Only few metulae arose from the vesicle and did not develop

according to the normal program. Frequently, secondary conidiophores grew out of the vesicle

(Fig. 5.17). In addition, the diameter of the conidiospores varied significantly (Fig. 5.16).

Recently, it was reported that in A. nidulans a MAP-kinase cascade is involved in the

adaptation of the fungus to high osmotic conditions (Han & Prade, 2002; Kawasaki et al., 2002).

Furthermore, it was found that deletion of the HogA MAP kinase, involved in osmoregulation,

resulted in abnormal hyphae when grown on high-salt medium. Therefore, the behaviour of the

steC mutant grown on media supplemented with different salt concentrations was studied. The

steC mutant strain was not more severely inhibited by the salt than the wild type. Curled hyphae

as in the hogA deletion strain were also not observed (results not shown). However, most of the

conidiophores (70-80 %) displayed the altered morphology described above (Fig. 5.16). This

phenotype was only observed in less than 10 % of wild type conidiophores.

Results

68

WT �steCA B

C

F

0102030405060

2.5-3.0 3.0-3.5 3.5-4.0 4.0-4.5 4.5-5.0

WT 4 DsteC

D

12

3

GWT�steC

05

101520253035404550

< 25 25-50 50-75 75-100 > 100

WT DsteC

WT�steC

2.5-3.0 3.0-3.5 3.5-4.0 4.0-4.5 4.5-5.0 µm

< 25 25-50 50-75 75-100 > 100 µm0

20

40

0

20

40

H

E

Fig. 5.16 Analysis of hyphae (A, B), conidiophores (C, D, E) and conidiospores (F, G, H) of a wild type (A, C, F) and a steC mutant (B, D, G). The numbers in (D) indicate three conidiophores of very different stalk-length. (E) The length of 200 conidiophores of wild type (black box) and steC mutant (white box) were measured and the values sorted into size categories. The numbers of conidiophores are given in %. The arrow in (G) points to an enlarged conidiospore. (H) The diameter of 300 spores of wild type (black box) and steC mutant (white box) were determined. The numbers of conidiospores are given in %.

Results

69

A B CWT �steC

Fig. 5.17 Role of steC during conidiophore development. Phase contrast picture of conidiophores of a (A) wild type and a (B, C) steC mutant strain grown on minimal medium supplemented with 0.6 M KCl. The scale bar represents 25 µm.

5.2.4 Deletion of steC inhibits heterokaryon formation and sexual development

During the course of several times of crossing experiments, it became evident that steC

mutant strains could not be crossed to other common laboratory A. nidulans strains (Fig. 5.18).

Since the first step for a successful cross is the fusion of the hyphae, the question arose whether

the failure of hyphae fusion or a later step was the cause for the observed sterility. To answer this

question, protoplast fusion experiments were performed. The cell walls of a wild type (RMSO11)

and a steC deletion strain (SWH51) were digested like for the preparation of a transformation, the

washed protoplasts was mixed in osmotically stabilized medium, treated them with polyethylene

glycol (PEG) to induce the fusion and plated them on minimal medium, on which neither the wild

type nor the deletion strain would be able to grow, because of their nutritional requirements. Only

heterokaryotic mycelium can grow. Colonies developed, which had the typical heterokaryotic

appearance with conidiospores of both colors of the parent strains (Fig. 5.18). The same

experiment was performed with two steC deletion strains (SWH51 and SWH33). Likewise,

heterokaryotic mycelia was obtained. However, whereas the heterokaryon derived from a wild

type and the steC deletion strain developed mature cleistothecia, the combination of two steC

deletion strains proved to be sterile, although Hülle cells were produced. Further development of

sexual structures, such as primordia or cleistothecia was never observed (Fig. 5.18).

Results

70

WT x WT WT x �steC

RMSO11 SWH51

SWH51X

SWH33

A

B

C

RMSO11 x

SWH51

Fig. 5.18 Heterokaryon formation and sexual development. (A) Cross of two A. nidulans strains (green and yellow color). (B) After crossing, the agar pieces at the border of two different strains were transferred to a selective medium. Wild type (WT) crossed to a wild type resulted in heterocaryontic mycelium growing a selective medium, but wild type crossed to a �steC mutant did not grow (C) Protoplasts were generated from a wild type (RMSO11) and two �steC strains (SWH51 and SWH33) and used for protoplast fusion in different combinations. The figures show heterokaryon of the indicated strains. Green and yellow conidiophores are visible. The frame indicates a ripe, black cleistothecium. The heterokaryon of two steC deletion strains does produce green and yellow conidiophores but no cleistothecia.

5.2.5 steC-transcription is developmentally regulated and induced in metulae and phialides

The phenotypic characterization of the steC deletion strains suggested several important

functions during the life cycle of A. nidulans. In order to answer whether the gene was

transcriptionally regulated during development. RNA was isolated at different time points during

Results

71

vegetative growth, asexual and sexual development and used for Northern blot analysis. It was

found that the steC transcript was more abundant during conidiophore development. After

asexual development was completed the signal decreased to a low level like in vegetative hyphae

(Fig. 5.18).

In order to further specify the spatial expression of the gene, a C-terminal GFP-fusion

construct was generated and introduced into a steC deletion strain. However, it did not

complement the mutant phenotypes, suggesting that the GFP protein interferes with the catalytic

function of SteC (results not shown). Fluorescence was detected only in the original

transformation plates but not after re-streaking on minimal media plates. This suggests that the

full-length fusion protein might be rather unstable. Because of this and the non-complementation

of the mutant, another translational fusion of SteC and GFP was constructed, where GFP was

fused before the catalytic domain (pHSRBgfp1) and introduced the plasmid into RMSO11.

Intense GFP-fluorescence in metulae, phialides and young conidiospores and no fluorescence in

conidiophore stalks was observed (Fig. 5.18). In older conidiophores, fluorescence was not

detectable anymore suggesting that the increase of steC expression was only transient. This

corresponds the results obtained in the transcript analysis described above. Promoter analysis

revealed several putative binding sites for the developmental regulators StuA and AbaA

(Andrianopoulos & Timberlake, 1994; Dutton et al., 1997). The fluorescence was not detectable

during sexual development.

Results

72

A

B

24 30 36 30 48 72 96hyphae asexual sexual

h

C

Fig. 5.18 Expression analysis of steC. (A) Northern blot analysis. RMSO11 was inoculated as a spore suspension on cellophane membranes on complete media and mycelium harvested after the time intervals indicated. 20 µg of total RNA was used for the Northern blot. A steC-specific fragment as a probe (32P-labelled by PCR) was used. Before hybridisation the membrane was stained with methylene blue to visualize rRNA as a loading control (lower panels). (B, C) Expression of a translational GFP fusion construct in RMSO11 and detection of fluorescence in conidiophores observed on an agar plate. (B) Low-magnification overview where several conidiophore heads are visible. (C) Mature conidiophore. Metulae, phialides and young spores are brightly stained.

Results

73

5.2.6 SteC activates at least two kinases

MAP kinase cascade signalling leads to the activation of MAP kinases by dual

phosphorylation of a tripeptide motif TXY. The question raised which MAP kinases would be

activated downstream of SteC during the life cycle of A. nidulans. For the detection of the

phosphoepitopes monoclonal antibodies are commercially available. These antibodies recognize

corresponding tripeptides in MAP kinases. Three different anti-phospho-antibodies (p44/42,

SAPK/JNK and p38) were used to detect MAP kinases in A. nidulans under different

physiological and developmental conditions. A wild type and a steC mutant strain for asexual

development were induced, the mycelium harvested after different time points was used for the

protein extracts in Western blot analyses. Signals were obtained with all three antibodies and one

signal with the apparent molecular mass of 30 kDa detected with p44/42 was dependent on SteC

(Fig. 5.19). A 42 kDa signal, detected with the p38 antibody, appeared to be increased upon

developmental induction but was not dependent on SteC. Using the anti-phospho-SAPK/JNK

antibody no reproducible change of the phosphorylation status was observed.

Since the SteC-dependent signal intensities obtained in the time-course experiment were rather

low, it was further tried to detect the phosphorylation under different physiological conditions. In

S. cerevisiae transient activation of MAP kinases can also be achieved after oxidative stress

application. Therefore, it was tested whether some MAP kinases of A. nidulans were transiently

phosphorylated under these conditions. Surprisingly, phosphorylation was detected not only after

application of H2O2 but also by changing the medium. Under both conditions, and in agreement

with the developmental phosphorylation pattern, phosphorylation of the kinase detected with

p44/42 was SteC-dependent. In addition, the phosphorylation of a kinase (49 kDa) detected with

the anti-phospho-SAPK/JNK antibody was stimulated by SteC (Fig. 5.19).

Results

74

A B

RMSO11

SWH33

(�st

eC)

RMSO11

SWH33

(�st

eC)

RMSO11

SWH33

(�st

eC)

RMSO11

SWH33

(�st

eC)

0 6 12 24 h

RMSO11

SWH33

(�st

eC)

H2O2

Anti-phospho-p44/p42

46 kDa

30 kDa

Anti-phospho-SAPK/JNK

49 kDa

42 kDaAnti-phospho-p38

Coomasie-stained gel

Fig. 5.19 SteC-dependent phosphorylation of MAP kinases. (A) Time course of asexual development. Protein extracts (RMSO11 and SWH33) were subjected to a western blot analysis with the antibodies indicated. The arrows in the first row of pictures point to the MAP kinase, which is SteC-dependent phosphorylated. (B) Induction of the phosphorylation by oxidative stress or change of the media (control). As a loading control the same amounts of protein were separated as used for the western blots on a SDS gel and stained it with Coomasie. The pictures are shown below the western blots. For further details see the methods section.

Discussion

75

6. Discussion

The fungus A. nidulans has two distinctive reproductive developmental processes: asexual

and sexual developments. Several genes involved in asexual development have been genetically

characterized and the interactions between them have been investigated (for a review, see

(Adams et al., 1998; Fischer, 2002). In contrast, the sexual developmental process has not been

well dissected. The initial step of sexual development is the aggregation of vegetative mycelia.

Some cells in the aggregates differentiate to Hülle cells and some to primodia. Cleistothecia are

later developed from primodia. In a cleistothecium, asci are formed in which eight ascospores are

produced by meiosis (for a review, see Zonneveld, 1977). Although some genes related to sexual

development have been successfully identified including veA (Champe et al., 1981; Kim et al.,

2002), tubB (Kirk & Morris, 1991), medA (Busby et al., 1996), stuA (Wu & Miller, 1997), nsdD

(Han et al., 2001) and steA (Vallim et al., 2000), lsdA (Lee et al., 2001), phoA (Bussink &

Osmani, 1998), dopA (Pascon & Miller, 2000), sakA (hogA)(Kawasaki et al., 2002; Han & Prade,

2002), the molecular genetic mechanisms controlling sexual reproduction of A. nidulans are still

unclear.

In this study mainly three genes mutA, hgtA and steC related to the process of sexual

development in A. nidulans were studied.

6.1 The carbon cycle during the sexual development in A. nidulans

6.1.1 MutA is expressed during sexual development in A. nidulans and mobilizes mutan

For many organisms, carbohydrates are important carbon and energy sources. In earlier

publications Zonneveld (1973) found that �-1,3 glucan (mutan) accumulates in A. nidulans

during vegetative growth and becomes metabolised during sexual development. He could show

that the presence of 2-deoxy-D-glucose (2-DG) inhibits the synthesis of mutan and sexual

development did not occur although there was no severe effect on vegetative growth. He

concluded that sexual development strictly depends on the formation and subsequent degradation

of mutan. In the later literature, mutan was considered as one of determined factors in

cleitothecium formation (Braus et al., 2002). In our molecular approach, surprisingly, I found that

the mutA gene is not essential for cleistothecium formation. Although it cannot be ruled out that

Discussion

76

in the absence of the MutA enzyme mutan is partly degraded by some other enzymes, it is found

that the degradation is greatly affected. Therefore it appears to be unlikely, that A. nidulans

possesses several enzymes with �-1,3-glucanase activity. A second reason why mutA deletion

strains are still able to form fruiting bodies could be that other carbon sources are consumed

during sexual development. Likewise, A. nidulans accumulates a variety of carbohydrate

polymers besides mutan during vegetative growth (Zonneveld, 1973). Recently, it was shown that

at the onset of sexual development many different cell wall lytic enzymes are produced and thus

many of the polymers are probably degraded (Prade et al., 2001). The degradation of these

alternative polymers could be also inhibited by 2-deoxy-D-glucose. Alternatively, the presence of

2-DG could also interfere with the pathways involved in sexual development. This would explain

why in earlier experiments sexual development was blocked in the presence of 2-deoxy-D-

glucose (Zonneveld, 1973).

Mutanase (�-1,3-glucanase) has been characterised in P. purpurogenum and T. harzianum.

Here it was found to be induced in the presence of mutan isolated from Streptococcus mutans

cultures (Fuglsang et al., 2000). The authors proposed a mutan binding domain in the C-terminal

of two mutanases of Penicillium and Trichoderma. However, this domain was not found in the

mutanase of A. nidulans. This might be due to the different roles of the enzymes in the different

organisms. In Penicillium and Trichoderma the enzyme is secreted to degrade extracellular

mutan. In contrast, it is believed that A. nidulans secretes the enzyme into the cell wall, where it

mobilizes the polymer. Since the mutan in the cell wall might be structurally different from

insoluble mutan it could be that the binding capability of MutA of A. nidulans does not rely on

the domain postulated in Penicillium. It could also be that the binding of A. nidulans MutA

requires another component present in the cell wall but not in Streptococcus mutan.

Compared with wild type, mutA deletion and overexpression strains did not show evident

differences with regards to vegetative growth, asexual development or sexual fruiting body

formation, the number of cleistothecia per cm2, the number of ascospores per cleistothecium, the

viability of the ascospores in normal minimal and complete media. But, the amount of “alkali-

soluble fraction” (mutan) in the wild type after 12 days of growth was always about 50 % of the

mutan in the mutA mutant, the amount of mutan in overexpression strain was slightly lower than

in wild type. This suggested that MutA indeed functions in mutan degradation. If approximately

estimating the mutan fraction as the amount of “alkali-soluble fraction” of a �mutA strain

minused by that of a mutA overexpression strain after longer growth, the mutan is just around 5-

Discussion

77

8% of the amount of supplemented carbon source such as glucose in normal minimal and

complete media. This explained why no evident phenotypes between �mutA and wild type and

mutA overexpression strains were observed in normal media supplemented with enough carbon

source, but evident phenotypes were observed in the medium just using the mutan fraction as

carbon source. In addition, MutA was specifically expressed in Hülle cells. This suggested mutan

was reutilized in the stage of sexual development, but appeared to play a minor role in the

contribution to fruiting body development. The higher density growth of overexpression and wild

type strain in the medium using mutan as carbon source, together with the signal peptide in the

N-terminus of MutA, suggested MutA is secreted to utilize the mutan. The gpd::mutA

constitutive overexpression strains of MutA did not show lysis of the mycelium. In addition,

MutA expression in wild type during sexual development did not show mycelium autolysis. This

suggests that the components of cell wall change dynamically during the life cycle of A. nidulans

and that mutan is probably not a fundemental skeletal component in A. nidulans cell wall.

Transcript and sgfp reporter construct analysis showed that the expression of mutA is highly

regulated and specifically induced in Hülle cells and some connecting hyphae. Thus, this gene

opens the possibility to study the regulatory elements as well as regulators. Three DNA-binding

protein regions of the putative regulatory factors were identified. A new method was used for

DNA-binding protein isolation. In the preliminary experiments, it was found that a 40 kDa

protein bound specifically to a 150 bp fragment at the –1.7 kb position of mutA promoter. This

protein needs to be identified using Mass-spectrum technology in the future study. Further studies

of regulatory elements which specifically bind will be of great importance in the analysis of the

transcription regulation mechanism of the mutA gene and other sexual pathway specific genes.

The experiments suggested another interesting aspect of the nutrition of a developing

mycelium. The consumption of cell wall polymers and the production of cell wall lytic enzymes

is a critical process. However, it was found that the expression of the mutanase gene in A.

nidulans is restricted to a certain type of cell, namely the Hülle cells. The cell walls of these cells

do not contain mutan (Zonneveld, 1977). Therefore it is likely that Hülle cells produce mutanase,

that other substrate hyphae are partly degraded and that the Hülle cells or the connecting hyphae

absorb the released carbohydrates. Hence, Hülle cells could provide the nutrition to the

cleistothecium and thus they could have a nurse cell function. This raises of course the question

about the uptake of the released hexose and how it is provided to the developing cleitothecium.

Discussion

78

To address this question, in the present study, a putative high-affinity hexose transporeter, found

in the SSH library (Scherer, 2001), was characterized.

6.1.2 hgtA encodes a high affinity glucose transporter and is expressed in ascogenous

hyphae

Bacteria and fungi can secrete some enzymes that degrade long chain carbohydrate polymers

from plant debris in the soil. The degradation leads to the release of hexose and other

monosaccharide. In order to reutilize those monosaccharides, it is necessary to transfer them over

the cell membrane by a group of hexose transporter proteins. In this work, a high-affinity hexose

transporter gene from A. nidulans was molecularly studied. hgtA encodes a 59 kDa protein with

substantial similiarity to hexose transporeters from other fungi. The hydrophobicity prediction

showed the presence of 12 putative transmembrane (TM) domains, a characteristic feature of the

major facilitator superfamily (Marger & Saier, 1993).

The deletion strain of hgtA didn’t show evident phenotypes. However, this might not be

surprisingly, given that at least 10 different hexose transporters were found in the A. nidulans

partial genome sequence (Weber, 2002). Therefore, it is likely that other transporters are able to

substitute the function of hgtA. In S. cerevisiae, HXT1-4 and HXT6-7 encode the major hexose

transporters. The expression of each of those genes in the MC996 background strain deleted for

HXT1-7 allows growth on glucose (Reifenberger et al., 1995). Likewise, in the yeast K. lactis,

two glucose transporters, the high-affinity transporter Hgt1 and the low-affinity transporter Rag1,

have been found (Wesolowski-Louvel et al., 1992; Billard et al., 1996). Although, no other Rag1

and Hgt1-related sequences have been found in the K. lactis genome, the deletion of both genes

did not completely prevent glucose transport, indicating that there are even more and probably

unrelated glucose transporters in K. lactis. The same might also be the case on P. stipitis

(Weierstall et al., 1999).

The hgtA gene was isolated as a gene expressed during sexual development and sgfp reporter

constructs revealed the expression in the cells inside the cleitothecium. Most likely, these cells

are the ascogenous hyphae. In contrast, Hülle cells showed no expression. In the shell of a

cleitothecium, a shining fluorescence was also be seen. This result suggested the carbon supply in

ascospore maturation and cleitothecium development occur through HgtA to transport the hexose

from the medium or MutA-released glucose (Fig. 6.1). It will be the challenge of the future

Discussion

79

research to unravel the exact nature of the carbon flow during the stage of sexual development of

A. nidulans.

MutACleistothecium

Hülle cells

Release and reutilization of glucose from cell wall

HgtA

glucose

Fig. 6.1 Model of MutA and HgtA functions. MutA is expressed in Hülle cells and secreted for degrading mutan in hyphae. HgtA transports the released glucose into the cleistothecium for supplying the carbon and energy source for the formation and maturation of the cleistothecium.

6.2 The MAPKK-kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in A. nidulans

In addition to search for the target genes that are involved in sexual development of A.

nidulans. The identification of the upstream factors, possibly involved in the regulation of the

expression of the target genes, were attempted. In this work I found that SteC and thus a MAP

kinase cascade is required for hyphal extension, conidiophore morphogenesis, heterokaryon, and

cleistothecium formation. Many of the observed phenotypes are similar to the ones described for

a corresponding MAPKK kinase mutant, nrc-1, of N. crassa (Kothe & Free, 1998). This related

fungus undergoes two developmental pathways, an asexual and a sexual one. Asexual spore

formation occurs after nutrient depletion. nrc-1 mutants grow slower, display a fertilization

defect and induce asexual development under non-starvation conditions (nrc = non-repressed

conidiation). In addition, the spores appear to have a developmental block and do not separate

Discussion

80

easily. The appearance of the chains of spores resembles the phenotype of abaA mutants of A.

nidulans, where phialides do not mature to produce conidia but elongate and swell in regular

intervals (Andrianopoulos & Timberlake, 1994). Despite the phenotypic similarities between the

nrc-1 mutant of N. crassa and the steC mutant of A. nidulans, a comparison to the results in S.

cerevisiae is more elusive to speculate about actions and interactions of SteC in A. nidulans.

6.2.1 Hyphal extension, conidiophore and conidial development

Deletion of steC impaired hyphal extension. Although it is not obvious which signals might

trigger hyphal elongation and might be transmitted by a MAP kinase cascade, there is more

evidence that phosphorylation events are important for this process. It has been shown that a

MAP kinase (MpkA) is involved in hyphal growth and thus SteC could be involved in the

regulation of the activity of this kinase although it is no experimental evidence for that (see

below) and the mpkA is much more severe than the steC mutation (Bussink & Osmani, 1999). In

addition to MpkA, several other kinases have been shown to be required for efficient cell wall

extension and SteC thus could also be upstream of those kinases (Chen et al., 2000; Navarro-

Garcia et al., 1998; Yarden et al., 1992).

Another effect of the steC mutation, is the disturbance of normal conidiophore and

conidiospore development. The height of the stalks and the diameter of the spores showed a

difference to wild type. This phenotype resembled the phenotype of the A. nidulans dopA mutant

(Castiglioni Pascon & Miller, 2000). The dopA encodes a leucin-zipper transcription factor. The

dopA mutant strains fail in addition to initiate cleistothecium formation, which also is comparable

to steC mutants. However, further experiments are needed for a detailed study of a possible

relationship between SteC and DopA. In addition to the spore and conidiophore stalk defects in

steC mutants, metulae do not differentiate properly and produce phialides, but instead re-

differentiate into hyphae or conidiophore stalks, resulting in secondary condiophores. Here,

comparison to S. cerevisiae could help to explain the role of a MAP kinase cascade. Metula

differentiation and phialide production resembles pseudohyphal development in yeast and

homologous regulators appear to be involved in the regulation of the two processes (Gavrias et

al., 1996; Mösch, 2002). Since this differentiation depends on a MAP kinase cascade in S.

cerevisiae, it is also likely to be the case in A. nidulans (Gustin et al., 1998). The MAP kinase

responsible for the final transmittance of the signal to a transcription factor could be the 30 kDa

Discussion

81

protein, which was identified. In S. cerevisiae two MAP kinase substrate transcription factors

Tec1 and Ste12 are involved in pseudohyphal growth regulation (Mösch, 2002). In A. nidulans

the homologue of Tec1 is AbaA, a transcription factor, which is involved in phialide

differentiation. Hence, this factor would be one good candidate for a target of the MAP kinase

cascade (Andrianopoulos & Timberlake, 1994). Although, the phenotypes of steC and abaA null

mutants are different, it could be that modulation of the AbaA activity in steC deletion strains

results in a different phenotype than deletion of the entire abaA gene. Similar phenomena were

reported for another important regulator of conidiophore development, BrlA (Prade &

Timberlake, 1993). In addition to the Tec1 homologue AbaA, a transcription factor with

similarity to Ste12 was also described in A. nidulans, but deletion of the gene had no obvious

phenotype on conidiophore development (Vallim, et al., 2000). Additional experiments will be

required to unravel the nature of interactions between those regulators and the MAP kinase

cascade in A. nidulans.

Since the defective phenotype of the steC mutant is found in a small percentage of

conidiophores, other cascades likely can substitute for the function of SteC. However, we

observed that under high osmolarity conditions the phenotype was much more severe. This might

be explained as follows: The transcription of the hogA gene is induced during conidiophore

formation and thus the protein amount is likely to be high during asexual development. High

osmotic pressure leads to an activation of HogA (= SakA) and this might interact with the

developmental program triggered by the 30 kDa MAP kinase (Han & Prade, 2002; Kawasaki et

al., 2002) Phosphorylated HogA could negatively influence the SteC pathway especially when

SteC is absent. Similarly, Hog1 appears to be involved in the regulation of morphogenesis in C.

albicans (Alonso-Monge et al., 1999). Such cross-talk between different MAP kinase pathways is

typical for these signaling modules and the analysis of the nature of its regulation is one

important field of research (Madhani & Fink, 1998; Madhani et al., 1997; Sabbagh et al., 2001).

Similarly, Kawasaki et al. (2002) suggested cross talk to explain precocious formation of sexual

structures in sakA mutants.

Despite an obvious effect of osmolarity on conidiophore development in steC mutants, we did

not observe an increased sensitivity of hyphal growth against high osmolarity, comparable to the

phenotype observed when the corresponding MAP kinase, HogA, is not functional (Han & Prade,

2002). This is not surprising because alternative, SteC-independent pathways might exist.

Likewise, in S. cerevisiae three pathways were described (O'Rourke & Herskowitz, 2002).

Discussion

82

6.2.2 Heterokaryon and cleistothecium formation

Heterokaryon formation could be compared to the mating reaction in S. cerevisiae. The latter

involves a pheromone-receptor system through which yeast cells of opposite mating type

recognize each other (Banuett, 1998). However, A. nidulans is a homothallic fungus and does not

require a mating partner of opposite mating type for a successful mating reaction (Coppin et al.,

1997). Nevertheless, there is mounting evidence that homothallic fungi harbour and express

mating type genes in the same hyphae and thus mimic the presence of a compatible partner. In

Sordaria macrospora it was shown that homologues of ascomycetous mating type genes exist,

and that they can complement corresponding mutants of the heterothallic species Podospora

anserina (Pöggeler & Kück, 2001). However, genes encoding a peptide-pheromone like in S.

cerevisiae have not yet been detected. Besides peptide-pheromones other low molecular weight

compounds could be involved in the signalling between hyphae. In A. nidulans a system of

interconvertible fatty acid derivatives, the PSI factors, have been described, which trigger

developmental decisions (Champe & El-Zayat, 1989; Champe et al., 1987). Application of one of

the compounds leads to a precocious induction of the sexual cycle. In addition, the reaction

requires a high density of cells, suggesting another sensing mechanism. The question about the

signal recognized by hyphae is certainly one of the most interesting ones to be solved in the

future. The fact that steC mutants of A. nidulans could not be crossed to other laboratory strains

with intact steC alleles could involve in such a communication system. Both partners have to

recognize each other and prepare the hyphae to fuse. This probably requires local lysis and

remodeling of the cell walls and subsequent fusion and resealing. The induction of the

corresponding genes or the triggering of the corresponding enzyme activities could be the role of

the MAP kinase cascade.

Another phenomenon, which might be explained with similar arguments, is the lack of

cleistothecium formation in steC mutant strains. These strains are still able to form Hülle cells,

but fail to continue with the development to produce mature cleistothecia. Since mating types are

also required for nuclear recognition in the ascus mother cell and for nuclear fusion, it could well

be that this process is inhibited in steC mutants (Coppin & Debuchy, 2000; Debuchy, 1999).

Whether the signals transmitted in this process in A. nidulans are the same as for heterokaryon

formation is unknown. In S. cerevisiae the downstream transcription factor for mating is Ste12.

Discussion

83

Similarly, it could be that a homologue of this protein is involved in mating and fruiting body

formation in A. nidulans. Recently, a gene encoding a protein with similarity to Ste12 was

characterized and indeed, deletion strains were sterile but produced masses of Hülle cells. The

mutation was recessive (Vallim, et al., 2000). However, the phenotype is distinct from the one of

steC deletion strains.

6.2.3 Identification of SteC targets

After discussing the different signals and environmental conditions, which might be SteC-

dependent transmitted, the question of the MAP kinase(s) downstream of SteC will be addressed.

Using antibodies against conserved phosphoepitopes, I detected several kinases and showed that

at least two were phosphorylated in a SteC-dependent manner. It was tried to detect

phosphorylation of specific proteins after heterokaryon formation, during asexual and during

sexual development. However, these processes are likely to be too slow in order to detect changes

of the phosphorylation status. In S. cerevisiae phosphorylation of Fus3 occurs within 5 minutes

after pheromone addition and decreases steadily thereafter (Sabbagh, et al., 2001). In comparison,

conidiophore development requires several hours and sexual differentiation even several days. In

addition, cultures are not well enough synchronized to detect such transient phosphorylation

events. However, after stress activation I found two kinases dependent on SteC. The sequences of

four different MAP kinases have been deposited in the databases, HogA (SakA) (Acc. no.

AF270498)(Han & Prade, 2002), MpkA (Acc. no. U59214)(Bussink & Osmani, 1999), MpkB

(Acc. no. 198118) and MpkC (Acc. no. AF195773). I detected one MAP kinase, which was

recognized by the anti-phospho antibody p42/p44. This antibody recognizes the tripeptide TEY,

which is found in MpkA and MpkB. The predicted molecular masses of these two kinases are

47.8 and 41.6 kDa, respectively. However, the SteC-dependent MAP kinase which is detected in

my experiments and which was phosphorylated during asexual development had an apparent

molecular mass of 30 kDa. This suggests that a new MAP kinase is involved in the regulation of

development. Indeed, MpkA is required for spore germination and hyphal growth (Bussink &

Osmani, 1999) and MpkB has not been functionally characterized yet although there is some

evidence for an involvement in sexual development (Jahng, Chonbuk, University of South Korea,

personal communication). The antibody SAPK/JNK recognizes the tripeptide TPY, which is not

found in any known A. nidulans MAP kinase. The p38 antibody detects the tripeptide TGY in a

Discussion

84

protein with an apparent molecular mass of 42 kDa. This peptide motif is found in MpkC and

HogA (SakA). The predicted molecular masses of MpkC and HogA are 39.5 and 43.2 kDa,

respectively. Thus one of them could be the one detected in our experiments. Although this

protein was induced upon development, its phosphorylation was not dependent on SteC. If it

were HogA, this result would not be surprising since three pathways for osmosensing exist in S.

cerevisiae, two of which are independent on Ste11 (Furukawa et al., 2002). Similarly, Kawasaki

et al. (2002) found transient activation of SakA upon induction of asexual development.

However, the time periods where the phosphorylated species of SakA was detectable varied in

my experiment from the reported data. This discrepancy may be explained by slightly different

experimental conditions. As it have been seen phosphorylation of SakA occurs very rapidly after

application of stress or changing of medium.

The steC mutant strain (SWH51) was also respectively transformed by several transcription

factors and target genes. A fluffy phenotype appeared similar in the transformants of steC mutant

and wild type strains after transformation by a rosA overexpression construct (Vienken, 2003), or

a fadA dominant mutant construct (Hicks et al., 1997). The bright fluresence also appeared in the

Hülle cells after the transformation of SWH51 by the construct of mutA promoter fused with sgfp

(Wei et al., 2001)(results not shown). Those results suggested RosA, FadA and MutA act in

different pathways with SteC. The identification of the SteC targets, as well as the discovery of

other unknown MAP kinases involved in the morphological transitions, will be one of the goals

for future research.

6.3 Outlook

The regulatory pathway or network of the genes that are involved in the asexual reproduction

process, from signaling of differentiation to completion of conidiation, is now well established

(Adams, et al., 1998). The signal transduction pathways that include fluG, fadA and flbA have

been shown to establish a balance between filamentous growth and development through

regulation of brlA expression (Adams, et al., 1998). Early brlA� expression initiates development

by activateing abaA expression. A positive feedback loop is established in which abaA enhances

brlA� expression and guarantees commitment to terminal differentiation (Mirabito et al., 1989;

Han et al., 1993; Prade & Timberlake, 1993). Correct spatiotemporal expression of both brlA and

abaA are required for asexual differentiation (Miller et al., 1992; Aguirre, 1993). Furthermore,

Discussion

85

the balance between brlA� and brlA� expression is particularly important throughout

development for correct conidiophore morphogenesis and conidial differentiation (Prade &

Timberlake, 1993; Busby, et al., 1996).

PheromonesLight

Air exposureMAP kinase cascade

Ste11

Ste7

Fus3Ste5

Ste12

G-protein

Ste20Signal

transductionTranscription

FactorsTarget genesMutA, HgtA...

StuA, VeA, RosAMedA,SteA...

Fig. 6.2 Scheme of the regulatory pathway in sexual development of A. nidulans and the positions of genes studied here (MutA, HgtA and SteC). The signals that initiate sexual development in A. nidulans are transferred by signal transduction pathways to some transcription factors that control the expression of target genes. A MAPK cascade from S.cerevisiae is used as a putative model of the signal transduction in the sexual development of A. nidulans.

However, information about the sexual cycle regulatory network and potential SteC target

genes is limited. Potential signaling molecules such as Aras, heteromeric G� and G� proteins and

RGS protein and a few developmental-specific components in MAP kinase cascade such as

MpkA (Bussink & Osmani, 1999), SteA (Vallim, et al., 2000) and HogA(�SakA) (Han & Prade,

2002; Kawasaki, et al., 2002) have been reported in A. nidulans. However, a complete signaling

pathway has not yet been described. Like other higher eukaryotes, A. nidulans uses multiple-

signal inputs to make developmental decisions leading to organogenesis and the formation of

multicellar structures. The A. nidulans life cycle requires the coordination of mitotic and meiotic

reproductive cycles. Regulatory functions of BrlA and AbaA are restricted to the asexual

developmental programme. By contrast, MedA, StuA and SteC are required during both

Discussion

86

reproductive developmental cycle (Vallim, et al., 2000; Wei et al., 2003). In recent years, the

number of reports about research in sexual development of A. nidulans has increased. Those

genes include genes involved in signal transduction, transcription factors and some target genes.

With the increasing number of genes involved in sexual development, a clear network of

regulaters is likely to emerge in the near future (Fig. 6.2).

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EUKARYOTIC CELL, Oct. 2002, p. 725–735 Vol. 1, No. 51535-9778/02/$04.00�0 DOI: 10.1128/EK.1.5.725–735.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Aspergillus nidulans Catalase-Peroxidase Gene (cpeA) IsTranscriptionally Induced during Sexual Development through the

Transcription Factor StuAMario Scherer, Huijun Wei, Ralf Liese, and Reinhard Fischer*

Laboratorium fur Mikrobiologie, Philipps-Universitat Marburg and Max-Planck-Institut fur Terrestrische Mikrobiologie,D-35043 Marburg, Germany

Received 1 March 2002/Accepted 9 July 2002

Catalases, peroxidases, and catalase-peroxidases are important enzymes to cope with reactive oxygen speciesin pro- and eukaryotic cells. In the filamentous fungus Aspergillus nidulans three monofunctional catalases havebeen described, and a fourth catalase activity was observed in native polyacrylamide gels. The latter activity isprobably due to the bifunctional enzyme catalase-peroxidase, which we characterized here. The gene, namedcpeA, encodes an 81-kDa polypeptide with a conserved motif for heme coordination. The enzyme comprises oftwo similar domains, suggesting gene duplication and fusion during evolution. The first 439 amino acids share22% identical residues with the C terminus. Homologous proteins are found in several prokaryotes, such asEscherichia coli and Mycobacterium tuberculosis (both with 61% identity). In fungi the enzyme has been notedin Penicillium simplicissimum, Septoria tritici, and Neurospora crassa (69% identical amino acids) but is absentfrom Saccharomyces cerevisiae. Expression analysis in A. nidulans revealed that the gene is transcriptionallyinduced upon carbon starvation and during sexual development, but starvation is not sufficient to reach highlevels of the transcript during development. Besides transcriptional activation, we present evidence for post-transcriptional regulation. A green fluorescent protein fusion protein localized to the cytoplasm of Hulle cells.The Hulle cell-specific expression was dependent on the developmental regulator StuA, suggesting an activat-ing function of this helix-loop-helix transcription factor.

Oxidative stress and the occurrence of reactive oxygen spe-cies is common to aerobically living organisms and might bedeleterious for living cells (10, 18). Reactive oxygen species aregenerated during normal cell metabolism and comprise super-oxide, hydroxyl radicals, hydrogen peroxide, and singlet oxy-gen. All aerobically living organisms employ one or severalsystems to cope with these toxic substances. Catalases andperoxidases are most commonly used to transform the harmfuloxygen compound H2O2 into harmless products. Catalases areheme-containing enzymes, which convert H2O2 into oxygenand water. Peroxidases are heme-containing enzymes as welland inactivate H2O2 by reducing it to water. In addition toheme-containing catalases and peroxidases, nonheme varietiesof these enzymes exist. Different cellular substrates can serveas electron donors for this reaction. Frequently, organisms usedifferent isozymes, which are expressed simultaneously or un-der developmental-stage- and environment-specific conditions(26, 35).

One good example for the employment of several catalasesand their differential regulation during the life cycle is thefilamentous fungus Aspergillus nidulans (15). A. nidulans is ableto grow as vegetative hyphae but then undergoes two develop-mental programs. After 20 h of vegetative growth it can enteran asexual reproductive pathway in which it generates thou-sands of single-cell, haploid conidiospores. In addition, it isable to reproduce itself with very durable sexually derived

ascospores (1). Both spore types are produced at or in specialmorphological structures, called conidiophores or cleistoth-ecia, respectively. The conidiophore consists of four differentcell types—a stalk, metulae, phialides, and conidia—and growsaway from the agar surface into the air. The asexual develop-mental pathway is very well characterized at the molecularlevel (1) and is triggered by a central cascade of transcriptionalactivators (3, 22). In an effort to characterize differentiallyexpressed genes during asexual development, a catalase gene(catA) was discovered (20) that is transcriptionally and post-transcriptionally regulated, and the protein accumulated inconidiospores (19). Using the catA sequence, a second cata-lase, catB, was isolated (16). This gene is developmentallyinduced during conidiophore formation, but the transcript isalmost absent in conidiospores. The catB expression, like thatof catA, also responds to different stress conditions (6, 16).Finally, Kawasaki and Aguirre, taking advantage of thegenomic sequencing project at Cereon Genomics LLC (Cam-bridge, Mass.), identified a third catalase gene, designated catC(15). The protein resides in peroxisomes and is constitutivelyexpressed. Interestingly, mutations in a single gene or in allcatalase genes did not have any detectable vegetative or devel-opmental phenotype, suggesting the presence of moreisozymes. Indeed, in native polyacryl amide gels a fourth cata-lase activity, catD, was observed (15). We have identified thecorresponding gene and found that it is a catalase-peroxidase.The expression of the gene is transcriptionally and translation-ally regulated upon carbon starvation and during sexual devel-opment. One important regulator is the transcription factorStuA.

* Corresponding author. Mailing address: Laboratorium fur Mikro-biologie, Max-Planck-Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany. Phone: 49-6421-178-330.Fax: 49-6421-178-309. E-mail: [email protected].

725

Molecular Microbiology (2003)

47

(6), 1577–1588

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 200347

615771588

Original Article

MAPKK kinase and sexual development in A. nidulansH. Wei, N. Requena and R. Fischer

Accepted 29 November, 2002. *For correspondence. E-mail [email protected]; Tel. (

+

49) 6421 178 330; Fax (

+

49)6421 178 309.

†

Present address: University of Tübingen, Departmentof Botany – Ecophysiology, Auf der Morgenstelle 1, D-72076 Tübin-gen, Germany.

The MAPKK kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus

Aspergillus nidulans

Huijun Wei, Natalia Requena

†

and Reinhard Fischer*

Department of Microbiology, University of Marburg and Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany.

Summary

Environmental signals can be transduced into intrac-ellular responses by the action of MAP kinase cas-cades. Sequential phosphorylation results in thetransient activation of a MAP kinase, which in turnactivates certain transcription factors and thus a setof pathway-specific genes. Many steps in this cas-cade are conserved, and homologues have been dis-covered from yeast to human. We have characterizedthe MAPKK kinase, SteC, a homologue of

Saccharo-myces cerevisiae

Ste11, in the filamentous fungus

Aspergillus nidulans

. The 886-amino-acid-long pro-tein shares the highest similarity to

Neurosporacrassa

Nrc-1. Deletion of the gene in

A. nidulans

results in a slower growth rate, the formation of morebranched hyphae, altered conidiophore morphology,an inhibition of heterokaryon formation and a blockof cleistothecium development. The gene is transcrip-tionally activated during asexual development andcontrols the phosphorylation of two putative MAPkinases.

Introduction

Mitogen-activated protein kinases (MAP kinases) areubiquitous among eukaryotes. MAP kinases are compo-nents of MAP kinase cascades, which are major signallingmodules by which cells transduce extracellular cues intointracellular responses (Gustin

et al

., 1998; Stork andSchmitt, 2002). Originally, they were described as proteinkinases, which were transiently activated by a variety ofmitogens, including insulin or growth factors, and are thus

implicated in cell proliferation and regulation of the cellcycle. Misregulation in animal cells leads to inappropriateactivation of cell division and may result in the develop-ment of cancer (Schramek, 2002). The basic mechanismof signal transduction appears to be very similar in differ-ent MAP kinase cascades, namely a sequential activationof protein kinases upon external stimulation with a signal.One early kinase after signal recognition is a MAP kinasekinase kinase, which phosphorylates a MAP kinasekinase at two amino acid residues. The latter kinase inturn activates a MAP kinase, again by dual phosphoryla-tion. The phosphorylated MAP kinase triggers the activityof transcription factors, and thus the external signal istransmitted from the surface of the cell into the nucleus.

The evolutionary conservation of MAP kinase signallingpathways allows the use of lower eukaryotes such as

Saccharomyces cerevisiae

or

Schizosaccharomycespombe

as models to unravel the molecular and biochem-ical functions of the components (Herskowitz, 1995). In

S.cerevisiae

, at least five different MAP kinase cascadesexist, which differ in the signals that are perceived andtransmitted, in the activation of the specific MAP kinaseand, ultimately, a specific transcription factor (Gustin

et al

.,1998; Hohmann, 2002). The cascades are involved inmating, nutrient sensing and pseudohyphal growth, osmo-regulation and stress adaptation, cell integrity andascospore formation. Thus, the cellular responses are asdiverse as the induction of mating upon pheromone per-ception and the synthesis of compatible solutes uponosmotic stress. Nevertheless, some signalling moleculesare used in different cascades. Likewise, the MAPKKkinase Ste11 is involved in mating, pseudohyphal growthand osmoregulation. The regulation of the specificity ofeach cascade and the prevention of cross-talk betweenthem are one important and largely unsolved questions(Sabbagh

et al

., 2001). In plant or human pathogenicfungi, MAP kinase cascades are involved in triggering thepathogenic programme and adaptation to the host-specific environmental conditions (Mayorga and Gold,1999; Müller

et al

., 1999; Lengeler

et al

., 2000; Xu, 2000;Mey

et al

., 2002).MAP kinase cascades are involved in major develop-

mental transitions in the life cycle of the unicellular fungus

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Acknowledgements

8 Acknowledgements

This work was done under the supervision of HD Dr. Reinhard Fischer in the Department of

Biochemistry in the Max-Planck-Institute for terrestrial Microbiology (Marburg, Germany) from

August, 2000 to March, 2003. The project was supported by the SFB 395, the Fonds der

Chemischen Industrie, the Deutsche Forschungsgemeinschaft (DFG) and the Max-Planck-

Institute for terrestrial Microbiology.

I would like to deeply thank

HD Dr. Reinhard Fischer for giving me the chance to work as a PhD student on this project,

for his support with ideas and advises, for his help in my working and living in Marburg.

Prof. Dr. Rudolf K. Thauer for providing me a working place and for the excellent conditions

in the Department of Biochemistry at the MPI for Terrestrial Microbiology (Marburg, Germany)

All members of our laboratory and members from PD Dr. Philipp Franken group and our

Department for their help. Sven Konzack, Daniel Mertens, Patricia Rischitor, Matthias Töws,

Kay Vienken, Katrin Koche, Jens Wagner, Prof. Dr. Unai Ugalde, Dr. Milton Roque, Dr.

M'Barek Thamasloukht, Dr. Philippe Rech, Beate Achatz, Ulf Grunwald, Astrid Klüver, Dr.

Daniela Rohdy, Maritha Lippman, Petra Mann. I especially thank Dr. Mario Scherer for

fundamental work and his help in the initial work of my study, and Dr. Niklas Schier, Kay

Vienken for technical help and discussion for my work. I am also indebted to Ralf Liese, Jochen

Scheld and Daniel Stöhr for their technical support. Reinhard Böcher and Dr. Manfred Irmler for

their help with the computer.

In addition, I would like to thank Dr. Steven Groot and Jan Bergervoet at Plant Research

International in the Netherlands for the project in their lab which contributed to come to Germany

to do my PhD work. Dr. Shengli Du, Dr. Dehua Ma and Dr. Haichun Jing in my original Institute

in China for their support.

Curriculum Vitae Name Huijun Wei Birthday 16 Feb. 1968 Nationality Chinese Family Status Married, 1 child Education 09/1987-07/1991 BS, Soil Science and Agrochemistry, Northwestern

Agricultural University, China 09/1991-07/1994 MS, Phytopathology, Northwestern Agricultural

University, China Experience 07/1994-09/1999 Associate Researcher, Tianjin Cucumber Research

Institute, Tianjin, China 10/1999-07/2000 Visiting Scholar, Plant Research International,

Wageningen, The Netherlands 08/2000-present PhD student, Laboratory of Microbiology, Faculty of

Biology, the Philipps University in Marburg, and Max-Planck-Institute for terrestrial Microbiology, Marburg, Germany

Marburg, Germany March, 2003

molecular analysis of sexual sporulation in aspergillus...

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