ISOLATION and CHARACTERIZATION of Pco-1, WHICH ENCODES a REGULATORY PROTEIN THAT CONTROLS PURINE DEGRADATION in NEUROSPORA CRASSA

ISOLATION AND CHARACTERIZATION OF pco-1, WHICH ENCODES A REGULATORY PROTEIN THAT CONTROLS PURINE DEGRADATION IN NEUROSPORA CRASSA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Ta-Wei D. Liu, M.S.

* * * * *

The Ohio State University 2003

Dissertation Committee: Approved by Professor George A. Marzluf, adviser

Professor Caroline Breitenberger Professor Donald H. Dean Professor Venkat Gopalan Department of Biochemistry

ABSTRACT

A feature of the nitrogen regulatory circuit in filamentous fungi is that pathway- specific control genes mediate induction of enzymes by substrates in specific pathways. The gene encoding a new pathway-specific factor involved in purine degradation pathway, pco-1, was isolated from Neurospora using a PCR-mediated

method. The open reading frame of the pco-1 gene is interrupted by two introns

which were identified by comparing the genomic DNA sequence and the cDNA

sequence obtained by RT-PCR. The predicted PCO1 protein contains 1101 amino

acids and appears to possess a single Zn(II)2/Cys6 binuclear-type zinc cluster. A

coiled-coil domain was predicted by computer-aided sequence analysis, suggesting

that PCO1 might function as a dimer. A chemical crosslinking assay indicated PCO1

does dimerize in vitro. Deletion of the coiled-coil domain completely abolished the

activity of PCO1. A loss of function pco-1 mutant was created by the rip procedure.

Analysis of pco1- strains revealed that PCO1 acts as a positive regulator of the purine degradation pathway. Results of mobility shift assays indicate that PCO1 specifically

binds to TCGG-N6-CCGA DNA sequences which exist in promoter regions of the

structural genes it regulates. The C-terminus of PCO1 features two glutamine-rich

regions which are commonly found in activation domains of transcription factors and

a polyglycine stretch. The PCO1 protein with one of the glutamine-rich regions

deleted was still partially functional. Removing both of them completely abolished

the activity of PCO1. This domain shows higher homology to NIT4, the Neurospora

pathway-specific factor in the nitrate assimilation pathway, than to UAY, its

counterpart in Aspergillus nidulans, suggesting transcription factors in N. crassa may

share similar activation regions.

iii

Dedicated to my parents

ACKNOWLEDGMENTS

I wish to thank my adviser, Dr. George A. Marzluf, for his instructions and constant encouragement with enormous patience, which made this dissertation possible. I also appreciate the advisory committee members for invaluable comments and suggestions. Thanks goes to the members of Marzluf’s lab, present and past, for suggestions, discussions, and support.

Special thank goes to all my family members for their consistent support to my research career. I also wish to thank Mr. Aaron D. Carter for helping me to handle various computer problems.

VITA

August 20, 1969...... Born – Taipei, Taiwan

1991...... B.S. Biology, National Chen-Kung University, Tainan, Taiwan

1993...... M.S. Biochemistry, National Yang-Ming University, Taipei, Taiwan

1996 – present...... Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

Research Publication

1. Mihlan M., Homann V., Liu T.D. and Tudzynski B. (2003) AREA directly mediates nitrogen regulation of gibberellin biosynthesis in Gibberella fujiluroi, but its activity is not affected by NMR. Mol. Microbiol., 47, 975-91.

2. Fong J.C., Chen C., Liu D., Tu M., Chai S. and Kao Y. (1999) Synergistic effect of arachidonic acid and cyclic AMP on glucose transport in 3T3-L1 adipocytes. Cell. Signal., 11, 53-8.

3. Liu D., Chen C., Chai S., Ho L. and Fong J.C. (1998) Arachidonic acid and protein synthesis inhibitor act synergistically to suppress insulin-stimulated glucose transport in 3T3-L1 adipocytes. Biochem. Mol. Biol. Int., 46, 681-8.

4. Fong J.C., Chen C.C., Liu D., Chai S., Tu M. and Chu K. (1996) Arachidonic acid stimulates the intrinsic activity of ubiquitous glucose transporter (GLUT1) in 3T3- L1 adipocytes by a protein kinase C-independent mechanism. Cell. Signal., 8, 179-83.

5. Low T.L., Liu D.T. and Jou J.H. (1992) Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver. Arch. Biochem. Biophys., 293, 32-9.

FIELDS OF STUDY

Major Field: Biochemistry

vii

TABLE OF CONTENTS

Page Abstract...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vi

List of Figures...... x

List of Tables ...... xii

Chapters:

1. Introduction ...... 1 The Biology of Neurospora...... 1 Neurospora Transformation and Gene Cloning...... 3 The RIP Phenomenon...... 7 Nitrogen Metabolism ...... 8 The Nitrogen Regulatory Proteins A. NIT2, a Global-acting Positive Regulator ...... 9 B. NMR, a Global-acting Negative Regulator...... 13 C. Pathway-specific Factors in Nitrogen Regulation Circuit ...... 14 Purine Metabolism...... 16 Use of Purines as a Secondary Nitrogen Source...... 17 The Pathway-specific Factors in Purine Catabolism...... 20 Research Goals...... 21

2. Materials and Methods ...... 23 N. crassa and E. coli Strains ...... 23 Neurospora Transformation ...... 24 E. coli Transformation and Plasmid Isolation...... 25 Protein Expression in E. coli and Purification...... 25 Electrophoretic Mobility Shift Assay...... 27 Isolation of Genomic DNA from Neurospora crassa...... 27 Southern Blot Analysis...... 28

viii

Western Blot Analysis...... 29 Pull-down Assay – in vitro Protein-Protein Interaction Study...... 29 RNA Preparation...... 30 RT-PCR cDNA Synthesis and PCR Amplification...... 31 Rapid Amplification of cDNA Ends (RACE) ...... 32 Xanthine Dehydrogenase Assay...... 33 Glutaraldehyde Protein Crosslinking...... 36 RIP Mutagenesis ...... 36 Site-directed Mutagenesis ...... 37 Filter-binding Assay...... 37 Equilibrium Dialysis...... 38

3. Isolation and Characterization of pco-1, which Encodes a Regulatory Protein that Controls Purine Degradation in Neurospora crassa ...... 39 Introduction ...... 39 Results ...... 42 Sequence Homology Search for Zn(II)2Cys6 Proteins...... 42 Disruption of pco-1 and the Phenotype of the Mutant...... 42 Gene Complementation...... 45 Nucleotide Sequence Analysis of pco-1 ...... 48 Determination of the 5’ and 3’ Termini of pco-1 Message ...... 49 Predicted Sequence of the PCO1 Peptide...... 54 DNA Binding Activity of PCO1 Protein...... 60 Binding Site for PCO1 Protein...... 60 Dimerization of PCO1 Protein in vitro...... 63 Deletion Analysis of PCO1...... 69 Detection of Protein-Protein Interaction between PCO1 and NIT2/NMR ...... 72 Ability of pco-1- Mutant Strain to Express Xanthine Dehydrogenase ...... 76 The Involvement of NIT2 and NMR in the Induction of Xanthine Dehydrogenase...... 76 The Homology between PCO1 and UAY Proteins ...... 82 Discussion...... 88

4. Study of Possible Cooperative DNA Binding of NIT2 and NIT4, and Study of Glutamine Binding Property of NMR protein...... 100 Introduction ...... 100 Results ...... 102 DNA Binding of NIT2 and NIT4 to nit-3 Promoter...... 102 Equilibrium Dialysis of NMR Protein against [3H]-Glutamine ...... 110 Discussion...... 113

5. General Discussion ...... 116

References ...... 122

LIST OF FIGURES

Figure Page

1 Neurospora crassa produces ordered tetrad ...... 4

2 General diagram of nitrogen regulatory gene action in N. crassa...... 11

3 Purine degradation pathway...... 18

4 Reactions of xanthine dehydrogenase...... 35

5 Sequence analysis of pco-1 rip mutant...... 46

6 Nucleotide sequence of pco-1 and its flanking regions...... 50

7 Similarity in the Zn(II)2Cys6 DNA-binding region of 15 fungal transcriptional activators ...... 55

8 Prediction of a PCO1 coiled-coil secondary structure...... 58

9 PCO1 fusion protein expression and electrophoretic mobility shift assay (EMSA) ...... 61

10 Identification of PCO1 binding site by EMSA ...... 64

11 PCO1 protein expression and crosslinking...... 67

12 Phenotype analysis of pco-1 deletion mutants ...... 70

13 Protein-protein interaction between PCO1 and the global-acting factors in nitrogen assimilation pathway...... 74

14 Induction of xanthine dehydrogenase by various nitrogen sources...... 77

15 Xanthine dehydrogenase assays of Neurospora nit-2 and nmr mutants...... 80

16 Alignment of PCO1 protein of Neurospora crassa and UAY of Aspergillus nidulans...... 83

17 The glutamine-rich regions in the carboxyl termini of PCO1 and NIT4 of Neurospora ...... 85

18 Proposed model for xanthine dehydrogenase induction ...... 93

19 Recycling of purines by the salvage pathway ...... 95

20 Equilibrium binding of NIT2 and NIT4 to nit-3 promoter ...... 103

21 DNA binding assays of NIT2 protein, with and without NIT4 ...... 105

22 Time course of the dissociation of protein-DNA complexes...... 108

23 Determination of glutamine binding property of NMR by equilibrium dialysis...... 111

LIST OF TABLES

Table Page

1 The ability of pco-1 rip mutant to grow in different nitrogen containing media...... 44

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CHAPTER 1

INTRODUCTION

The Biology of Neurospora

Neurospora crassa is commonly found in warm, humid climates. This fungus had been described in the mid-1800s as a contaminant of French bakeries and was known as the red bread mold. However, the genus Neurospora was first named and characterized by Shear and Dodge in 1927 (Shear and Dodge, 1927). Since then, the genetics and biochemistry of Neurospora, particularly N. crassa have been well studied, and much literature has been published on the topic.

N. crassa is classified in the fungal class Ascomycetes. Vigorous growth can be achieved by supplying a carbon source, mineral salts, and biotin, an essential vitamin. N. crassa can use monosaccharides, such as glucose, mannose, fructose, and xylose; disaccharides, such as sucrose, maltose, cellobiose, and trehalose, and other carbon sources, such as acetate, succinate and glycerol. Ammonium ion is the preferred nitrogen source, though nitrate, nitrite, several amino acids, urea, various amides, and purines can also be utilized. Growth takes place at pH values from 4 to 7.5, and optimal growth occurs between 30 and 35°C (Davis and deSerras, 1970; Metzenberg, 1979).

Neurospora cells are enclosed by double membranes and cell walls. Organelles include mitochondria, an endoplasmic reticulum, peroxisomes, vacuoles, and nuclei. The haploid nucleus contains seven chromosomes, linkage group I - VII, each of which is about the size of the Escherichia coli genome, 27,000 Kb in total (Krumlauf and Marzluf,

1980; Perkins and Barry, 1977). In the vegetative stage, Neurospora grows as mycelia composed of multinuclear, branched filamentous elements called hyphae. The hyphae are divided into compartments by septa with holes. Cytoplasm, along with nuclei and other organelles can flow through the holes. In optimal growth conditions, asexual vegetative growth thrives. Aerial hyphae grow upwards from culture media and bear large orange masses of asexual spores called macroconidia, or simply, conidia.

Macroconidia are multinucleate, containing an average of 2.5 nuclei, and are used for inoculating vegetative cultures or for sexual crossing (Springer, 1993). In addition to macroconidia, Neurospora crassa produces another type of asexual spore, microconidia.

Microconidia are extruded directly from the branched stationary-phase hyphae. They are relatively small in size and germinate with lower efficiency than macroconidia. However, microconidia are important for mutational analysis because of their uninucleate nature

(Lowry et al., 1967; Maheshwari, 1991).

When vegetative growth is not permissible during starvation, such as nitrogen or carbon limitation, the cells switch to sexual reproduction (Westergaard and Hirsh, 1954).

The sexual cycle requires that parents be of different mating type, determined by alternative forms of the genetically complex mating-type region, mat A and mat a

(Metzenberg, 1979). Either strain may serve as a female parent or male parent. The

female parent forms a multicellular fruiting body, the protoperithecium. The male

fertilizing agent is normally a conidium of the opposite mating type. From the gametic

cell in the protoperithecium, one or more specialized hypha called trichogynes emerges

and responds to a pheromone emitted by conidia of the opposite mating type by growing

toward them until contact and cell fusion occurs (Bistis, 1983). Upon fusion, a nucleus of

the conidium is transported into protoperithecium through a trichogyne. The dikaryotic fertilized cell carries out 10 or more rounds of nuclear division but without nuclear fusion

(Raju, 1992). Then the nuclei from both parents pair and undergo two meiotic divisions simultaneously (Iyengar et al., 1977; Rossen and Westergaard, 1966). Each of the four meiotic products goes through another mitotic division and yield eight sexual spores called ascospores packed inside a long, tubelike ascus. Each ascospore can be activated by a short heat shock, and upon germination, a haploid vegetative culture can be obtained.

An especially nice feature about Neurospora crassa is that the results of meiosis are kept compartmentalized in a linear sequence within an ascus (Figure 1) (Beadle, 1946;

Davis and Perkins, 2002). The resulting ascospores are in a linear order that reflects the events in meiosis. Therefore, tetrad dissections (spore isolation in order) of Neurospora asci have been used as a powerful tool in analyzing meiotic segregation and crossing over.

Neurospora Transformation and Gene Cloning

An effective transformation technique is required for many genetic studies. For

example, for direct cloning of genes by complementation of mutant strains and for

functional studies of genes by introducing in vitro manipulated genes back into the cells.

Figure 1. Neurospora crassa produces ordered tetrad. In the sexual cycle,

Neurospora goes through an extra round of mitosis after the two meiotic divisions;

this yields an octad of four spore pairs. Inside an asci, the spores are kept in a linear order inside preserving the events in meiosis.

aa AA

a a A A

a a a a AAAA

Adapted from Davids and Perkins (2002)

The efficient transformation of Neurospora was first achieved by Case in 1979 (Case et

al., 1979) and further modified by Akins and Lambowitz in 1985 (Akins and Lambowitz,

1985). The procedure uses the enzyme preparation, novozyme 234, to partially digest germinated conidia to form spheroplasts. Alternative systems such as lithium ion treatment and electroporation of the conidia have also been successfully used for DNA- mediated transformation (Chakraborty and Kapoor, 1990; Dhawale and Marzluf, 1985).

Unlike E. coli, Neurospora does not have stable, freely replicating plasmids. In

DNA-mediated transformation of N. crassa, DNA integration into genome is generally

non-homologous, and often multiple copies will be inserted (Paietta and Marzluf, 1985;

Timberlake and Marshall, 1989). The transforming DNA may be integrated into the

genome randomly in different ectopic sites. Homologous recombination occurs

infrequently, usually in less than 5% of the transformants (Dhawale and Marzluf, 1985).

Ectopic integration, although much more frequent, can create problems when interpreting

data. Since individual transformants may have varying copy numbers of the transforming

DNA integrated into the genome in different sites, the results are difficult to standardize

and comparison between transformants cannot accurately be made.

To make valid comparisons, a system was developed to select for homologous

recombination, ensuring that the transformed DNA is integrated into the genome in a

particular manner. The system, first described by Ebbole (Ebbole and Sachs, 1990), uses

a Neurospora strain in which the his-3 gene has a point mutation, resulting in a mutated

his-3 product. The his-3 mutant cells cannot grow without supplemental histidine. The

plasmid used for transformation contains a portion of his-3 gene. Only homologous

recombination will restore the functional full-length his-3, and the transformants can be

selected for histidine prototrophy. The vector targets to his-3 locus, thereby

standardizing DNA integration in each of the transformants, and makes accurate

comparisons possible.

The RIP Phenomenon

An important and unusual phenomenon known as RIP (Repeat Induced Point

mutation) has been discovered in Neurospora (Cambareri et al., 1989; Selker, 1990), and

has been proved to be valuable for in vivo studies. The phenomenon is commonly seen in

the cross progeny of normal × ectopic transformants. When a second copy of a host

resident gene is introduced into genome by transformation, both copies of the duplicated

sequence are often heavily mutated during the sexual cycle of N. crassa (Irelan et al.,

1994; Selker, 2002; Selker and Garrett, 1988). The progeny of the cross are selected by

the lack of function for the gene of interest. It is not uncommon that ripped genes in the

progeny’s genomic DNA cannot be visualized in Southern blot analysis using original

DNA sequence as probe. The RIP-induced mutation is characterized by multiple G-C to

A-T transitions in the duplicated segment (Selker, 1990; Selker, 1999). The severity of

the effect is correlated with the length of the ectopic, homologous sequence and its

proximity to another copy. Tandem duplications over a few hundred base pairs are

highly susceptible to the RIP process, while unlinked homologous sequences are affected to a lesser extent (Romano and Macino, 1992).

By performing tetrad dissection, the timing of the RIP process can be determined.

The alterations take place in the nuclear divisions right before nuclear fusion (Selker,

2002).

The mechanism for this phenomenon is yet uncertain, but it was proposed by

Selker that methylation of the C::G pairs followed by deamination of cytosine to uracil might be the cause for G::C to A::T transition (Selker, 1990; Selker, 1999).

Using RIP, a specific DNA sequence can be severely mutated, ensuring that there will be no background activity of the gene in question. By transforming a DNA fragment carrying any gene of interest into wild type Neurospora cells, the resulting strain containing an extra copy of that gene can be obtained, and then crossed with another mating type to inactivate that gene via RIP. Many of the Neurospora knock-out mutants of specific genes involved in this study were created by colleagues and myself in our laboratory using the RIP technique.

Nitrogen Metabolism

A fungus faces two fundamental questions regarding to regulating nitrogen metabolism: what nitrogen sources are available and which of them should be used preferentially? The availability of a specific nitrogen source is usually monitored by regulatory proteins that control the expression of genes that associated with the uptake

and metabolism of a specific compound. These pathway-specific factors are usually

members of the zinc binuclear cluster family (Marzluf, 2001; Todd and Andrianopoulos,

1997). The second regulatory factor, known as nitrogen metabolite repression (NMR),

determines the availability of preferred nitrogen sources and represses the utilization of poorer nitrogen sources, which usually require more energy to be metabolized. In most fungi, these second components are usually transcription factors of the GATA family

(NIT2 and AREA in N. crassa and A. nidulans, respectively) (Lowry and Atchley, 2000;

Marzluf, 2001). These nitrogen regulatory factors act globally, monitor the cell’s nitrogen status, and regulate a large number of structural genes in different pathways.

In most cases, each of the signals mentioned above is not sufficient to activate transcription of a structural gene by itself. Both the global-acting and pathway-specific factors are generally required to achieve expression.

The Nitrogen Regulatory Proteins

A. NIT2, a Global-acting Positive Regulator

Ammonia and glutamine (Gln) are excellent nitrogen sources which fungi prefer

- to use. Other nitrogen sources, such as NO3 , purines, acetamide, various amino acids

+ and proteins, are metabolized to NH4 or Gln for biosynthetic reactions in the organism.

+ The presence of sufficient levels of extracellular NH4 or Gln represses the metabolism of

those alternative nitrogen sources. This is so called nitrogen metabolite repression and

appears to relate directly to intracellular levels of both Gln and Glu (See general diagram,

Figure 2) (Margelis et al., 2001; Morozov et al., 2001; Wiame et al., 1985).

Reinert and Marzluf (1975) showed that the N. crassa regulatory gene nit-2 is

+ required to utilize a wide range of nitrogen sources other than NH4 and Glutamine.

Mutations of nit-2 exhibit the repressed phenotype i.e., the inability to utilize secondary nitrogen sources (Marzluf, 1997; Reinert and Marzluf, 1975).

Genetic studies of nit-2 mutants, which are unable to use several different nitrogen sources and also exhibit a broad loss of nitrogen utilizing enzymes, indicated that nit-2 might encode a positive trans-acting regulatory protein. The molecular cloning and characterization of nit-2 revealed that the nit-2 transcript is present in nitrogen- limited cells and in cells growing in nitrogen-repressed conditions (Tao and Marzluf,

1999).

The NIT2 protein is comprised of 1036 amino acid residues and contains a single

Cys-X2-Cys-X17-Cys-X2-Cys zinc finger DNA binding domain homologous to the mammalian GATA factors’ zinc finger domain (Tsai et al., 1989). It has two acidic and basic regions which might be relevant for transcriptional activation (Fu and Marzluf,

1990a). Mobility-shift assays and footprinting experiments with recombinant NIT2 zinc finger domain fusion protein revealed three potential NIT2 binding sites in the promoter region of nit-3 gene, which encodes nitrate reductase, with a core consensus sequence

TAGATA (Fu and Marzluf, 1990b). Site-directed mutagenesis studies in the zinc-finger motif and the immediately downstream basic region showed that these two regions together constitute a DNA-binding domain, and both regions are critical for transactivating function in vivo and specific DNA-binding in vitro (Fu and Marzluf, 1990c).

Pathway-Specific Grobal-Acting Nitrogen Repression (+) (+) (-)

Glutamine

+1 NIT-4 NIT-2 sites sites nit-3 Nitrate Reductase

Figure 2. General diagram of nitrogen regulatory gene action in N. crassa.

Further mutation studies indicated that specific amino-acid residues within the NIT2

zinc-finger loop region are important for DNA-binding activity, while other residues

affect the specificity for DNA binding site recognition (Xiao and Marzluf, 1993).

The counterpart of nit-2 gene in A. nidulans is areA (Arst and Cove, 1973). Both

genes appear to function in a similar manner (Marzluf, 2001). A wide range of mutations

has been isolated in areA gene. Loss of function mutations areA- leads to nitrogen-

repressed phenotype (Arst and Cove, 1973). The second class exhibits a gain of function,

mutants show a differentially altered function so that they appeared derepressed for some

specific activities and repressed for others (Arst and Scazzocchio, 1975; Hynes and

Pateman, 1970). In addition, a few dominant mutations were also identified, which lead

to derepression of most activities under areA regulation, i.e., the mutant strains could

+ express genes involved in various metabolic pathways even in the presence of NH4 and

Gln (Cohen, 1972). Such a wide range of mutations has not yet been isolated in the nit-2 locus.

Extensive deletion analysis has revealed the activation domains of NIT2 and

AREA. In NIT2 there are two acidic domains. Internal deletions that disrupt either lead to partial loss of function while the deletion of both domains fully disrupts NIT2 activity

(Pan et al., 1997). An acidic region has also been identified in AREA, and deletion of this domain led to loss of function (Kudla et al., 1990). However, in Aspergillus oryzae, another fungus has been used for centuries in the fermentation industry, homologous protein deleted of an acidic domain similar to that of NIT2 and AREA retained significant biological function (Christensen et al., 1998). These studies of both NIT2 and

AREA suggest that various regions within each are involved in activation. However, there are also considerable segments of the NIT2 and AREA protein which can be deleted with little or no affect upon the protein function.

B. NMR, a Global-acting Negative Regulator

A second global regulatory protein in N. crassa is NMR. Unlike the null phenotype of nit-2 mutants, mutations of nmr were selected on the basis of derepressed nitrate reductase activity in the presence of Gln (Premakumar et al., 1980; Tomsett et al.,

1981). Disruption of the nmr gene yielded strains which were insensitive to metabolite repression, resulting in constitutive expression of various nitrogen catabolic enzymes under repressed conditions (Jarai and Marzluf, 1991). Molecular cloning and characterization of nmr gene revealed that nmr is expressed constitutively. The transcript is about 1.8 kb, yields a polypeptide of 488 amino acid residues (Fu et al., 1988). NMR has been shown to bind to NIT2 using the yeast two-hybrid system as well as pull-down assays in vitro (Pan et al., 1997; Xiao et al., 1995). Two α-helical regions of NIT2, one located within the NIT2 zinc finger, directly interact with NMR (Pan et al., 1997). The analysis of nmr mutants has failed to identify any domain within the NMR protein that is related to the negative regulation. It has been suggested that the NMR protein interferes with the DNA-binding or activation ability of NIT2 (Xiao et al., 1995), and inhibits the transactivating function of NIT2 (Figure 2). Thus, structural genes which encode nitrogen assimilatory proteins are not expressed during nitrogen repression, a time when their products are not needed.

An NMR homologue in A. nidulans, NMRA, which shows ~60% identity to

NMR at the amino acid level has been identified (Andrianopoulos et al., 1998). The X- ray crystal structure of NMRA reveals similarity to the short-chain dehydrogenase/ reductase family (Stammers et al., 2001). To date, how NMR monitors the nitrogen status of the cell and the actual mechanism in establishing nitrogen repression is still not completely understood.

C. Pathway-specific Factors in Nitrogen Regulation Circuit

Besides the global positive and negative factors that mediate nitrogen metabolite repression, expression of the enzymes and transporters needed for utilizing secondary nitrogen sources also requires specific induction in the particular pathway. This induction signals the availability of a particular compound. One of the best-characterized pathway-specific factors is NIT4, which is required for using inorganic nitrate as a nitrogen source. Nitrate uptake is mediated by nitrate permease. Inside the cell, nitrate is first converted into nitrite by nitrate reductase, and then nitrite reductase further reduces nitrite to ammonium. It was found that both a functional nit-2 gene and nit-4 gene are essential for expression of nitrate assimilation enzymes, since mutations at either locus completely abolished the expression of nitrate and nitrite reductase (Fu and Marzluf,

1988; Marzluf, 1997).

Unlike the global factor NIT2, NIT4 is only required for the nitrate assimilation pathway. NIT4 encodes a polypeptide of 1090 amino acid residues. A putative GAL4 type Zn(II)2Cys6 zinc cluster DNA binding domain was identified in the N-terminal of

NIT4. Mutagenesis analysis revealed the zinc cluster is essential for NIT4 function.

Substitution of cysteine residues or certain basic amino acids in zinc cluster completely

eliminated NIT4 function (Yuan et al., 1991). A coiled-coil dimerization region was found immediately after the zinc cluster. Crosslinking assay indicates NIT4 forms homodimers in vitro (Fu et al., 1995).

The C-terminus of NIT4 contains a glutamine rich region and a polyglutamine stretch, a feature commonly observed in transcription factors such as SP1 (Courey and

Tjian, 1988; Feng and Marzluf, 1996). The transactivation function of NIT4 C-terminal was confirmed with the yeast one-hybrid system. Fusion of NIT4 C-terminal regions to the GAL4 DNA binding domain restores GAL4 activation function in yeast. In addition, a novel leucine-rich, acidic domain was also identified in the C-terminal of NIT4, which functions as a minimal activation motif in yeast cells (Chiang and Marzluf, 1995). These

results support the positive role played by NIT4 in regulating the nitrate assimilation pathway. NIRA, the counterpart of NIT4 in Aspergillus, shows significant sequence

similarity to NIT4. In evidence obtained so far, both proteins share similar features and

characteristics, such as DNA-binding properties and dimerization (Feng et al., 1995;

Strauss et al., 1998).

The studies of nit-3 promoter region in N. crassa revealed two elements that serve

as the binding site for NIT4, and several elements for the global-acting NIT2 binding.

All of the binding sites are required for full expression. It is particularly significant that

neither NIT2 nor NIT4 alone allows any detectable transcription of the nit-3 gene. Only

when both functional NIT2 and NIT4 are present, could nit-3 be turned on and result in

high level of expression (Chiang and Marzluf, 1995; Fu and Marzluf, 1987). Specific protein-protein interaction between NIT2 and NIT4 has been proven to be required for optimal expression of nit-3 gene. Besides NIT2, at least five other GATA factors have been identified in Neurospora with overlapping DNA binding activities, yet each controls a distinct set of structural genes. It has long been questioned how the specificity of each

GATA factor is established. The essential interaction between NIT2 and NIT4 for nit-3 gene expression strongly suggests that pathway-specific factors play a role in defining the specificity of each GATA factor through protein-protein interaction (Feng and Marzluf,

1998; Marzluf, 2001).

Purine Metabolism

Purines are the foundational molecules of nucleotides; therefore they are of particular importance in many biochemical processes. (1) They are the precursors of

DNA and RNA. (2) They are the building blocks of high-energy intermediates ATP and

GTP. ATP is a universal currency of energy in all known life forms, while GTP powers many movements of macromolecules. (3) Adenine nucleotides are components of the major coenzymes such as NAD+, FAD, and CoA. (4) In signal transduction pathways, cyclic AMP and its analog cyclic GMP are common mediators and involve in many hormonal functions (Munch-Petersen, 1983).

The importance of purines in cellular metabolism is indicated by the observation that nearly all cells can synthesize them from basic compounds. However, de novo synthesis of purines is complicated and expensive in terms of energy cost and the number

of enzymes involved. Therefore, purines are recycled from the degradation products of

nucleic acids by a salvage reaction. Free purine bases are formed by the hydrolytic

degradation of nucleic acid and nucleotides. New nucleotides can be synthesized directly

from these preformed bases thus yielding a saving in energy and resource (Murray, 1971;

Zalkin and Dixon, 1992).

Use of Purines as a Secondary Nitrogen Source

Filamentous fungi Neurospora and Aspergillus are able to use purines as a nitrogen source when other preferred sources are not available. As discussed above, purines play important roles in all cells, and their metabolism is another example of a highly regulated nitrogen catabolic pathway in fungi (Diallinas and Scazzocchio, 1989;

Lee et al., 1990; Nahm and Marzluf, 1987; Reinert and Marzluf, 1975; Suarez et al.,

1991a; Suarez et al., 1991b). This catabolic process is accomplished in a multi-step

pathway as follows: hypoxanthine → xanthine → uric acid → allantoin → allantoate →

urea and glyoxylate → ammonia (Figure 3) (Reinert and Marzluf, 1975; Scazzocchio and

Darlington, 1968). Xanthine dehydrogenase (XDH; EC 1.2.1.37) is of particular

importance since it catalyzes the first two steps of purine degradation. XDH will mediate the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. In N. crassa,

XDH, as well as other enzymes in this pathway are subject to regulation at the level of

enzyme synthesis. Xanthine dehydrogenase activity increases at least six fold in wild-

type N. crassa mycelia grown on uric acid, compared to XDH levels in mycelia grown on

4 NH Xanthine Dehydrogenase Allantoicase Xanthine Dehydrogenase Guanine Uricase Allantoinase Adenylyate Deaminase Figure 3. Purine degradation pathway. Purine degradation 3. Figure

ammonium as a sole nitrogen source. In addition to this unusual induction by the product, uric acid, XDH synthesis is also subject to nitrogen repression by ammonium or glutamine, a phenomenon termed nitrogen metabolite repression (Marzluf, 1981; Perkins et al., 1982; Reinert and Marzluf, 1975).

Xanthine dehydrogenase exhibits a wide range of substrate specificity, oxidizing purines, aldehydes, pteridines, and NADH. The enzyme is also able to employ a variety of electron acceptors including molecular oxygen, methylene blue, cytochrome c, and

NAD+, as well as prosthetic groups including nonheme iron/sulfur centers, flavin, and

molybdenum (Lyon and Garrett, 1978a; Massey et al., 1969).

Xanthine dehydrogenase has been isolated from N. crassa and purified to

homogeneity. The native enzyme (estimated molecular weight, 357,000) upon sodium

dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a single

band of molecular weight 155,000, suggesting that the holoenzyme is a homodimer

(Lyon and Garrett, 1978a). The molybdenum found in the pterine cofactor in xanthine

dehydrogenase is common to nitrate reductase (Pateman et al., 1964), sulfite oxidase,

formate dehydrogenase, and aldehyde oxides (Nason et al., 1970; Nichol et al., 1985).

Mutational analysis of N. crassa by selecting for strains which grow on uric acid but not

hypoxanthine as the sole nitrogen sources identified a locus, xdh-1 (Reinert and Marzluf,

1975). Subsequent studies established that xdh-1 mutants lack xanthine dehydrogenase

activity (Tomsett and Garrett, 1981).

The Pathway-specific Factors in Purine Catabolism

In A. nidulans, a pathway specific regulatory protein UAY has been identified

(Philippides and Scazzocchio, 1981; Sealy-Lewis et al., 1978). The uaY gene is needed

for the induced expression of at least nine unlinked gene coding for enzymes or

permeases involved in purine catabolic pathway, including adenine deaminase, xanthine

dehydrogenase, urate oxidase, allantoinase, allantoicase, and xanthine-urate permease

(Scazzocchio and Darlington, 1968; Scazzocchio et al., 1982). The product of uaY gene

is a positive acting transcription factor that mediates induction by uric acid, which is the

only physiological inducer in this pathway (Scazzocchio, 1973; Sealy-Lewis et al., 1978).

The polypeptide derived from cDNA nucleotide sequence indicates that UAY protein

contains a typical zinc binuclear cluster domain and a dimerization domain; these features

resemble the Saccharomyces cerevisiae transcription factor PPR1, which regulates

pyrimidine biosynthesis (Suarez et al., 1995a).

Uric acid and some analogues (e.g., 2-thiouric acid) induce UAY activity. The

other purines induce the system indirectly through uric acid to a lower extent. The uaY

gene is constitutively expressed and the protein binds to a consensus sequence TCGG-N6-

CCGA. The same consensus binding element is shared by the transcription factor PPR1

of S. cerevisiae. All genes controlled by the UAY protein are also subject to nitrogen

metabolite repression, i.e., are repressible by ammonium and glutamine. The GATA-type

transcription factor coded by the areA gene is required for efficient induction. Sequences that conform to the UAY recognition element have been found in the promoters of at

least five genes regulated by UAY, hxA, hxB, uapA, uapC and uaZ (Amrani et al., 1999;

Suarez et al., 1995a). Detailed studies have shown that the UAY mediated induction of

uapA (coding for urate-xanthine permease) and uaZ (coding for urate oxidase) is

completely dependent on a functional areA gene product (Gorfinkiel et al., 1993;

Oestreicher and Scazzocchio, 1993).

Research Goals

I have been focusing on understanding nitrogen regulation in Neurospora. My research is constituted of two parts. The first goal of my research was to identify and clone a predicted pathway-specific factor for purine metabolism in Neurospora, and

examine its characteristics and biological role. The project began with a search for other

possible Zn(II)2Cys6 zinc clusters in the genomic database of Neurospora, and a possible

locus, pco-1, was identified. The cDNA sequence of pco-1 was obtained by RT-PCR in

which two introns were identified. The encoded protein reveals similarity to the UAY

protein which regulates purine metabolism in A. nidulans. A pco-1- knock-out strain was

created by rip mutagenesis and the phenotype indicates the mutant is impaired in utilizing

purines as nitrogen sources. In vitro studies were also conducted to characterize this protein. The possibilities of dimerization, DNA binding properties to promoters, and protein-protein interactions with other factors were examined.

The second part of my research was to further examine the regulatory circuit on the induction of nitrate reductase. The requirement of both DNA binding proteins NIT2 and NIT4 in the expression of nit-3 gene raises the question whether these proteins bind to the nit-3 promoter independent of each other, or whether the interaction between the

two increases their respective DNA binding affinity in a synergistic/cooperative manner.

A filter-binding assay was used to test DNA binding when both proteins were present.

Another question I investigated was the mechanism by which NMR protein monitors the nitrogen status of the cell. I tested the possibility that the NMR protein is a glutamine sensor by using equilibrium dialysis. The results indicated NMR protein does not bind glutamine.

CHAPTER 2

MATERIALS AND METHODS

N. crassa and E. coli Strains

The Neurospora crassa wild type strain, 74-OR8-1a (FGSC #988) and the his-3- mutant strain Y234M723 (FGSC #6103) used for targeted transformation, were obtained from the Fungal Genetics Stock Center, Kansas City, Kansas. All the other mutant strains used in this study were created previously in the laboratory. The wild type strain, and the pco-1 rip, nmr rip, and nit-2 rip mutants were grown on Vogel’s minimum medium. The his-3- mutant was grown on Vogel’s minimum medium plus histidine.

The culture techniques, growing and sexual crossing of Neurospora strains were according to Davis and deSerres, 1970 (Davis and deSerras, 1970).

Escherichia coli DH5α (Invitrogen) and TOP10 (Invitrogen) were used for plasmid propagation, and BL-21(DE3) pLysS (Stratagene) was used for protein expression.

Neurospora Transformation

The polyethylene glycol (PEG) protoplast method was used for all transformation

experiments with Neurospora. For preparation of protoplasts, conidia were obtained

from cultures grown in 500 ml flasks containing solid Vogel’s medium. Conidia were harvested from 7-10 day old cultures by filtration. After washing with water, the conidia were resuspended in 1X Vogel’s medium with proper supplements and shake at 30°C for

4-6 hours to germinate. Germinated conidia were collected and resuspended in 20 ml of

1 M sorbitol containing 4-20 mg of Novozyme 234. After incubating at 30°C for 20-40 minutes, protoplasts released were collected and washed twice with 1 M sorbitol and once with STC buffter (1M sorbitol, 50 mM Tris, pH 8.0, and 50 mM CaCl2). Protoplasts

were finally resuspended in 15 ml STC. 200 µl of DMSO and 4 ml of PTC (40%

PEG4000, 50 mM Tris, pH 80, and 50 mM CaCl2) were added. After thorough mixing,

protoplasts were aliquoted and stored at –70°C or used immediately. The critical step in

preparation of protoplast competent for transformation is the digestion by Novozym 234.

Over digestion or under digestion both compromise the quality of the protoplast for high

transformation efficiency.

Transformation was accomplished by incubating 1.0 µg of plasmid DNA with 25

µg of heparin in a total volume of 10 µl. Protoplasts (100 µl) were then added and the

mixture was incubated on ice for 30 minutes. Then, 1.0 ml of PTC was added and the

sample was incubated at room temperature for another 20 min. The mixture was then

resuspended in 10-15 ml top agar (500 mM MgSO4, 0.05% fructose, 0,05% glucose, 2%

sorbose, 0.6% agar, and 1x Vogel's minimal medium supplemented with biotin, amino acids or antibiotics) prewarmed to 55°C and immediately poured onto petri dishes containing solid sorbose Vogel’s medium with proper supplements or selective agent.

The composition of this medium was identical to the top agar except that the

concentration of agar is 1.5% and no MgSO4 was added. These plates were incubated at

30°C and colonies that represent the transformation products were observed after three

days.

E. coli Transformation and Plasmid Isolation

Competent E. coli cells were prepared following the standard procedure in

Molecular Cloning by Sambrook et al. (Sambrook et al., 1989). Transformation was

accomplished by incubating 0.1 µg plasmid DNA and the competent cell mixture on ice

for 30 min. Then the mixture was heat shocked at 42°C for 90 sec, followed by

incubation at 37°C for 1 hour in SOC medium. The cells were spread on LB plates with

ampicilin selection.

Isolation of plasmid DNA was done by the alkaline method as described by

Sambrook et al. (Sambrook et al., 1989), with an additional RNaseA digestion of the

DNA samples.

Protein Expression in E. coli and Purification

The expression of the HA / His6-tagged PCO1 proteins was done using the pHB6 vector (Roche) and the BL-21(DE3) pLysS E. coli strain. To express PCO1 or NMR

GST fusion proteins, the relevant gene was subcloned into the pGEX-2T or 3X vector

(Pharmacia). His6-tagged NIT2 was expressed using pRSET-A from Invitrogen. Fresh

transformants were inoculated into 5 ml of LB medium supplemented with 50 µg/ml of

ampicillin and incubated overnight at 37°C. The culture was then added to 250 ml of

fresh LB medium containing ampicillin and incubated at 37°C until the OD600 reached

0.4, which usually took 3-6 hours. The inducer, IPTG, was then added to a final concentration of 0.5 mM. After further incubation for 6 hours, the cells were collected by centrifugation and resuspended into buffers appropriate for the affinity resins. The cells were lysed by sonication, with microscopic inspection to insure breakdown of the majority of the cells. After centrifugation at 15,000 x g for 20 min, the supernatant was collected for affinity purification. For His6-tagged proteins, the Ni-NTA (nickel-

nitrilotriacetic acid) agarose resin (Qiagen Cat No.1000630) was used according to the

manufacturer’s instructions. Elution of the bound protein was accomplished with 250

mM imidazole. For GST fusion proteins, a glutathione-agarose resin (Sigma Cat. No. G-

4510) was added to the supernatant and incubated on ice for 0.5-2 hours with slow

agitation to keep the resin in suspension. The resin was then washed three times with the binding buffer (potassium phosphate, pH 7.0, 1% NaCl, 5 mM -mercaptoethanol, 1 mM

PMSF). The bound protein was eluted with Tris-HCl pH 8.0 containing 5 mM reduced

glutathione.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays (EMSA) were performed according to Hope and Struhl (Hope and Struhl, 1987)with modifications. Purified proteins were prepared as described above. Radioactive DNA probes were 3’ end-labelled with DNA

Polymerase I Klenow fragment and [α-32P] dNTP, depending on the specific 3’-terminal sequence of the DNA fragment. The binding reaction contained the end-labeled DNA probe, 2 µg poly(dI:dC) and 0.10 – 2.0 µg of protein in binding buffer (12 mM Hepes pH

7.9, 2 mM DTT, 3 mM MgCl2, 0.5 mM EDTA and 50 mM KCl, 15% glycerol). The mixture was incubated at room temperature for 25-30 minutes, and then loaded onto a 5

% polyacrylamide gel and electrophoresed in 0.25x TBE buffer. The gel was then dried and exposed to an X-ray film (Kodak BioMax MS).

Isolation of Genomic DNA from Neurospora crassa

DNA samples used for PCR and Southern analysis were prepared using the method developed by Leach et al, 1986 (Leach et al., 1986). Briefly, a loopful of conidia was inoculated into 5-20 ml of Vogel's complete medium and incubated overnight by shaking at 60 rpm. The mycelia were collected and placed into a 13 x 100 mm glass test tube. 0.7 ml of LETS buffer (0.1M LiCl, 10 mM EDTA, pH 8.0, 10 mM Tris-Cl, pH 8.0,

0.5% SDS) and 1 g of glass beads were added, and the tube was vortexed at top speed for

1-2 minutes. One ml of PCA (phenol:chloroform:isoamyl alcohol, 25:24:1) was then

added and vortexed for an additional 20 seconds. The aqueous layer containing the DNA

was separated from the organic phase by centrifugation, and the DNA was then

precipitated by ethanol and dissolved in 40 µl TE buffer.

Southern Blot Analysis

Genomic DNA was digested with selected restriction enzyme(s) and fractionated

using agarose gel electrophoresis. After electrophoresis following standard procedures,

(Sambrook et al., 1989) the DNA in the gel was denatured in 1.5 M NaCl / 0.5 M NaOH

for 30 min with gentle shaking. Then the gel was neutralized with 1.5 M NaCl / 1.0 M

Tris-HCl, pH 8.0 with one change of buffer.

Transferring DNA from the gel to a nylon filter (Hybon d-N, Amersham Biotech) was accomplished with 10x SSC as the transfer buffer. The derived nylon filter was UV cross-linked and treated with pre-hybridization buffer (50% deionized formamide, 6x

SSC, 0.25% dehydrated milk) for 2-5 h. In the meantime, a radioactive DNA probe was prepared as described above in the EMSA procedure. After the labeled probe was added, hybridization was performed at 42°C overnight in a rotary incubation oven. The blot was rinsed twice in 2x SSC with 0.1% SDS for 10 min at room temperature with gentle shaking. The blot was then washed with 0.1x SSC and 0.1% SDS at 68°C until the background radioactivity of the filter was very low, as checked with a Geiger counter.

The filter was wrapped with Saran wrap and exposed to X-ray film at -70°C overnight or longer, depending on the strength of the signal on the filter.

Western Blot Analysis

Western blots were done according to the procedure in Current Protocols in

Molecular Biology (1990). Protein samples were first resolved in an SDS-PAGE gel.

Transfer was carried out using the Bio-Rad TransBlot Cell apparatus and power supply

(Model 160, Bio-Rad Labs) for 2 hours at 65 volts or overnight at 25 volts. A nitrocellulose membrane was used for the blotting. After transfer, the nitrocellulose filters were blocked for one hour at room temperature or overnight at 4°C using 5% dried non-fat milk in TBS-T buffer, following the manufacture’s instructions of the Enhanced

Chemiluminescence (ECL) Kit (Amersham Corp). After three washes with TBS-T buffer, the rabbit primary antibody diluted in TBS-T was added and incubated at room temperature for 1 hour. The dilutions used in this research were 1:1,000 for anti-NIT2 monoclonal antibody, 1:500 for Anti-HisG (Invitrogen Cat. No. R940-25), and 1:3300 for anti-hemagglutinin (HA) antibody 3F10 (Roche Cat. No. 1867423). After three washes, an anti-rabbit horseradish peroxidase-conjugated secondary antibody was added at a

1:2,000 dilution and the incubation was continued for one additional hour. After extensive washing, the ECL Kit was used to reveal specific bands. The results were visualized by exposing to Kodak BioMax MS film.

Pull-down Assay – in vitro Protein-Protein Interaction Study

To investigate a possible interaction between PCO1 and the global-acting regulatory proteins NIT2 and NMR, GST fusion proteins were used.

In the study of a potential PCO1-NIT2 interaction, the gene encoding PCO1 N-

terminal 220 residues was fused to GST in the pGEX-2T vector, expressed in BL-21(DE3) pLysS, and purified as described previously. The gene encoding NIT-2 was cloned into the pRSET vector, expressed, and the His6-NIT2 fusion protein was purified.

Approximately 50 µl of glutathione-agarose resin was used for each interaction assay.

The resin was equilibrated first with PBS buffer, and incubated with purified GST-PCO1

dissolved in PBS buffer at 4°C for 20 min. After the GST-PCO1 was absorbed to the

resin, purified NIT-2 protein was allowed to interact in a microcentrifuge tube at 4°C for

2 hours. The resin was washed three times with PBS buffer. Proteins bound to the resin

were then eluted as described earlier. The protein samples were analyzed by Western

blot using Anti-HisG (Invitrogen, Cat. No. R941-25) to detect the presence of His6-NIT2.

In the study designed to examine a possible PCO1-NMR interaction, the protein-

protein binding and Western blot procedures were identical to the ones used in PCO1-

NIT2 interaction. Full-length NMR was expressed as a GST fusion protein; the truncated

gene encoding PCO1 1~220 residues was cloned into the pHB6 vector and expressed as a

fusion protein with an N-terminal hemagglutinin (HA) epitope and a His6 tag on carboxyl

terminus. The antibody utilized in Western blot analysis to detect PCO1 was the anti-HA

antibody 3F10 from Roche.

RNA Preparation

Conidia were used to inoculate liquid cultures in Vogel’s medium which were

incubated in a reciprocal shaker at 37°C for 24-30 h. Mycelia were transferred to fresh

Vogel’s medium containing uric acid 5 mM as the sole nitrogen source and incubated for

6 h. The mycelia were ground to a fine powder with a mortar and pestle in the presence

of liquid nitrogen, and total RNA was extracted with the Trizol reagent (Invitrogen). To

verify the RNA integrity, 10 µg of RNA from the preparation was then fractionated in an

agarose gel, stained with ethidium bromide and then visualized with UV light. The

presence of intact 28S and 18S rRNA bands was used as the indication of RNA integrity.

The RNA concentration was calculated by the OD 260/280 absorbance.

RT-PCR cDNA Synthesis and PCR Amplification

The primers used in RT-PCR and PCR reactions were designed from the genomic

DNA sequence with the information of putative introns predicted by NetGene Server

(The Technical University of Denmark). To determine the locations of the two putative introns, two gene specific primers PCORT and PCORT2 were used for first strand cDNA synthesis. Two pairs of primers were then used in PCR reactions to amplify the cDNA.

The sequences of the primers were as follows:

Primers for determining the first intron: PCORT 5’- TACCGACGACTGCACCGCAG -3’ PCOPCR1-F 5’- CGTAGGATCCATGCCGGCGCACACTCCTCCGTCCC -3’ PCOPCR2-R 5’- GTCAGAATTCCCGAGGTAGATCCCAGATAGCGAGC -3’

Primers for determining the second intron: PCORT-2: 5’- GTTGCTGGTGCTGTTGGATG -3’ PCOPCR-8F 5’- GTGGGCTTGGTCAACTATACC -3 PCOPCR-8R 5’- GATCAATGGAGGTCTCGCCTAC -3’

The RNA preparation was first treated with DNaseI to remove any DNA contamination before RT-PCR. A 20 µl reaction mixture for reverse transcription

contained 5 µg of total RNA, 1x first strand buffer, 10 mM dithiothreitol, 0.5 mM dNTP

(each), 10 pmol of PCORT or PCORT2 primer, and 40 units RNaseOUT Recombinant

Ribonuclease Inhibitor (Invitrogen). Before addition of SuperScript II reverse

transcriptase (Invitrogen), the mixture was incubated at 72°C for 10 min and rapidly

cooled on ice. After adding reverse transcriptase, the mixture was incubated for 1 h at

42°C. Then, 3 µl of the RT-PCR reaction mixture was used as template for PCR

amplification with Platinum Taq Polymerase (Invitrogen). The reaction mixture for PCR

contained 1x PCR buffer, 0.3 mM dNTP (each), 1.5 mM MgCl2, primers (0.2 µM each),

and Platinum Taq polymerase (5 units) in a total volume of 50 µl. PCR amplification was done for 35 cycles under the following condition: after an initial 2 min at 94°C to activate the antibody-bound polymerase, denaturation was at 94°C for 30 sec, annealing

at 63°C for 30 sec, and extension at 72°C for 1 min 30 sec. The amplified DNA

fragments were analyzed on an agarose gel.

Rapid Amplification of cDNA Ends (RACE)

Total RNA from mycelia of wild type strain 74-OR8-1a was isolated with Trizol

(Invitrogen) and mRNA was purified with an Oligotex mRNA mini kit (Qiagen).

To obtain full-length 5´ ends of the pco-1 cDNA, RNA-ligase-mediated rapid

amplification of cDNA ends (RLM–RACE) was performed with the GeneRacer kit

(Invitrogen). This kit ensures the amplification of only full-length transcripts by

eliminating truncated message from the amplification process. The procedure was

performed as described in the manufacturer's protocol by using mycelia mRNA and a

gene specific primer PCORT downstream of the translation start site 5’-

TACCGACGACTGCACCGCAG -3’. To eliminate the possibility of artifacts, nested

PCR was performed to increase the specificity and sensitivity of RACE products.

GeneRacer 5’ Nested forward primer and a gene-specific reverse nested primer

PCO5TR-R 5’-CAGACCCGTCAAGTAGAAGCTC-3’ were used in the nested PCR reaction with 1 µl of the original amplification reaction as a template. DNA fragments resulting from the RACE PCR were analyzed by electrophoresis on an agarose gel, cloned with a TOPO TA Cloning kit (Invitrogen) and sequenced.

In 3’-RACE, the GeneRacer Oligo dT primer was used to prime the first strand cDNA synthesis in the RT reaction. The cDNA fragments were amplified by PCR using

a gene specific primer PCO3TR-FL 5’-CATAGCAACGCAAGCTCCATTGCG-3’ and

GeneRacer 3’ primer. Nested PCR was also performed to enhance specificity by using

PCO3TR-FS 5’-GGGACAGATGGATCTAGGCTTTGG-3’ and GeneRacer 3’ Nested

reverse primers. The analysis and sequencing of 3’-RACE products were done in the

same manner as in 5’-RACE.

Xanthine Dehydrogenase Assay

Neurospora mycelia were prepared by growing in complete Vogel’s medium

overnight. The culture was then transferred to minimal medium with appropriate

supplements and incubated for 6 h. The mycelia were harvested by filtration, thoroughly

washed, and stored at -80°C. To prepare crude cell extract, frozen mycelia were

homogenized with 0.1 M sodium phosphate buffer, pH 8.0 (1.2 ml/g mycelia) in a bead beater (Bio-Spec Products, 4200 rpm, 30 sec x 2 at 4°C). Phenylmethylsulfonylfluoride

(PMSF) (2 mg/ml) was added prior to homogenizing to inhibit proteolytic digestion. The homogenate was centrifuged at 15,000 x g for 20 min at 4°C. Since xanthine dehydrogenase displays the phenomenon of excess substrate inhibition, the supernatant recovered was passed over a Sephadex G-25 column that had been equilibrated with 0.1

M sodium phosphate buffer, pH 8.0 to remove any residual purines. Determination of

protein concentration was done using the BioRad protein reagent with bovine serum

albumin as the standard. Aliquots of extract were stored at -80°C.

A fluorometric xanthine dehydrogenase assay, described by Glassman and

Mitchell (Glassman and Mitchell, 1958)and modified by Lyon and Garrett, (Lyon and

Garrett, 1978b) was used to determine the enzymatic activity. The reaction is based on the oxidation of pteridines, which are purine homologues, to fluorescent molecules

(Figure 4).

The assay was done in borosilicate test tubes with 0.1 M sodium phosphate buffer, pH 8.0, 10 µM 2-amino-4 hydropteridine, 200 µM of NAD+, and 50 µl of crude extract in

a total volume of 2 ml. The rate of fluorescence production was measured by a

spectrofluorometer, (FluoroMax-3, Jobin Yvon Ltd. USA) which provides the index of

XDH activity. Excitation wavelength was set at 326 nm and emission wavelength at 412

nm. XDH oxidizes 2-amino-4-hydroxypteridine to isoxanthopterin, which is fluorescent.

Figure 4. Reactions of xanthine dehydrogenase.

Glutaraldehyde Protein Crosslinking

Samples of a region (N-terminal 1-220 a.a.) of the PCO1 protein expressed in pHB6

vector were incubated with 0.005% Glutaraldehyde (Sigma G-6257) at room temperature

in 40 µl of potassium phosphate buffer, pH 7.0. After various reaction times, 10 µl of 5x

SDS-PAGE sample loading buffer was added to each sample, and the mixtures were

heated at 95°C for 5 minutes prior to loading into SDS-PAGE gels. The protein bands

were visualized by using Simply Blue SafeStain (Invitrogen Cat. No. LC6060).

RIP Mutagenesis

A 2.0-kb DNA fragment carrying the pco1 gene from the beginning with its

coding region was linked to a truncated his-3 gene in the pDE I vector. The construct was transformed into his-3- spheroplasts. Only the transformants in which the plasmid

DNA targeted to the his-3 gene locus can grow in the selective medium without histidine.

Genomic DNA was isolated from the putative transformants and Southern blots were used to check the copy number of the introduced DNA fragment in each transformant. A transformant which had one copy of the targeted DNA fragment was crossed with a wild type strain of the opposite mating type. Ascospores were collected from cultures after three weeks at 30°C, heat treated at 68°C for 30 min and spread on Vogel’s medium after dilution. Single colonies were picked and tested for growth on different media to determine their phenotype (Davis and deSerras, 1970; Singer et al., 1995).

Site-directed Mutagenesis

PCR based mutagenesis was performed with the QuickChange XL site-directed mutagenesis kit (Stratagene). To delete the putative coiled-coil region of the pco-1 gene in the pDE1 plasmid, the following oligonucleotides (single-stranded sense and anti- sense oligonucleotides) were used as primers: 5'-

CGTGCTGTCCCAGTTATATCAACAATATCACTTTCCCG-3' and 5'-

CGGCGGGAAAGTGATATTGTTGATATAACTGGGACAGC-3'. In accordance with the manufacturer's instructions, the PCR reactions were performed with wild-type pco-1 gene as template; the PCR products, digested by DpnI, were used to transform E. coli.

The presence of expected mutations was verified by sequencing. Plasmids containing the mutations were chosen for large-scale DNA preparation and used in transfecting

Neurospora pco-1 rip mutant spheroplasts.

Filter-binding Assay

Filter-binding experiments were performed with 25 mm nitrocellulose filters with a mean pore size of 0.45 µm in a Hoefer 10-place filter apparatus. The XbaI ~ NarI DNA fragment from the nit-3 promoter which contains both the NIT2 and NIT4 binding sites and flanking sequences was radiolabeled with [γ-32P]ATP by T4 polynucleotide kinase.

Binding reactions were performed in a 60 µl total volume in binding buffer (12 mM

Hepes pH 7.9, 2 mM DTT, 3 mM MgCl2, 0.5 mM EDTA, 50 mM KCl, and 10% v/v glycerol). Samples were allowed to equilibrate for 30 min at room temperature before being passed through a nitrocellulose filter pre-wetted with binding buffer. The filters

were washed 3 times with 10 ml binding buffer, air dried, and counted for radioactivity.

The concentration of radiolabeled DNA fragment was 1 nM; protein concentrations of

GST-NIT4 (1-200) and His6-NIT2 (710-1036) were as indicated. For the protein-DNA dissociation experiment, complexes between labeled DNA and purified proteins were formed as described above. At 20-fold excess of unlabeled DNA identical to the labeled fragment was added into the preformed DNA-protein complex and incubation continued for additional 1-60 min as indicated.

Equilibrium Dialysis

Dialysis was performed in a clear acrylic equilibrium-type dialysis cell (Bel-Art

Products). The cell was divided into two chambers by a piece of dialysis membrane, which had a molecular mass cutoff of 12,000-14,000 daltons (Spectra/Por, Spectrum

Companies), and each chamber had a 1.0 ml capacity. GST-NMR fusion protein in 600

µl phosphate buffer saline pH 7.3 was added to one chamber at a concentration of 0.5 mM. The same volume of [3H]-glutamine in the same buffer was added to the opposing

chamber at a concentration of 1.0 mM. Dialysis was carried out at 4°C for up to 72 h with gentle agitation. At various time points, 50 µl samples of were withdrawn from

each chamber with a microsyringe and their radioactivity was measured.

CHAPTER 3

ISOLATION AND CHARACTERIZATION OF pco-1, WHICH ENCODES A REGULATORY PROTEIN THAT CONTROLS PURINE DEGRADATION IN NEUROSPORA CRASSA

INTRODUCTION

DNA binding is a critical property of many proteins with a wide range of

functions, including transcriptional control, DNA packaging, DNA repair, recombination,

DNA replication, as well as restriction and modification. The studies of the DNA

binding domains of transcription regulatory factors have revealed many distinct structures

which are able to interact with DNA. Each class of DNA-binding domain is

characterized by a particular protein sequence motif which is highly conserved among

other members of the class. DNA-binding motifs include helix-loop-helix, helix-turn-

helix, homeodomain, basic region-leucine zipper (bZIP), zinc fingers (GATA factors and

the Zn(II)2Cys6 binuclear clusters represent two subgroups) (Murre et al., 1994; Reddy et al., 1992; Treisman et al., 1992). Within a given class, DNA-binding motifs and their corresponding binding sites generally show conservation (Jamieson et al., 1994; Merika and Orkin, 1993; Suzuki et al., 1994).

The six-cysteine Zn(II)2Cys6 binuclear cluster DNA binding domain was first discovered and characterized in the yeast GAL4 protein, which is required for catabolism of galactose and melibiose (Pan and Coleman, 1990). More than 80 proteins in this class have been identified. To date, this class of DNA binding motif is unique to fungi.

Zn(II)2Cys6 proteins are involved in a wide range of processes, including metabolic pathways, drug resistance, and meiotic development. Almost all proteins of this class with known function are regulatory proteins; most of them are transcriptional activators.

The Zn(II)2Cys6 binuclear cluster DNA-binding motif has a conservative CX2CX6C-X5-

16-CX2CX6-8C arrangement and coordinates two zinc(II) ions. Like many other classes of

transcriptional activators, Zn(II)2Cys6 proteins are composed of distinct DNA binding

and activation domains. The DNA-binding Zn(II)2Cys6 motifs are usually close to the

amino termini of the proteins, while the activation domains often occur in their carboxyl termini (Parsons et al., 1992; Qui et al., 1991; Sze et al., 1993; Yuan et al., 1991).

Examination of all known Zn(II)2Cys6 proteins reveals that a large number of these have predicted leucine zipper-like motifs C-terminal to their zinc cluster. These motifs have been shown to form a coiled-coil structure which is involved in protein- protein interaction (Lupas, 1996). The crystal structures of yeast GAL4 and PPR1 showed both proteins dimerize via their coiled-coil region and bind to DNA as homodimers (Marmorstein et al., 1992; Marmorstein and Harrison, 1994). However, a number of Zn(II)2Cys6 proteins lack an obvious coiled-coil motif and may function as monomers (Anderson et al., 1995; Todd and Andrianopoulos, 1997).

In N. crassa, three Zn(II)2Cys6 proteins NIT4, ACR2, and QA1F have been identified. NIT4 is required for induction of enzymes needed for nitrate assimilation;

QA1F is essential for expression of enzymes of quinate/shikimate utilization; while

ACR2 regulates the enzymes for acriflavine resistance. They are all regulatory proteins, show DNA binding activities and possess a predicted coiled-coil dimerization domain.

NIT4 has been found to dimerize in vitro (Akiyama and Nakashima, 1996; Geever et al.,

1989; Yuan et al., 1991).

The generally small size of fungal genomes makes sequencing of the entire genome of a number of fungi economically feasible. With the near completion of the N. crassa genome sequencing project, as well as the discovery of conserved Zn(II)2Cys6 cluster containing genes in Neurospora and other fungi, it became possible to identify unknown Zn(II)2Cys6 proteins by searching the genome database.

At least eight possible Zn(II)2Cys6 proteins were discovered when the zinc cluster sequence of NIT4 was used to search for homologous proteins in the Neurospora genome database. In this chapter, I report the search leading to the identification of

PCO1, which controls the expression of the genes necessary for purine catabolism. By creating a knock-out strain, I demonstrated PCO1 is required for Neurospora to grow in media containing purines as the sole nitrogen source. The ability of PCO1 to interact with the promoter of the genes it regulates and the possible interactions with other regulatory proteins was also investigated. My results also indicated that PCO1 can dimerize in vitro and that its predicted coiled-coil region is essential to its function.

RESULTS

Sequence Homology Search for Zn(II)2Cys6 Proteins

This research to identify and characterize pco-1 started with a homology search,

when the Neurospora genome sequence database hosted at Whitehead Institute for

Biomedical Research, Cambridge, Massachusetts, was made available in 2001. Using the

peptide sequence of the NIT4 Zn(II)2Cys6 zinc cluster as a reference to search against

the database (TBLASTN), seven sequences encoding peptides exhibiting significant

similarity to the NIT4 zinc cluster were found. None had previously been identified. The

genomic DNA sequences of the seven segments containing the hypothetical Zn(II)2Cys6

clusters were downloaded from the Neurospora Database, and translated in all possible

reading frames by computer. The sequences of predicted peptides were again used as

templates to perform a homology search against the GenBank nr (non-redundant)

database (BLASTX) for known proteins. One of the seven coding regions exhibited high

homology (43% identity) to the amino terminal region of the UAY protein, which

regulates expression of enzymes in purine catabolic pathway in Aspergillus nidulans.

This sequence mapped to chromosome IV of Neurospora, closely linked to arg-2 and

arg-14 loci. Due to its high homology to UAY, this hypothetical Neurospora

Zn(II)2Cys6 protein was predicted to encode a regulatory protein in purine metabolism,

and was tentatively named purine control-1 (pco-1).

Disruption of pco-1 and the Phenotype of the Mutant

To determine the function of pco-1, the RIP (Repeat Induced Point mutation)

process was employed to generate pco-1 mutant strains in which the resident pco-1 gene

is severely damaged. A 2-kb DNA fragment, which contains the sequence coding for the

amino-terminal region of PCO1 protein including the Zn(II)2Cys6 cluster, was inserted between the Eco RI and Xba I sites of the pDE1 vector. pDE1 contains a 5’-truncated

his-3 gene which can be used as a selectable marker when transformed into his-3 mutant strain Y234M723 (mating type: A, FGSC #6103). The point mutation in the resident his-

3 gene of the mutant strain can be replaced by the wild type sequence to form a functional his-3 gene only when the transformed plasmid targets at the his-3 locus by homologous recombination. This will also result in the integration of the pco-1 gene near the his-3 locus. After transformation, genomic DNA was isolated from the transformants and a Southern blot was used to identify progeny carrying one extra copy of pco-1.

These transformants were than crossed with a wild type strain of the opposite mating type

74-OR8-1a (mating type: a, FGSC #988) as described in Materials and Methods. Several hundred ascospores were spread on solid Vogel’s minimal medium and colonies were picked for further analysis after incubation at 25°C for five days.

One hundred and thirty four colonies from the RIP cross were tested in Vogel’s liquid medium containing 5 mM hypoxanthine as the only nitrogen source, among which

14 progeny (~10%) displayed the phenotype that they could not grow on the selection medium while the other progeny behaved similar to the wild type parents. These 14 strains were selected and further tested on other selective media. Table 1 shows that pco

1 rip mutants cannot grow with hypoxanthine, xanthine or uric acid as the sole nitrogen source. The results indicate that pco-1 encodes a protein which is essential for

WT pco-1-

Hypoxanthine + -

Xanthine + -

Uric Acid + -

Urea + +

Ammonium + +

Nitrate + +

Table 1. The ability of pco-1 rip mutant to grow in different nitrogen- containing media. The concentration for all the nitrogen sources was 5 mM except ammonium chloride, for which 25 mM was used.

Neurospora to utilize purines as nitrogen sources. It is not certain if the pco-1 rip strain is able to use allantoin as nitrogen source since the allantoin used may be contaminated by trace amount of nitrogen compounds such as ammonium. Such a contamination is indicated by the unexpected growth with allantoin of the negative control strains, Aln- and alc-, which are defective in the structural gene encoding allantoinase and allantoicase respectively. The pco-1 rip mutants grew normally in medium containing nitrate as a sole nitrogen source demonstrating that pco-1 is not required in the nitrate assimilation pathway.

DNA sequencing was carried out to examine the nucleotide changes in the pco-1 rip mutant. Genomic DNA of a pco-1 rip mutant along with DNA from the wild type strain was prepared and the coding region of the pco-1 gene was isolated by PCR and sequenced. As shown in Figure 5, the sequence of the pco-1 gene from the rip mutant revealed a massive amount of G-C to A-T mutations when compared with the sequence of the wild type strain. Fifteen percent of the G::C pairs in the region sequenced were altered in the rip mutant, while the sequencing result from the wild type strain confirmed the published genomic DNA sequence in the database.

Gene Complementation

To further test the function of pco-1, it was of interest to determine whether the cloned pco-1+ gene could complement the loss-of-function pco-1 rip mutant. A N. crassa pco-1+ gene construct, a 5.0 kb fragment containing approximately 500 bp of the

Figure 5. Sequence analysis of pco-1 rip mutant. The pco-1 gene was amplified from chromosomal DNA of the rip mutant by PCR and the sequence was analyzed.

A portion of sequencing result is shown here in comparison with wild-type; wild-

type sequence is in the upper line. About 15% of G::C pairs were altered to A::T

by the rip process and are indicated as highlighted bases.

WT CACGCCAAACCGGACCGCACTGCGAGACCGGTCACACAGGGACAGCGGGA pco-1- CACGCCAAATCGGACCGCATTGCGAGACCGGTCATACAGGGATAGTGGGA

CTGATTGGAGGTGCTCTACTGAGCAGCCTGGCATAATGCCGGCGCACACT CTGATTGGAGGTGCTCTACTGAGCAGCCTAGTATAATGCCGGCGTATACT

CCTCCGTCCCCGAACCACCACCCAAACAAGCGACCGCGCCAATCGTCCCC CCTCCGTCCCCGAACCACCACCCAAATAAGCGACCGTGCTAATCGTCCCC

GGAACGAGACTCTCCGGCTTCGGTTCCGTCTGGGGAAGCCCCGTCTCCCG GGAACGAGATTCTCTAGCTTCGGTTCTATCTGGGGAAGCCCCGTCTCCTG

CTAACGAGCTTCTACTTGACGGGTCTGGCGCCGCTCATGGAGGCCAGGGA CTAACGAGCTTCTATTTAACGGGTCTGGCGCCGCTTATGGAGGCTAGGGA

GGCGGGTCGACGGGGGGCAAGGCCGGACAAAGCAGCAGCTTCCGCAACGT GGCGGGTCGACGGGGGGCAAGGCCGGACAAAGCAACAGTTTCCGTAATGT

GAGCGCTTGTAACCGATGTAGGCTTCGCAAAAACCGCTGTGACCAGAAGC AAGCGTTTGTAACCGATGTAGGCTTCGCAAAAACCGCTGTGATTAGAAGC

TACCGAGTTGTGCCAGTTGCGAAAAGGCCAATGTTGCCTGCGTTGGCTAT TACCGAGTTGTGCCAGTTGCAAAAAGGCCAATGTTGCCTGCGTTGGCTAT

GATCCCATCACCAAGAGAGAGATCCCGAGGAGGTCAGACCCCCCACCCGG GATCCTATTACTAAGAGAGAGATTCCGAGGAGGTTAGACCCCCCACCCGG

CCGGGACCCAATCATTGAGGTTCCCGCCATAACCCATCTTCACCAACGCC CCGGGACCTAATCATTGAGGTTCCCGCTATAACCTATTTTTACTAACGCT

TCGCTTCCTGCCTGTGAATAACAAAAGTACTGACGCCGTGCTGTCCCAGT TCGCTTCCTGCCTGTGAATAATAAAAGTACTAACGCCGTACTGTCCTAGT

TATATCTTTTACCTCGAGAAACGAGTAGAGCAACTGGAACACCTGCTCAA TATATCTTTTACCTCGAGAAACGAATAGAGCAACTGGAATACCTACTCAA

GGACAACAATATCACTTTCCCGCCGGCCGAAAATTTGGACTACTGTTCCA AGACAATAATATTACTTTCCCGCCGGCCGAAAATTTAGACTACTATTCTA

AGAAGGGAGATAGGCCGCGCCATGTCCACTCGACCCAGGTGGACACGAGC AGAAGGGAGATAGGCCGCGCCATATCTACTCGACCTAGGTAGACACGAAT

GGGCATCCCTCACAACTAGAAACACCAGATAGCATCAACAATCAAAGACC GGGCATCCCTCACAACTAGAAATACTAGATAGTATTAATAATTAAAGACT

promoter region and the entire coding region were transformed into pco-1 rip mutant spheroplasts. Conidia of the transformants were inoculated in liquid medium with 3 mM uric acid as the only nitrogen source, in parallel with wild-type N. crassa as well as pco-1 rip mutant as controls. After incubation at 30°C for 4 days, successful transformants as well as wild-type Neurospora crassa showed vigorous growth and were starting to conidiate. In contrast, pco-1 rip mutants could not utilize purines and thus only showed minimal growth, and no conidiation occurred. This result demonstrated that the purine utilization auxotropic phenotype of the rip mutant could be complemented to prototropy by the 5-kb fragment containing the pco-1+ gene.

Nucleotide Sequence Analysis of pco-1

A genomic DNA sequence, including the coding region and flanking sequences of

pco-1 gene, was retrieved from Neurospora genome database. Computer-assisted six

frame translation analysis revealed an open reading frame of 235 amino acids which

specifies a protein region with a typical Zn(II)2Cys6 cluster. The existence of introns

was postulated since all three reading frames following the zinc cluster region contained

a series of stop codons. Furthermore, the average length of known Zn(II)2Cys6 proteins

is around 600 to 1000 residues. Automatic gene prediction was performed by the

NetGene2 prediction server (Brunak et al., 1991; Hebsgaard et al., 1996). Due to the

lack of species-specific information for Neurospora; algorithms designed for other

eukaryotic organisms were used as reference. The results strongly suggested a 117-

nucleotide intron located immediately after the coding sequence for the Zn(II)2Cys6

cluster when parameters for either human, C. elegans or A. thaliana were applied

(confidence value > 0.9). The prediction for a possible second intron about 2 kb downstream from the first one was less conclusive, and the three algorithms noted above yielded different predictions of splice sites with a low confidence level.

RT-PCR was used to determine whether pco-1 contains introns and to identify

their splicing sites. The full-length pco-1 cDNA was expected to be longer than 3.0 kb.

A few attempts to obtain the complete cDNA through a single RT-PCR reaction were

unsuccessful. Two sets of primers were designed to determine the exact locations of

introns. Each pair covers about 1.0 kb of sequence; these RT-PCR reactions were

successful. RT-PCR with PCR primers PCOPCR1-F and PCOPCR2-R produced an

amplicon of expected size (656 bp), whereas PCR using the same primers, and genomic

DNA as template revealed amplicons of 773 bp in size. Similarly, PCR primers

PCOPCR-8F and PCOPCR-8R produced a smaller amplicon when cDNA was used as template compared with the product from genomic DNA (Figure 6). Two introns of 117

and 70 nucleotides were confirmed by comparing the cDNA sequences resulting from

RT-PCR with their genomic sequences. The complete pco-1 nucleotide sequence is

displayed in Figure 6.

Determination of the 5’ and 3’ Termini of pco-1 Message

The transcriptional start site and the 3’ end of pco-1 mRNA were determined by

RACE (Rapid Amplification of cDNA Ends) using mycelial RNA extracted from a wild-

type strain grown under fully inducing conditions, and two sets of gene-specific primers

Figure 6. Nucleotide sequence of pco-1 and its flanking regions. This figure

comprises the results of sequencing both the genomic and cDNA clones of the

pco-1 gene. Numbers at the top of the sequence indicate nucleotides from the start

of the sequence and numbers on the bottom indicate amino acids after the first

ATG. The putative Zn(II)2Cys6 zinc cluster is underlined. Two introns are

indicated in lower case letters. The vertical arrows indicate the start and stop

points of the pco-1 transcript.

-594 ACCT CTCCACTTGC TATCGACGTC CGCTATCCAC CTGCCCTCAA TACTAGGCCC

-540 ATTATCCACA CTCACTACAC CTGCGCCGTT GCCGCTCTCC GCGTGCCGCC CGCCTTTGGT TCGTTCCTCT TGCATCGCAT CATAGATAAA

-450 AGTCAGTCGA GTTTCAATAA TGATGCTACC GTCCGTCAGT CTAGTGTGAC CCGCGACGAA ACCCGGACGT ATTCGACCCT GCCCGCGCCA

-360 ACAAGGATTG TAGCTATTTT TTTCACGGCT CGACCGCGCA AACCAACAAC GGCCCTGACG GCCGACCATC ACGAGGGTCG CTTCTAGGGT

-270 CCTCCTCCGT CTCGGTTACC ACTCACTACT CTAGGTACAC TACACTACCG TTCATTCAGG CCACTCATTG TCGGTCAAGC CTCGGATCCG

-180 GATTCGCATT CCCCAACAAA AGAAACCAAC CAACGCAACA GCCCAGCAGC CGGAGAGGTA GCAGACGCGC ACTTGATCCA CGCGCCCACG

-90 CCTCGCACGC CAAACCGGAC CGCACTGCGA GACCGGTCAC ACAGGGACAG CGGGACTGAT TGGAGGTGCT CTACTGAGCA GCCTGGCATA

1 ATGCCGGCGC ACACTCCTCC GTCCCCGAAC CACCACCCAA ACAAGCGACC GCGCCAATCG TCCCCGGAAC GAGACTCTCC GGCTTCGGTT 1 M P A H T P P S P N H H P N K R P R Q S S P E R D S P A S V

91 CCGTCTGGGG AAGCCCCGTC TCCCGCTAAC GAGCTTCTAC TTGACGGGTC TGGCGCCGCT CATGGAGGCC AGGGAGGCGG GTCGACGGGG 31 P S G E A P S P A N E L L L D G S G A A H G G Q G G G S T G

181 GGCAAGGCCG GACAAAGCAG CAGCTTCCGC AACGTGAGCG CTTGTAACCG ATGTAGGCTT CGCAAAAACC GCTGTGACCA GAAGCTACCG 61 G K A G Q S S S F R N V S A C N R C R L R K N R C D Q K L P

271 AGTTGTGCCA GTTGCGAAAA GGCCAATGTT GCCTGCGTTG GCTATGATCC CATCACCAAG AGAGAGATCC CGAGGAGgtc agacccccca 91 S C A S C E K A N V A C V G Y D P I T K R E I P R R

361 cccggccggg acccaatcat tgaggttccc gccataaccc atcttcacca acgcctcgct tcctgcctgt gaataacaaa agtactgacg

451 ccgtgctgtc ccagTTATAT CTTTTACCTC GAGAAACGAG TAGAGCAACT GGAACACCTG CTCAAGGACA ACAATATCAC TTTCCCGCCG 117 Y I F Y L E K R V E Q L E H L L K D N N I T F P P

541 GCCGAAAATT TGGACTACTG TTCCAAGAAG GGAGATAGGC CGCGCCATGT CCACTCGACC CAGGTGGACA CGAGCGGGCA TCCCTCACAA 142 A E N L D Y C S K K G D R P R H V H S T Q V D T S G H P S Q

631 CTAGAAACAC CAGATAGCAT CAACAATCAA AGACCACAGA ATGAGGGAGA TGATGTGGTC AGACTACAGA AACTGGTGTC CAAGTCTGAC 172 L E T P D S I N N Q R P Q N E G D D V V R L Q K L V S K S D

721 CTTGGTGGCG TATCCGCAAC GGCAAAAGCT CGCTATCTGG GATCTACCTC GGGAATATCT TTTGCACGCA TTGTTTTCGC TGCGGTGCAG 202 L G G V S A T A K A R Y L G S T S G I S F A R I V F A A V Q

811 TCGTCGGTAT CCGATCAGAA GTCCACCTCG GACAAAGCTG GCATCCGACC CTACAAGCCT GCTCCCAACA ATGGCCCCGC CACCGCCGGA 232 S S V S D Q K S T S D K A G I R P Y K P A P N N G P A T A G

901 ACGTCTATGA GAGACTCGTT CTTTGGCTTG CACACAAAAC CAACGATTCA TCCGGCAACA TTCCCCAGCA GGGCTTTGGG CGAAAAGTTG 262 T S M R D S F F G L H T K P T I H P A T F P S R A L G E K L

991 ATGTCGCTTT ATTTTGAGCA TGCGAACCCC CAAATGCCTG TGCTCCACAA AGGCGAGTTC CTGGAAATGT TTGAGCAAGC CTATGCCGAA 292 M S L Y F E H A N P Q M P V L H K G E F L E M F E Q A Y A E

1081 GAAAACCGCG TACGGGGGCC ACGAGAACTT TACATGTTGA ACATGGTCTT CGCGATAGGC GGCGGCATCA TTGTGGGAGA ATCGACCAAG 322 E N R V R G P R E L Y M L N M V F A I G G G I I V G E S T K

1171 GCCAGTGGAT CGGCAGAAGC ACCAGGTAAA ACTGATGACA ACACAAGACA GTGCCAGCCA GAAGAATATC ATGCTAGTGC CATTGTTCAT 352 A S G S A E A P G K T D D N T R Q C Q P E E Y H A S A I V H

1261 CTTGAGGCTT GTCTTGGCAA CAGTGCCGGT GGTTTGGAAG AGTTACAGGC CGTCTTGCTT CTCGCCAACT TTGCCTTATT GAGGCCAGTT 382 L E A C L G N S A G G L E E L Q A V L L L A N F A L L R P V

1351 CCTCCTGGGC TGTGGTACAT CGTCGGCGTC GCCATCAGGT TGGCGGTCGA TCTCGGCCTA CACTACGAAG ACGGTAAGGA CATTGAATCC 412 P P G L W Y I V G V A I R L A V D L G L H Y E D G K D I E S

Continued

Figure 6 continued

1441 GGTCTAGGGA TCGGAGACCA GAGCGAAGCA TTCTCGCGGG AAAGGGGAAG AAGAGAGTAC ATGCGTGATC TCAGGCGGCG TCTGTGGTGG 442 G L G I G D Q S E A F S R E R G R R E Y M R D L R R R L W W

1531 TGCACCTATT CACTCGATCG CCTCGTCAGT GTTTGCGTCG GCAGGCCATT TGGCATATCA GATCAAGTCA TTACCACAGA GTTCCCCTCT 472 C T Y S L D R L V S V C V G R P F G I S D Q V I T T E F P S

1621 CTCCTAGATG ATCGATTCAT TACGCCAAGC GGCTTGCTTG AACCACCCCC CGACGTAATA GGACCGACTT ACAAACTCAT TGCACATCAC 502 L L D D R F I T P S G L L E P P P D V I G P T Y K L I A H H

1711 TACTTCCGTC TTAGGCTGCT CCAGTCAGAA GTTCTTCAAG TCTTGCAGTT CCAGCAATCA CAGCTTGCGC GGGCCTCTGG ACTGAACCAG 532 Y F R L R L L Q S E V L Q V L Q F Q Q S Q L A R A S G L N Q

1801 AAGAACCCTT ACATGCATAC ATCTCTTCCA TCACCGTTCT TGTCACAGTT CGAAACCTTC CGAGCATGGC GCATTGATAT CGACAGGAGA 562 K N P Y M H T S L P S P F L S Q F E T F R A W R I D I D R R

1891 CTATGGGAGT GGAAAAATTC CGCACCAACA CGGCAGCAGA CTGGCGTACA ATTTTCGCCT GAGTTTTTTG ACCTCAACTA CTGGCAGGCT 592 L W E W K N S A P T R Q Q T G V Q F S P E F F D L N Y W Q A

1981 GTCATTATGC TCTATCGCCA AAGTCTAAGT GTGCCAGCCT TGTTCGAAGG TGAATATCAC ACGTCAAAGG AGGTCAACAG CCCGACAATG 622 V I M L Y R Q S L S V P A L F E G E Y H T S K E V N S P T M

2071 TTCAACATGG AGCTTCGGGA GGATGAAGAC CGCGTGTATC TCAAGGTCGC CGAGGCAGGC CAGCGTATCT TGCGCCTCTA TCGCCAATTG 652 F N M E L R E D E D R V Y L K V A E A G Q R I L R L Y R Q L

2161 CACCGTGTGG GCTTGGTCAA CTATACCTAT TTGGCCACGC ACCATTTGTT CATGGCCGGA ATATCTTATC TTTATGCGAT CTGGCATTCC 682 H R V G L V N Y T Y L A T H H L F M A G I S Y L Y A I W H S

2251 CCTATCGTGA GGAGCAGACT Tgtaaggaca cacaactttt attgctgatt attttactgt ttttcacagg ctaacgcgca ccccctttta 712 P I V R S R L

2341 gACCGTGGAT GAAGTCGACT TCACGATTCT GGCTGCCAAA TCCGTTTTTA CAGACCTGAT AGATAAATGT CCGCCAGCAG AAGCTTGTCG 719 T V D E V D F T I L A A K S V F T D L I D K C P P A E A C R

2431 AGACGCCTTT GATCGCACCG CCAAAGCAAC CATCAAAATG GCAAACTCGA CTGGAGGCTT CGGCCAAGGC CAGGACTTGT CCGGCGGATA 749 D A F D R T A K A T I K M A N S T G G F G Q G Q D L S G G Y

2521 TGGAAACAAC ACCCACATAA GACGTCCTGG AGGAAGTATT GATCAGCGTC TCGACTGGAG CTCACAGAGC GATTCTGCCG CTTCTAGTTT 779 G N N T H I R R P G G S I D Q R L D W S S Q S D S A A S S L

2611 GCAACACTAT AGAATGCAGC AACAACAACA ACAGCACCAG CAGCATCAGC AGCAGCAACA TCGCCAGCAC CAACAACAAC AACACCGCCA 809 Q H Y R M Q Q Q Q Q Q H Q Q H Q Q Q Q H R Q H Q Q Q Q H R H

2701 TCGCCCGCCT ATGCTCCGAA ACCCTTCGTC ACAGTACAGC GATCTCGCCT CCGACGCCTA CTCCGTCTCA TCAGCATCTC AGCTTTCCGC 839 R P P M L R N P S S Q Y S D L A S D A Y S V S S A S Q L S A

2791 CTTCCAACAG GCCCAACAGT TCCGCATGTC CGGTGCCGCT GCCGCCGCCG CGATCAAGAG CGAGCAGGAA GGCGGCTTCT CTCTCATGCG 869 F Q Q A Q Q F R M S G A A A A A A I K S E Q E G G F S L M R

2881 AAACCCACCT CCGCCACCAC ATAGCAACGC AAGCTCCATT GCGGAAGCCG CCTCTGGCGG CGGGGGACCG ATGGCTCAAA GCCCAGTAGG 899 N P P P P P H S N A S S I A E A A S G G G G P M A Q S P V G

2971 CGAGACCTCC ATTGATCCGA CCTTGATGCC CTCGCCGAGC GCCATCCAAC AGCACCAGCA ACGGCAAGGA CAAGGACTTA GTAACCCCTT 929 E T S I D P T L M P S P S A I Q Q H Q Q R Q G Q G L S N P L

Continued

Figure 6 continued

3061 AACGCCGCCT GCTCAGATGG GTGGATCACC GATTATCGGG GCCAACTCAC AACAGCAACA ACAACGCGGA GGGACGCCAA ATGACTATCT 959 T P P A Q M G G S P I I G A N S Q Q Q Q Q R G G T P N D Y L

3151 ACAGCAACAG CAACAACAGC AACAACAACA ACAAGCATAC TCCAATAGTC CTGGCACACT AAGCTTCAGT GATCTACAAG GCCTAGAGTT 989 Q Q Q Q Q Q Q Q Q Q Q A Y S N S P G T L S F S D L Q G L E F

3241 CTTGCAAAGT GTTGATGGCG GCGCCGCTGG TGGTATGGAC ATTAGTAATG GAGGAGATCT GGCGAATTTG AACGTGAACC TGAATCCGGC 1019 L Q S V D G G A A G G M D I S N G G D L A N L N V N L N P A

3331 TGAGGTAGCG GGACAGATGG ATCTAGGCTT TGGTATCGGG TGGGAAGGGA ACCATCATGA TTTTAGCGAT GGACAGCAGT TAGATCTGTT 1049 E V A G Q M D L G F G I G W E G N H H D F S D G Q Q L D L F

3421 TGAAGGATTC TTTTTTGGTG GGCAGCAGGG TGGTGGCGGT GGAGGAGGAG GTGGTGGTGC TGGTGGGAAC TAGAAAGTAG GGAAACTTGG 1079 E G F F F G G Q Q G G G G G G G G G G A G G N *

3511 GATAGGATTG ATGAGGCTGA TATATATATT ATACCACGAG GAGAAGAAGG GAAGAGGTGG GAAGGAATGA GAAGGGTGTG GGCAAGATAC

3601 GGAGAGGGAA AATGGAGGAC TCATTATTAT AGGAGGCCTC ACCATTTAGG AACATCTACG CGACACGCAG TTGCCTAGCA TTTGCCCTCC

3691 TTCAGGTTGG GAGAAGAGTA TGAATGAGTT GATGGGGGAT GTTAAGAAAC AAATATGAAG AAGAGTCGAG AAGCTGATAA CGGGTTGTGG

3781 GGCTTCGATT ACCACGGGGA CGATCTAGTT CAGATGGAAG AAAATACAGC TTTCTGGACA TTGATCGACA AGTGAGATCA GATGGAGTCA

3871 TTCATGGTGA GTGTTTACTC ATCCTTTGTT TTTCAAGCAA GGAGTTCAGA CTTAGGTCAA CATGGCTATC AACGGACAGA TATCGGAGCA

3961 TGCTACCAGG ATTCGCATCT TACAACACGA AAATGTCTAT GTGCGATTCA TGTTGAAACT CGAAGAGATC AGTGAATATG TCATGATGGA

4051 ATTTTCAAAA ACAGTGAACG AAAATCTTCT AAAGTCGAGA TGGCCCCAAT TCTTTTCCTT ATACAAATGA AAATCAGAAG TACGCCCCCA

4141 GCCCCCCAGC TCCAGTCCCG AATATGTCTA GGTGTGCCCC AGTCCCAGTC CCATTTCTTG AACAAAAATT CTTCTCCTCT CTTGTGACCA

4231 CGGAACTAAA AAAGACTGTA CTACCCCTCT TTTGTGTGTT GCCCGCGCTG TCTATCATAA CATTTCCATA GACAACAAGC CACCCCAATG

4321 TATGGAAACA AAAAGAAACT CCGGCCTTGC TTTTCCCGTG ATAATTATGC TATAGTACAG ATGGTATCAA TGTCGAACAG TACGGAAAGG

4411 AAATACATGG GGGACCCGAT ACCACGGTTT TTGAGAAGGT GTCTGTCTCG CTCAGCAGAC AAAACCCCGC AAGCATCTTC TTTCAGCGGG

4501 AAAAACCAAA TAACCCATTC GTCGCCAATG CACAGTTTGA CCCAGCTCTT AGTTTAGAAG CTGGCCTGCT GGCGACGGGT GGGACGGCGA

4591 TCGAGATCCT GAGCCCTG

situated within the first exon (for 5’-RACE) and last exon (for 3’-RACE). Nested PCR reactions using a second pair of primers (nested primers) which bind within the first PCR products were performed on both 3’ and 5’ ends to eliminate non-specific products or artifacts. Amplified 3’-end and 5’-end cDNAs obtained with RACE reactions were cloned into pCR4-TOPO vector; the resulting clones of each reaction were subjected to sequence analysis. The 5’ and 3’ termini of the pco-1 transcript revealed by sequence results are indicated in Figure 5. Noticeably, the 3’ noncoding region does not contain the typical polyadenylation signal, AATAAA, as already reported in the case of other fungal genes (Centis et al., 1996).

Predicted Sequence of the PCO1 Peptide

The size of pco-1 transcript based on 5’-end and 3’-end mapping and excluding the two introns is about 3.7 kb. The exons of the pco-1 gene encode a polypeptide that is

1,101 amino acids long, with a calculated molecular weight of 123,456 Dalton.

A binuclear zinc cluster extends from residues 75 to 102 (from cysteine 1 to cysteine 6). Compared to known Zn(II)2Cys6 proteins, the six cysteine residues of PCO1 are arranged in the typical CX2CX6CX5-16CX2CX6-8C formation. This zinc cluster motif and the immediate downstream basic region represent the DNA-binding domain in this class of DNA-binding regulatory proteins. The six cysteine residues are absolutely conserved among known Zn(II)2Cys6 proteins, while other residues within the six- cysteine region are also largely conserved, but are not absolute (Figure 7).

Figure 7. Similarity in the Zn(II)2Cys6 DNA-binding region of 15 fungal transcriptional activators. The list contains all the identified Zn(II)2Cys6 proteins in Neurospora crassa along with similar proteins from Aspergillus and several well characterized GAL4-like proteins from yeast and other fungi. Conservations are shown above the sequence. N. cra, Neurospora crassa; A. nid, Aspergillus nidulans; A. nig, Aspergillus niger; S. cer, Saccharomyces cerevisiae; K. lac,

Kluyveromyces lactis.

Conservations +C C+ + +C+ P C C C PCO1 (N.cra) ACNRCRLRKNRCDQKLPSCASCEK-ANVAC UAY (A.nid) ACNRCRQRKNRCDQRLPRCQACEK-AGVRC

NIT4 (N.cra) ACIACRRRKSKCDGALPSCAACASVYGTEC ACR2 (N.cra) ACYNCHRKRLRCDKSLPACLKCSI-NGEEC QA1F (N.cra) ACDQCRAAREKCDGIQPACFPCVS-QRGSC NIRA (A.nid) ACIACRRRKSKCDGNLPSCAACSSVYHTTC QUTA (A.nid) ACDSCRSKKDKCDGAQPICSTCAS-LSRPC FACB (A.nid) ACDRCRSKKIRCDGIRPCCTQCAN-VGFEC XLNR (A.nig) ACDQCNQLRTKCDGQHP-CAHCIE-FGLTC GAL4 (S.cer) ACDICRLKKLKCSKEKPKCAKCLK-NNWEC PPR1 (S.cer) ACKRCRLKKIKCDQEFPSCKRCAK-LEVPC ARGR2(S.cer) GCWTCRGRKVKCDLRHPHCQRCEK-SNLPC HAP1 (S.cer) RCTICRKRKVKCDRLRPHCQQCTK-TGVAC MAL63(S.cer) SCDCCRVRRVKCDRNKP-CNRCIQ-RNLNC LAC9 (K.lac) ACDACRKKKWKCSKTVPTCTNCLK-YNLDC

A number of Zn(II)2Cys6 proteins contain leucine zipper-like heptad repeat motif

C-terminal to the zinc cluster. These motifs have been shown to form coiled-coil

structures involved in protein-protein interactions (Cohen and Parry, 1990; Lupas, 1996;

Todd and Andrianopoulos, 1997). Figure 8 shows the prediction of a coiled-coil domain

obtained by NPS@ for the PCO1 protein (Combet et al., 2000; Lupas et al., 1991).

Residues 121-134 of PCO1 showed a high probability of being a coiled-coil domain. The crystal structures of yeast GAL4 and PPR1 showed that their coiled-coil domains mediate

dimerization, (Marmorstein et al., 1992; Marmorstein and Harrison, 1994) suggesting

PCO1 might also exist as homodimer.

The carboxyl terminus of PCO1 contains two distinctive glutamine-rich domains that are typical of an extensive family of transcriptional activators conserved across multicellular organisms (Freiman and Tjian, 2002). Residues 809 to 835 contain 18 glutamines out of 27 amino acids (67%), and 16 of 25 residues (64%) from position 975 to 999 are also rich in glutamine. A study of human SP1 transcription factor revealed that its glutamine-rich domain mediates specific interaction between SP1 and a subunit of

TFIID, TAFII130. Disruption of the interaction between SP1 and TAFII130 greatly

reduced transcriptional activation of target genes containing G/C boxes (Saluja et al.,

1998).

The two introns in the open reading frame of pco-1 do not seem to split any

obvious putative functional domains. One is positioned between the coding regions for

the zinc cluster and coiled-coil domain. The second intron is located upstream of the

coding regions of the two glutamine-rich domains. Typical SV40-like or bipartite

Figure 8. Prediction of a PCO1 coiled-coil secondary structure. The protein is depicted as a plot of probability for the formation of coiled-coils calculated by the

NPS@ Network Protein Sequence Analysis (Combet et al., 2000).

nuclear localization sequences are not present in PCO1. However, residues 15-18 (KRPR) resemble a nuclear localization sequence consisting of a few basic residues followed by a proline in several proteins (Dingwall and Laskey, 1991).

DNA Binding Activity of PCO1 Protein

Electrophoretic mobility shift assays (EMSA) were performed with a purified

GST-PCO1 fusion protein. To obtain PCO1 protein, a DNA sequence encoding the

amino terminal 220 residues of PCO1 (1~220) was inserted into expression vector

pGEX-2T (Amersham Biotech). The construct was sequenced to confirm that the correct

reading frame was obtained. The GST-PCO1 fusion protein was then expressed in E. coli

strain BL-21(DE3) pLysS, purified by affinity chromatography using glutathione-agarose

resin, and visualized on SDS-PAGE. A dominant band of anticipated molecular weight

was obtained (Figure 9A). To maintain comparable binding conditions, an identical

amount of fusion protein was used in all binding assays. The DNA binding results,

shown in Figure 9B, demonstrate that PCO1 can specifically bind to the promoter of the

xanthine dehydrogenase gene. On the other hand, PCO1 showed only very weak binding

to unrelated control DNA sequences under identical condition.

Binding Site for PCO1 Protein

Like many other DNA binding protein families, Zn(II)2Cys6 DNA binding

domains interact with similar DNA binding sites. The binding sites for a particular

protein may differ from those of other members of the Zn(II)2Cys6 class, therefore

Figure 9. PCO1 fusion protein expression and electrophoretic mobility shift assay

(EMSA). A. GST fused to the N-terminal 220 residues of PCO1 (GST-PCO1)

after affinity purification. B. Results of mobility shift experiments show that

GST-PCO1 fusion protein binds to the promoter (-282 ~ -563) of the xanthine dehydrogenase gene. The amounts of protein used in lanes from left to right are 0,

0.1, 0.5, 1.0, 2.0 µg. The arrow identifies the free DNA probe.

A B XDH promoter

1 O C P T- T S PCO1 G GS KDa 114 80 64 50

allowing the specificity of action of individual proteins. A core binding sequence of this class of proteins has been identified which consists of conserved terminal trinucleotides, usually in a symmetrical inverted configuration, spaced by a sequence of variable length.

Many proteins in this family bind to a core sequence of CGG-N6-11-CCG (Todd and

Andrianopoulos, 1997). A putative binding site CGGggcacaCCG (-369 ~ -380), which is a perfect match with the consensus binding site, was found in the promoter region of the gene encoding xanthine dehydrogenase. A double-stranded DNA oligonucleotide containing the above sequence was synthesized to test binding with PCO1, along with another oligonucleotide in which nucleotides of the putative binding site were changed to a random sequence but with a similar G/C content (Figure 10A). Mobility shift assays showed that PCO1 specifically binds to the ds-DNA oligo with the wild type consensus sequence but only showed weak binding to the mutated one (Figure 10B).

Dimerization of PCO1 Protein in vitro

The Zn(II)2Cys6 cluster of PCO1 near its amino-terminus is followed by a possible coiled-coil dimerization motif. A coiled-coil protein prediction program suggested that residues 116-135 are very likely to form a coil-coiled motif based on heptad repeats (Figure 8). To examine the possibility that PCO1 exists as a dimeric protein, an N-terminal fragment of PCO1 containing the putative coiled-coil domain was

Figure 10. Identification of PCO1 binding site by EMSA. A. The sequence of

synthesized ds-DNA probes containing the wild-type (xdh-w) and the mutated

(xdh-m) PCO1 binding site of the xanthine dehydrogenase promoter (only one

strand of each probe is shown). B. Mobility shift of the xdh-w and xdh-m probes

using GST-PCO1 fusion protein. The amounts of protein used, in lanes left to

right, are 0, 0.1, 0.5, 1.0, 2.0 µg. The arrow identifies the free DNA probe.

A xdh-w 5’-GTACCTGATCGGGGCACACCGACCTCAAGG-3’ xdh-m 5’-GTACCTGAGACCTGCACCAGTGCCTCAAGG-3’

B xdh-w xdh-m

PCO1

subject to a crosslinking experiment. Glutaraldehyde was chosen as a crosslinking

reagent because it efficiently forms covalent bonds between lysine residues on the surface

of proteins. Cross linkage stabilizes oligomeric forms of proteins allowing subsequent

visualization by SDS-PAGE.

The cDNA encoding the N-terminal 220 residues of PCO1 was inserted into

pHB6 vector (Roche) in frame. The expressed PCO1 fusion protein has a human

influenza hemagglutinin (HA) epitope at its N-terminus and also a His6 tail at the C-

terminal end. This construct was sequenced to confirm that the correct reading frame

was obtained. The fusion protein was then expressed in E. coli strain BL-21 (DE3)

pLysS and purified by affinity chromatography using Ni-ATA-agarose resin (Figure

11A). Purified PCO1 protein was treated with the crosslinking agent, glutaraldehyde.

SDS-PAGE revealed that this chemical crosslinking procedure resulted in the formation of SDS-resistant dimers which, upon gel electrophoresis, migrated at the position expected. The amount of this dimeric species increased with incubation time while the monomeric species decreased accordingly (Figure 11B) Significantly, other multimers were not observed under these conditions. Western blot analysis of the crosslinking reaction confirmed that the dimeric species can be recognized by anti-HA monoclonal antibody (Figure 11C).

Figure 11. PCO1 protein expression and crosslinking. A. Expressed PCO1 amino terminal 220 residues fused with HA epitope (N-terminus) and His6 tag (C-terminus)

after affinity purification. The position of the expressed PCO1 is indicated by an arrow.

B. Glutaraldehyde crosslinking of PCO1 protein. 5 µg of PCO1 was incubated with

0.005% glutaraldehyde for 0, 1, 2, 3, 6 and 10 min. The arrow identifies the crosslinked dimer; the lower band which decreases in intensity with time is the monomer species. C.

Western blot analysis of the crosslinking product probed with 12CA5 antibody to detect

HA-epitope tagged proteins. The arrow indicates the position of the crosslinked dimer.

A KDa 12 182 115 84 62 51 38

B C Time KDa KDa 200 182 115 116 84 97 62 66

51 55 37 36 31 26 21 14 20

Deletion Analysis of PCO1

The carboxyl terminus of PCO1 contains two distinctive glutamine-rich domains that are typical of an extensive family of transcriptional activators conserved across multicellular organisms (Freiman and Tjian, 2002). To investigate weather the glutamine-rich regions are essential for PCO1 activity, I constructed a series of deletion derivatives with various numbers of C-terminal residues deleted. These truncated genes each with an intact promoter were inserted into pDE1 vector and used to transform the pco-1 rip mutant. Transformants were then selected on Vogel’s minimal medium with 3 mM uric acid as the only nitrogen source (Figure 12). The integrity of a C-terminal polyglycine region is important for optimal function of PCO1. The CD1 deletion strain, in which the last 32 amino acids, including the polyglycine region, were deleted but in which the two glutamine-rich activation domains were still intact, showed a significant decrease in the vigor of growth relative to wild-type. However, little difference was discerned between CD1, CD2, and CD3 strains in which different amounts of the glutamine-rich region were deleted. The CD4 strain, in which both glutamine-rich regions are deleted, showed a poor-growth phenotype similar to its parental strain, the pco-1 rip mutant. The results suggest that the C-terminal tail (32 amino acids) of PCO1 plays an important role to provide maximal activity of PCO1 and that at least one of the glutamine-rich domains is sufficient for its function.

Figure 12. Phenotype analysis of pco-1 deletion mutants. The open box indicates

the PCO1 protein-coding region. The 5’ untranslated region is shown with a solid

line. The introns are indicated as small black boxes, and the Zn(II)2Cys6 zinc

finger, near the amino terminus, is represented by a dotted box. Two glutamine-

rich domains near the carboxyl terminus are shown as hatched boxes. A gray box

indicates the putative coiled-coil dimerization domain. The number of amino

acids left in each pco-1 deletion clone is shown. Functional analysis of

transformants is indicated by the +/- symbols.

PCO1-FL 1101 ++++

DECOIL 1085 -

CD1 1010 +

CD2 941 +

CD3 837 +

810 - CD4

An internal deletion of 15 amino acids of the putative coiled-coil region was

created by site-directed mutagenesis. A growth test showed that the putative coiled-coil

dimerization domain is critical for pco-1 activity, suggesting PCO-1 might function as a

dimer (Figure 12).

Detection of Protein-Protein Interaction between PCO1 and NIT2/NMR

Direct protein-protein interactions have been shown to be crucial in the nitrogen

regulatory circuit in filamentous fungi. Global-acting transcription factors NIT2 (positive) and NMR (negative) are involved in the regulation of many nitrogen related genes

(Marzluf, 2001). In the case of NIT4, the pathway-specific Zn(II)2Cys6 factor in nitrate assimilation, a specific protein-protein interaction with NIT2 was detected and the domain involved in the interaction has been mapped to the DNA-binding domains of both proteins. No interaction between NIT4 and NMR was discovered (Feng and Marzluf,

1998). To investigate if PCO1 could interact with NIT2 or NMR, GST Pull-down assays with E. coli expressed proteins were employed.

1. Interaction with NIT2

To test for an interaction between PCO1 and NIT2, the PCO1 N-terminal 220

residues were expressed as a fusion protein with GST by means of the pGEX-2T

prokaryotic expression vector. A NIT2 KpnI-EcoRI fragment (encodes a.a. 710-1036,

and contains from the Zn finger to the C-terminal end) was inserted into the pRSET-C

vector and expressed as a His6 tagged protein. The His6-NIT2 fusion protein was then

incubated with GST-PCO1 fusion protein bound to glutathione-Sepharose beads. As a

positive control, His6-NIT2 was incubated in parallel with GST-NMR (full-length)

protein also immobilized on glutathione-Sepharose beads. After extensive washes in

high stringency buffer containing 200 mM NaCl, the proteins were eluted from the

Sepharose beads. The presence of NIT2 in the eluates was then detected by Western bolt

using polyclonal antiserum specifically against NIT2. The results reveal His6-NIT2

protein was pulled down by GST-NMR as shown in an earlier study, but not by GST-

PCO1 under the same conditions (Figure 13A).

2. Interaction with NMR

I next analyzed whether or not PCO1 would physically interact with NMR by

pull-down experiments. PCO1 (1-220) with a His6 tag was incubated with GST-NMR

fusion protein bound to glutathione-Sepharose beads. A His6-NIT2 fusion protein was

also incubated with the same amount of GST-NMR fusion protein in parallel as a positive

control. Western blot of the eluates using anti-HisG monoclonal antibody showed His6-

PCO1 (N220) was not pulled-down by GST-NMR (Figure 13B).

I conclude that recombinant PCO1 amino terminus does not interact in vitro with either NIT2 or NMR alone. This property is noticeably different from its homolog NIT4, whose zinc cluster domain not only binds to the promoter of nit-3 gene but also specifically interacts with the global-acting factor NIT2 (Feng and Marzluf, 1998).

Figure 13. Protein-protein interaction between PCO1 and the global-acting factors in nitrogen assimilation pathway.

A. Pull-down assay of protein-protein interaction between PCO1 and NIT2. The

Western blot was probed with the anti-NIT2 antiserum. Lane 1, eluate from

the incubation of His6-NIT2 and GST-PCO1 (N220) immobilized on

glutathione-agarose resin. Line 2, eluate from the incubation of His6-NIT2 and

GST-NMR immobilized on glutathione-agarose resin.

B. Protein-protein interaction between PCO1 and NMR. The Western blot was

probed with the anti- His6 antibody. Lane 1, eluate from the incubation of His6-

PCO1 (N220) and GST-NMR immobilized on glutathione-agarose resin.

Lane2, eluate from the incubation of His6-NIT2 and GST-NMR immobilized

on glutathione-agarose resin. Lane 3, blank. Lane 4, affinity purified His6-

PCO1 protein. Lane 5, affinity purified His6-NIT2 protein.

A. B. 12 12345 KDa KDa

84 84 64 64 50 50 NIT2 NIT2 37 37 PCO1

26 26 20

Ability of pco-1- Mutant Strain to Express Xanthine Dehydrogenase

To further examine the role of PCO1 on regulation of purine metabolic genes, the ability of the pco-1 rip mutant to express xanthine dehydrogenase was tested. Mycelia of

the pco-1 mutant were induced by purines under nitrogen derepression (no glutamine or

NH4 available) or nitrogen repression (presence of glutamine or NH4) conditions. Then

cell lysates were prepared and assayed for xanthine dehydrogenase activity. The results

(Figure 14) showed that xanthine dehydrogenase activity only showed a basal expression level upon purine induction under either a repressed or derepressed condition. In contrast, xanthine dehydrogenase activity is induced readily to greater than five times the basal level under the derepressed condition in wild type Neurospora. We conclude that pco-1+ is essential for the expression of xanthine dehydrogenase. Noticeably, purine induction of xanthine dehydrogenase is clearly subjected to nitrogen repression. In the presence of ammonia alone with the inducer uric acid (Uric Acid + NH4Cl), the induction by uric acid

is drastically reduced in the wild type strain (Figure 14).

The Involvement of NIT2 and NMR in the Induction of Xanthine Dehydrogenase

The assay of xanthine dehydrogenase activity in pco-1 rip mutant (Figure 14) showed that the induction of XDH is subject to nitrogen repression, i.e. synthesis of the enzyme was suppressed when a “preferred” nitrogen sources, e.g., ammonia, was

Figure 14. Induction of xanthine dehydrogenase by various nitrogen sources.

Wild type and pco-1- strains grown in Vogel’s complete medium for 30 h were

transferred for 6 h incubation in medium containing the appropriate nitrogen

source. The concentration for all the nitrogen sources was 10 mM except

ammonium chloride and potassium nitrate, for which 50 mM was used. Crude cell

extracts were prepared from mycelia and enzyme activity was assayed

fluorometrically. The results of xanthine, hypoxanthine and uric acid induction are

the averages of three separate experiments.

available, despite the presence of an inducer. The result prompted a study of the

involvement of NIT2 and NMR, which have been shown to be global-acting regulatory

proteins which mediate nitrogen repression in the nitrogen regulation circuit.

Neurospora crassa wild-type and mutant mycelia were grown on Vogel’s complete medium containing ammonium chloride as a nitrogen source and then transferred to a derepressed-induced condition (Uric acid as the sole nitrogen source), or

a repressed-induced condition (Glutamine + Uric acid) for 6 h. Xanthine dehydrogenase

activity was measured in crude extracts prepared from nit-2 rip mutant, nmr rip mutant,

and wild-type strains. As shown in Figure 15, after 6 hours of induction (uric acid), XDH

activity of nit-2 rip mutant can be induced to five times the basal level, similar to the

induction level of wild type strain. This indicates that a functional nit-2 is not required

for XDH induction. However, the nitrogen repression mechanism is still intact in nit-2 mutant since when 25 mM of glutamine was present during induction, XDH activity was reduced about 50 percent. However, the nitrogen repression effect was abolished in the nmr rip mutant. This result confirms the negative role of NMR in nitrogen repression, and that it can exert its effect even in the absence of the NIT2 protein. Moreover, the nmr

rip mutant showed higher XDH activity than the wild-type strain after induction,

suggesting that in wild type Neurospora, a basal activity of NMR may exist even under the derepressed condition.

Figure 15. Xanthine dehydrogenase assays of Neurospora nit-2 and nmr mutants.

The xanthine dehydrogenase activities of nit-2 and nmr rip mutants are compared with wild-type, under induction with uric acid in the presence or absence of glutamine. Mycelia from 30 h culture were transferred to 5 mM uric acid or 5 mM uric acid plus 25 mM glutamine for an additional 6 h. Crude cell extracts were then prepared for enzyme assay. The results of wild-type and nit-2 rip mutants are from three separate experiments.

The Homology between PCO1 and UAY Proteins

The uaY gene of Aspergillus nidulans, which corresponds to pco-1 of Neurospora,

has been cloned and sequenced, (Suarez et al., 1995b) which makes possible a direct

comparison of the PCO1 and UAY regulatory proteins. The UAY protein consists of

1,060 amino acids and thus is somewhat shorter than PCO1, which contains 1,101

residues. Interestingly, in nitrate catabolic pathway, the pathway-specific regulatory protein NIT4, which is also a Zn(II)2Cys6 protein, is slightly longer than its counterpart,

NIRA, in Aspergillus as well. PCO1 is the longest protein among those Zn(II)2Cys6

factors which have been identified in Neurospora in comparision to NIT4 (1,090 a.a.),

QA1F (816 a.a.), and ACR2 (595 a.a.). PCO1 shows significantly higher degree of

overall homology with UAY (46% identical, Figure 16) than with other proteins in this

class which suggests functional conservation. Of particular significance is the fact that

the highest amino acid identity between PCO1 and UAY occurs in zinc cluster and

immediate adjacent residues. Alignment of known Zn(II)2Cys6 cluster motifs reveals an absolute conservation of the six cysteine residues (Figure 7). The carboxyl terminus of

PCO1 shows a much lower degree of homology with UAY. Interestingly, the glutamine-

rich regions close to the C-terminus of PCO1, a feature shared with a number of

transcription factors of Neurospora, such as NIT4 and WC-1, do not exist in UAY. Thus

the similarity between PCO1 and NIT4 in the carboxyl terminus is marginally higher than

that between PCO1 and UAY (Figure 17). However, the overall homology between

PCO1 and NIT4 is just 28 %. Another feature of pco-1 is the location of the first intron,

Figure 16. Alignment of PCO1 protein of Neurospora crassa and UAY of

Aspergillus nidulans. Sequences were aligned using SIM algorithm (Huang and

Miller, 1991). Dashes have been inserted to optimize the alignment. * indicates

an exact match between the two proteins.

Figure 17. The glutamine-rich regions in the carboxyl termini of PCO1 and NIT4

of Neurospora. Glutamine residues are highlighted.

PCO1 778-YGNNTHIRRPGGSIDQRLDWSSQSDSAASSLQHYRMQQQQQQ HQQHQQQQHRQHQQQQHRHRPPMLRNPSSQYSDLASDAYSVS SASQLSAFQQAQQFRMSGAAAAAAIKSEQEGGFSLMRNPPPP PHSNASSIAEAASGGGGPMAQSPVGETSIDPTLMPSPSAIQQ HQQRQGQGLSNPLTPPAQMGGSPIIGANSQQQQQRGGTPNDY LQQQQQQQQQQQAYSNSPGTLSFSDLQGLEFLQSVDGGAAGG MDISNGGDLANLNVNLNPAEVAGQMDLGFGIGWEGNHHDFSD GQQLD-1076

NIT4 717-YSHPTPPHLGPESISDPGMSPNIVTFSDLPDPSAPIIPQQQQ NMQAISSLSQNNMLHQHHHHLQNQHQPQQPHHNHMTYQQQQH NLLTHPVSASSMSMSDTLATITAWGIPTSSPGNNNNNNIVSQ HPHHQKQPQQQQQPQAQRYPTVGSVGTNTVKPPAAATQTFTP AQLHANNLATATRSTASNHKSVGRHVSPSSIYAIDGQDWYLK DGVTWQQGFQGWDLEGGGAGTATSTGGIGDGGGPTGGAGDSM ARLAPRGNIGGGGGGGGGSTGQRQQQQQRQQQQQQQQQQQQQ QQQQQQQQQQQQQQEANMFAYHHGAERGGGGIESTGMGMTTV GGGGGGFDPIGGSGLLDDLVGLDELGSLDGLGHLPGL-1089

which is adjacent to the Zn(II)2Cys6 cluster coding region, whereas there is no intron splitting the open reading frame in the same region of uaY. The second introns of both

genes are located at the identical position (Suarez et al., 1995b).

DISCUSSION

Sequence analysis of the pco-1 gene revealed it encodes an 1,101 residue polypeptide containing a GAL4-type Zn(II)2Cys6 binuclear cluster motif followed by a linker and a series of leucine zipper-like heptad repeats near the amino terminus. In addition, glutamine-rich regions, which may be involved in transcriptional activation, were identified near the carboxyl terminus of the protein. These features suggest that

PCO1 functions as a transcriptional activator. Knock-out of pco-1 by RIP mutagenesis showed a phenotype that xanthine dehydrogenase activity could not be induced, and also that the mutant had lost the ability to utilize various purines as a nitrogen source.

Transforming pco-1 gene back into the rip mutant strain restored its ability to utilize purine as a nitrogen source, indicating a functional pco-1 gene product is essential to metabolize purines.

The PCO1 protein contains heptad repeats C-terminal to the zinc cluster, which may form a coiled-coil structure like those shown to be involved in dimerization in other proteins (Cohen and Parry, 1990; Lupas, 1996). A chemical crosslinking experiment showed that PCO1 is able to dimerize in vitro (Figure 11). Homodimerization has been demonstrated for several proteins in this class, including NIT4, GAL4, and PPR1 (Feng and Marzluf, 1996; Marmorstein et al., 1992; Marmorstein and Harrison, 1994). The crystal structures of DNA bound GAL4 and PPR1 showed that these proteins bind to

DNA as dimers. Deletion of the heptad repeat sequence of PCO1 abolished PCO1 function (Figure 12), suggesting that PCO1 also functions as a homodimer. Almost all

Zn(II)2Cys6 proteins are regulatory proteins most of them transcription factors, implying dimerization might be critical for DNA binding for zinc cluster proteins (Todd and

Andrianopoulos, 1997). However, not all the Zn(II)2Cys6 proteins possess a heptad repeat motif (Anderson et al., 1995; Masloff et al., 2002). These proteins may function as monomers or dimerize through other protein-protein interaction motifs.

Like other transcription factors, Zn(II)2Cys6 proteins are composed of separable

DNA binding and activation domains (Feng and Marzluf, 1996; Parsons et al., 1992; Qui et al., 1991; Sze et al., 1993). In most cases, the Zn(II)2Cys6 cluster motif is at the N- terminal end of the protein and is completely conserved among all the members. The activation domains, on the other end, usually show significant variation between proteins.

Transcriptional activation domains which are rich in glutamine have been identified in many transcription factors (Courey and Tjian, 1988; Feng and Marzluf, 1996; Ulmasov et al., 1999; Xiao and Jeang, 1998). Deletion of both glutamine-rich domains on PCO1 C- terminal end destroyed the function of the protein, suggesting these domains may actually be involved in transcription activation. Detailed studies on NIT4, a Zn(II)2Cys6 protein which also has two glutamine-rich domains, demonstrated that those domains are critical for transcriptional activation and also are functionally separable. A chimeric protein containing the NIT4 DNA binding domain and the activation domain from NIRA of

Aspergillus, which is completely different from NIT4, is functional in Neurospora (Yuan et al., 1991). In Neurospora, both Zn(II)2Cys6 factors related to nitrogen regulation,

NIT4 and PCO1, have glutamine-rich domains as their activation domain; whereas their counterparts in Aspergillus, NIRA and UAY respectively, have a different type of

activation domains. Interestingly, QA1F and ACR2, the other two Zn(II)2Cys6 factors of

Neurospora in non-nitrogen related regulation, lack similar domains rich in glutamine.

This finding suggests Neurospora Zn(II)2Cys6 factors involve in nitrogen related regulation might share similar functional domains. The C-terminal tail of PCO1 (32 a.a.), which includes a polyglycine stretch, appeared to be critical for the maximal function of

PCO1. The biological function, if any, of the polyglycine repeat is still unclear.

However, studies on the androgen receptor (AR), a transcription factor that has both polyglutamine and polyglycine regions in its activation domain, revealed that the length of the glycine repeats correlates with the transactivation activity of AR (Edwards et al.,

1999; Suzuki and Ito, 1999).

The pco-1 rip mutation resulted in the loss of inducibility of xanthine dehydrogenase by purines, but a constitutive basal level of enzyme activity is still present in all the culture conditions tested. A previous study showed that in xdh-1 mutants, in which the structural gene encoding XDH is mutated, absolutely no enzyme activity could be detected under either repression or inducing conditions (Griffith and Garrett, 1988).

The results suggest that the basal expression of XDH is not dependent on a functional pco-1 gene product. The continuous low level expression might be maintained by some constitutive expression of the xdh gene or be due to an unidentified transcription factor acting at xdh promoter.

NIT2 is viewed as a global-acting factor required for activation of the enzymes / permeases needed to utilize secondary nitrogen sources in Neurospora crassa. A well studied example is its involvement in the induction of nit-3 and nit-6, the genes which

encode nitrate reductase and nitrite reductase, respectively. The specificity of induction is achieved by protein-protein interaction between NIT2 and the pathway-specific factor

NIT4. A functional nit-2 gene product was also found to be essential in the expression of many enzymes needed for utilizing other secondary nitrogen sources (Marzluf, 2001).

However, induction of xanthine dehydrogenase activity appeared to bypass a requirement for a nit-2 (Figure 15). While a nit-2 mutant was reported to retain the ability to express

XDH, (Griffith and Garrett, 1988) it was interpreted as a mutation with a partial loss of

NIT2 function. With the availability of three different strains of nit-2 rip mutants, in which the nit-2 genes were fully destroyed by the rip process, this research has demonstrated that nit-2 is indeed not required for XDH induction. On the other hand, no observable protein-protein interaction between NIT2 and PCO1 could be detected in the pull-down assay with expressed proteins. Together, these findings suggest that NIT2 is not involved or at least not completely responsible for the induction of xanthine dehydrogenase.

Furthermore, the results reported here demonstrate that xanthine dehydrogenase activity is glutamine repressible, even in nit-2 mutants. This repression appeared to be dependent on nmr, but not nit-2 (Figure 15). The NMR protein lacks any obvious DNA binding domain and does not bind to the xdh promoter in mobility shift assays (data not shown). Moreover, NMR also failed to interact with PCO1 in a pull-down assay (Figure

13). Thus the manner by which NMR mediates nitrogen metabolite repression in the regulation of XDH is still in question.

Based on all the experimental evidence and the above analysis, I propose the existence of at least one unidentified transcription factor that is involved in the regulation of the xanthine dehydrogenase gene. This Factor X could be related to NIT2 or belong to a different class of transcription factors, but it is postulated to bind to the promoter independently and contribute to XDH expression. The NMR protein might interact with

Factor X and convey the nitrogen repression signal to the promoter as it does with NIT2 in nit-3 regulation. Factor X might also interact with NIT2 (Figure 18). Another possibility is the existence of an unknown negative regulatory protein that signals nitrogen repression and inhibits PCO1 function by some fashion, such as preventing

PCO1 from binding to DNA or inhibiting the transactivating activity of PCO1.

The constitutive basal level of xanthine dehydrogenase activity present even in mycelia grown in ammonia shows that nitrogen repression of the system is not complete

(Figure 14). The requirement of xanthine dehydrogenase at all times even when preferred nitrogen sources are present implies the enzyme might not function just to provide an alternative nitrogen source. Xanthine dehydrogenase might be important to some other crucial cellular processes. A steady level of XDH activity may be required for the operations of nucleotide degradation and purine recycling by purine salvage pathway. Most organisms recycle purine nucleotides due to the high energy cost and complicated steps involved in de novo synthesis. The free purine bases formed by the hydrolytic degradation of nucleic acid and nucleotides, namely adenine, guanine and hypoxanthine, can be reconverted to their corresponding nucleotides by phosphoriboxylation (Figure 19). However, a certain fraction of these nucleotides is

Figure 18. Proposed model for xanthine dehydrogenase induction. PCO1 is the pathway-specific regulator of the purine degradation pathway; NMR is the regulator mediates nitrogen repression. Factor X is the proposed factor that allows xdh expression and also mediates NMR function. The + and – signs indicate positive action and the negative role played by the regulatory genes, respectively.

The double-headed arrows indicate protein-protein interactions. The arrows pointing to the promoter indicates direct DNA binding. Blue arrows show the proposed functions of Factor X.

Pathway-Specific Nitrogen Repression (+) (-) pco-1 nmr

PCO1 (inactive) Glutamine Uric Acid Basal Expression (+) NMR PCO1 Factor X (active)

+1+1 PCO1 Factor X site site xdh Xanthine Dehydrogenase

Figure 19. Recycling of purines by the salvage pathway. The black arrows summarize the degradation of AMP and GMP to uric acid. The light and broader arrows indicate the recycling loops. XDH, xanthine dehydrogenase; PRPP, 5- phosphoribosyl-1-pyrophosphate.

actually degraded and a basal XDH activity is needed to prevent the intermediate, xanthine, from accumulating. Perhaps due to its particular biological importance in nucleotide metabolism, xanthine dehydrogenase is controlled differently from other enzymes in nitrogen assimilation and is not regulated by nit-2, which governs the acquisition of secondary nitrogen sources.

An increase of observed enzyme activity can result from an increased quantity of the enzyme, or an increased intrinsic activity of each enzyme molecule, or a combination of both. A previous study discovered that the addition of cycloheximide blocked the increase in xanthine dehydrogenase activity in uric acid-induced cells (Lyon and Garrett,

1978a). Furthermore, the result from an immunoblot analysis revealed de novo synthesis of XDH occurred after uric acid induction (Griffith and Garrett, 1988). Therefore, the change in XDH activity observed in this research has been interpreted to result from a change in gene expression. In fungi, transcriptional regulation has already been found as the main regulatory mechanism controlling the levels of the enzymes in a variety of metabolic processes, such as pathways in nitrogen assimilation, carbon catabolism, sulfur metabolism and ethanol catabolism (Felenbok et al., 2001; Fu and Marzluf, 1988; Haurie et al., 2001; Struhl, 1995; Thomas and Surdin-Kerjan, 1997).

Since the discovery of the first Zn(II)2Cys6 protein - GAL4 of S. cerevisiae in late 1989, the Zinc cluster proteins have been found throughout the ascomycetes class, such as Saccharomyces, Neurospora, Aspergillus, Kluyveromyces, Schizosaccharomyces,

Candida, Pichia, and Cochliobolus. More recently, Zn(II)2Cys6 cluster proteins were also found in the basidiomycete (mushroom-like) Lentinus edodes (Endo et al., 1994;

Kaneko et al., 1998)suggesting that the Zn(II)2Cys6 motif may have arisen before the divergence of the two major fungal groups. Zn(II)2Cys6 proteins are typically transcriptional activators and regulate a wide range of biological processes, including

primary and secondary metabolism, drug resistance, and meiotic development. Selective

pressure might be a factor leading to the wide usage of this class of DNA binding domain.

Analyzing the complete sequence of the Saccharomyces cerevisiae genome has revealed

56 Zn(II)2Cys6 genes, (Todd and Andrianopoulos, 1997) the function of the majority still remain to be determined. In Neurospora, a total of 10,082 genes have been predicted according to current genomic database, which is approximately double the number of genes in S. cerevisiae. The functional conservation revealed from known zinc cluster proteins among species implies a comparable number or even more genes in the

Zn(II)2Cys6 protein family can be expected in N. crassa. To date, only four

Zn(II)2Cys6 proteins have been identified and characterized in N. crassa, namely pco-1, nit-4, qa-1F and acr-2. The eight candidates discovered from my unrefined homology search in Neurospora genomic draft sequence did not include qa-1F and acr-2, indicating more Zn(II)2Cys6 genes could be found when search criteria are optimized. The finalized sequence of the Neurospora crassa genome is expected to be available in the near future, and can be expected to reveal all the potential zinc cluster proteins.

Phylogenetic comparison of undefined proteins with known ones from S. cerevisiae,

Aspergillus and other fungi could have the homologous regulatory genes quickly assigned and identified. The prevalence of Zn(II)2Cys6 proteins across the fungal kingdom will make the information gained from analyzing the genome of Neurospora

valuable. A wealth of information on the metabolic and developmental processes controlled by Zn(II)2Cys6 proteins in a model system such as Neurospora could contribute to an understanding of other fungi with medical or industrial importance.

CHAPTER 4

STUDY OF POSSIBLE COOPERATIVE DNA BINDING OF NIT2 AND NIT4, AND STUDY OF GLUTAMINE BINDING PROPERTY OF NMR PROTEIN

INTRODUCTION

Among the secondary nitrogen sources Neurospora crassa can utilize, nitrate assimilation has been subject to extensive investigation and is one of the best understood metabolic pathways. Inorganic nitrate is a good nitrogen source, but it is not used unless the cells lack the favored nitrogen sources. Utilization of nitrate requires the de novo synthesis of nitrate reductase, which is encoded by nit-3 gene. The expression of nit-3 is controlled by a dual-signal regulatory system to ensure that it is turned on only when both nitrogen derepression and nitrate induction conditions are satisfied. The first signal comes from the DNA binding protein NIT2, a GATA factor which mediates nitrogen repression / derepression. The repression signal is relayed to NIT2 by a negative regulatory protein, NMR, which does not bind to DNA. The second signal, nitrate induction, is mediated by the pathway-specific positive regulatory protein NIT4. NIT4 is a member of the GAL4 family of fungal transcription factors which possess a

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Zn(II)2Cys6 domain that provides sequence-specific DNA binding. Neither NIT2 nor

NIT4 alone is able to turn on expression of the nit-3 gene. To active nit-3 expression, both of these signals need to be presented. Though both proteins bind to the nit-3 promoter at their specific sites, a detailed study showed physical interactions between

NIT2 and NIT4 are critical for optimal expression of nit-3 gene (Marzluf, 2001).

Several questions remain to be answered concerning nitrogen metabolic regulation. The requirement of both DNA-binding proteins NIT2 and NIT4 in the expression of nit-3 gene raised the question whether these proteins bind to the nit-3 promoter independent of each other, or whether the interaction between the two enhances their DNA bind affinity. A vacuum filtration experiment was used to test DNA binding activities when both proteins were present. My results suggest that there is no cooperative binding of NIT2 and NIT4 at the nit-3 promoter.

Another fundamental question which puzzled researchers involved in the study of fungal nitrogen regulation is how negative control (repression) is initiated in the presence of preferred nitrogen sources. Glutamine appears to be the critical metabolite which exerts nitrogen catabolite repression. The signaling pathway and the elements involved in sensing the presence of glutamine are still unclear. The NMR protein has shown to be a negative regulator which binds to NIT2 and inhibits its transactivating function. It is tempting to speculate that NMR binds to glutamine to initiate the repressing process. My study used sensitive equilibrium dialysis to test this hypothesis and revealed that NMR does not bind to glutamine.

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RESULTS

DNA Binding of NIT2 and NIT4 to nit-3 Promoter

Purified NIT2 and NIT4 proteins expressed by E. coli were investigated for their binding properties to the nit-3 promoter region. The 334-bp Xba I ~ Nar I DNA fragment from the nit-3 promoter (-917 ~ -1251) containing both the NIT2 and NIT4 binding sites and flanking sequences was labeled and used in filter binding assays. To determine their binding properties, various amounts of the individual proteins were incubated with DNA at 30°C for 30 min and absorbed onto nitrocellulose membrane; then thoroughly washed and the membranes were counted for radioactivity (Figure 20).

Both proteins at low concentrations showed significant binding to the nit-3 promoter. At 7 nM and 18 nM, NIT2 bound to about 20% and 65% available binding sites, respectively. As for NIT4, 36% and 67 % binding sites were occupied at the concentrations of 8 nM and 16 nM, respectively. To test if NIT2 and NIT4 bind to DNA cooperatively, the XbaI ~ NarI DNA fragment of the nit-3 promoter was incubated with

NIT2 and NIT4 simultaneously. Cooperative binding would be expected to result in an increase of binding affinity, thus the extent of DNA binding would be higher than the binding of the individual proteins combined. The concentrations of both proteins chosen for this study were below that required to saturate the available binding sites as determined earlier (Figure 20). The results revealed that DNA binding of NIT2 and NIT4 was additive. The combination of NIT2 and NIT4 did not increase their apparent affinity for DNA binding (Figure 21).

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Figure 20. Equilibrium binding of NIT2 and NIT4 to nit-3 promoter. A. 1 nM of

[32P]-labeled Xba I ~ Nar I DNA fragment of nit-3 promoter was incubated with the indicated concentrations of NIT2 for 30 min as described under “Materials and

Methods.” Samples were loaded onto nitrocellulose filters, thoroughly washed, and the filters were counted for radioactivity. B. An identical experiment was performed using NIT4 protein.

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A 16000

14000

12000

10000

8000 CPM 6000

4000

2000

0 0 7 18 70 700 NIT2 (nM) B 16000

14000

12000

10000

8000 CPM 6000

4000

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0 0 8 16 80 800 NIT4 (nM)

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Figure 21. DNA binding assays of NIT2 protein, with and without NIT4. NIT2 protein (7 nM) was incubated with 1 nM nit-3 promoter for 30 min with various

concentrations of NIT4 as indicated. Samples were filtered through nitrocellulose

membranes, and the filters were counted for radioactivity. Filled bars show the

DNA binding by NIT2 and NIT4 combined. Empty bars indicate the binding by

NIT4 protein alone.

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14000

NIT4 + NIT2 (7 nM) 12000 NIT4

10000

8000

CPM 6000

4000

2000

0 0 5 10 15 NIT4 (nM)

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An enhancement of DNA binding affinity may be reflected by the stability of the ternary complex if cooperative DNA binding occurs between NIT2 and NIT4.

Competition binding assays were used to access the stability of the complexes. In the assay shown in Figure 22, proteins and probe DNA were incubated until equilibrium was reached, and a 20-fold molar excess of unlabeled probe DNA was then added. After various times, the mixtures were examined with the filter binding assay to determine the amount of the protein-probe complex that was still present. The time required for half of the complex to dissociate, t1/2, indicates the stability of the complex. However, the t1/2

and dissociation rate constants for these protein-DNA complexes could not be determined

by the above filter binding method, because at the shortest time possible almost all the

labeled complexes had already dissociated.

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Figure 22. Time course of the dissociation of protein-DNA complexes. NIT2-

DNA, NIT4-DNA, and NIT2-NIT4-DNA complexes were preformed by incubation at 25°C for 5 min. At time 0, excess unlabeled DNA (20 X) was added to serve as the specific competitor. After the addition of the cold DNA, the incubations were continued for the indicated times before filter binding assays were performed.

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90 NIT2 NIT4 NIT2 + NIT4 80

50 %

0 0123510203060 TIME (min)

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Equilibrium Dialysis of NMR Protein against [3H]-Glutamine

Equilibrium dialysis was used to detect a possible glutamine binding property of

purified NMR protein. This method permits low affinity binding to be measured without

disturbing the equilibrium. The dialysis cell was separated into two equal sized chambers

by a semipermeable membrane. One chamber was filled with 0.5 mM purified NMR and

dialyzed against 1 mM [3H]-glutamine (Figure 23A). Aliquots from each side were taken for radioactivity measurements at various time points. The sensibility of this method would be able to detect binding even if as little as 5% of the glutamine was bound to

NMR. However, Figure 23B shows the system reached equilibrium at 20 h, and no difference in radioactivity between the two chambers was observed. Similar results were

obtained in duplicated experiments extended to 48 and 72 hours (data not shown). Thus,

equilibrium dialysis failed to detect any binding between NMR and glutamine.

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Figure 23. Determination of glutamine binding property of NMR by equilibrium dialysis. A. The setup of equilibrium dialysis. The apparatus was separated in two chambers by a semipermeable membrane. At time 0, 0.5 mM NMR was at one side of the membrane and 1 mM [3H]-glutamine in the other. N, NMR; Q,

glutamine. B. Plot of radioactivity in the two compartments over the course of

dialysis.

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Sampling sites

Time *QN N*QN *Q *Q Dialysis membrane Time 0 Equilibrium

4500 NMR 4000 3H-GLN 3500

3000

2500

CPM 2000

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0 0 5 10 15 20 25 TIME (h)

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DISCUSSION

The regulation of gene expression is mediated by protein factors binding to promoter and enhancer elements. Proper control of gene expression requires that these factors bind with sufficient affinities and specificities. Many eukaryotic transcription factors do not appear to have the required affinity for specific DNA sequence by themselves. Interactions between multiple proteins are needed to achieve the required

DNA binding affinity and specificity for their DNA binding sites required for proper control of gene regulation. Protein-protein interaction between NIT2 and NIT4 has shown to be essential for optimal nit-3 expression, but cooperative binding was not observed in the DNA binding of these two proteins. Though the dissociation constant cannot be accurately determined by the setup of the experiment, each of the proteins binds to the nit-3 promoter with good affinity.

Five GATA factors, including NIT2, have been identified and characterized in

Neurospora to date. They regulate distinct areas of cellular function. However, they overlap in DNA-binding activities. In the case of nit-3 expression, the specificity of

activation of NIT2 comes from physical contact with pathway specific factor NIT4. An

intriguing question remains how the protein-protein interaction between NIT2 and NIT4

translate into the synergistic activation of nit-3. In vivo, DNA is tightly packed with chromatin proteins as nucleosomes. Promoters packed in nucleosomes are in a repressed state and not readily accessible. Even though in vitro both NIT2 and NIT4 individually bind strongly to their cognate binding sites, it is still possible NIT2-NIT4 interaction is

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required for efficient DNA binding in vivo. NIT2 may help open up chromatin to allow

itself and NIT4 to bind to DNA. Another possibility is both proteins are required for effective recruitment of general transcription factors and RNA polymerase to the promoter. NIT4 possesses a glutamine-rich putative activation domain, similar to that of human transcription factor SP1. The glutamine-rich activation domain of SP1 mediates direct interaction with TFIID and is essential for optimal activation (Saluja et al., 1998).

It is possible that NIT4 functions in the similar manner and requires NIT2 for effective recruitment of the elements needed for assembling the initiation complex.

The mechanism for sensing and initial signaling of the availability or quality of the nitrogen source in Neurospora is still not understood. Glutamine has been identified as the probable metabolite which exerts nitrogen metabolite repression in Neurospora

(Premakumar et al., 1979; Wang and Marzluf, 1979). The results from equilibrium dialysis, capable of detect low-affinity ligand binding without disrupting equilibrium, suggest that NMR is not able to bind glutamine. A similar study revealed that NIT2 also does not bind to glutamine (unpublished result). The identities of the element or signaling pathway that recognizes the level of glutamine concentration remain to be

determined. It is possible that a protein complex such as NIT2/NMR homodimer

recognizes glutamine. Another possibility would be the existence of an unknown protein

or a factor of other nature that detects the glutamine status and conveys the repressing

signal to a global-acting factor such as NMR or NIT2. A glutamine tRNA was reported

to signal nitrogen status for regulation of selective developmental processes in

Saccharomyces cerevisiae (Beeser and Cooper, 1999; Murray et al., 1998). It is not clear

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if a similar mechanism exists in N. crassa. Understanding the mechanisms how

Neurospora sense this environmental cue and responds is an important goal for the future research.

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CHAPTER 5

GENERAL DISCUSSION

In the living environment of fungi, nitrogen is generally a limited resource. A fungus faces two basic questions when it comes to nitrogen regulation: what nitrogen sources are available and which of those should be used preferentially. Versatile metabolic systems therefore have evolved in fungi to efficiently utilize a wide range of compounds as their source of nitrogen in different conditions, which inevitably requires precise expression of the many genes involved in nitrogen assimilation. Similar regulatory systems also occur in the utilization of other biologically important compounds such as carbon, sulfur, and phosphorus.

In nitrogen regulation, the availability of a specific nitrogen source is generally monitored by regulatory proteins that have a narrow range of activity. Their activities are essential but not sufficient to activate the expression of genes in specific pathways. The regulatory proteins involved in these pathway-specific processes usually belong to the zinc binuclear cluster family, a class of transcription factors specific to fungi.

A second regulatory component, known as nitrogen metabolite repression, determines that the availability of primary (preferred) nitrogen sources, then represses the

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utilization of secondary (poorer) nitrogen sources. This component is a system that

monitors the nitrogen state of the cell, i.e. the cell’s requirement for nitrogen, and in turn

controls transcription of many structural genes. In most fungi, this process is mediated

primarily by a transcription factor of the GATA family, such as NIT2 and AREA in N.

crassa and A. nidulans respectively. This signal is also not sufficient to activate

transcription in itself. Signals from both of the above components are required to

successfully initiate transcription of specific genes.

Studies in other metabolic systems, such as utilization of secondary carbon or

sulfur sources, revealed similar regulatory schemes to nitrogen regulation. Carbon metabolism has been well studied in Aspergillus nidulans. CREA plays a central role in

carbon catabolite repression to ensure that primary carbon sources, e.g., glucose, are

preferentially used. Secondary carbon source, e.g., ethanol, quinic acid, proline, acetate,

or acetamide are only used when primary sources are not available. Like nitrogen

regulation, expression of the genes which encode specific enzymes for utilization of the

secondary carbon sources requires both the lifting of carbon catabolite repression and

pathway-specific induction. In the sulfur regulatory circuit, methionine is the preferred

sulfur source. The enzymes and permeases needed for utilizing secondary sulfur sources

are also regulated at the transcriptional level by both positive and negative regulatory

factors.

There are a number of compounds that can be used both as nitrogen and as carbon

sources by fungi. In these cases, the structural genes are generally subject to both

nitrogen and carbon metabolite repression, as well as specific induction. A well studied

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example is the prn gene cluster involved in proline utilization in A. nidulans. There are five genes included in the prn cluster, prnA, prnB, prnC, prnD and prnX. prnA encodes a pathway-specific regulatory protein PRNA, which regulates the expression of structural genes prnB, prnC and prnD required for transporting and metabolizing proline. Besides

PRNA, transcriptional regulation of these genes also involves AREA of the nitrogen regulatory circuit as well as the transcriptional repressor, CREA, from the carbon regulatory system. Additionally, the gene cluster also responds to amino acid starvation, and the stage of development. Therefore, at least five regulatory circuits have been found to act at the central control region of the prn gene cluster (Sophianopoulou et al., 1993).

How these signals from different regulatory circuits converge in the regulatory element of prn cluster is still poorly understood, given that each of the five systems is itself complicated and still under scrutiny. Just between the best studied nitrogen and carbon regulatory systems, it had been speculated that CREA represses PRNA activity and AREA in turn neutralizes the repression mediated by CREA. However, when the

CREA binding sites at prn regulatory region were deleted, instead of leading to depression of the genes as anticipated, carbon metabolite repression was no longer operative and gene expression became fully dependent on AREA. Other possibilities for the integration of the regulatory circuits have been proposed, but they still remain to be investigated (Gonzalez et al., 1997).

The example of proline metabolism in A. nidulans illustrates the complexity and high degree of integration among metabolic regulatory systems, which almost certainly occurs in other fungal systems. Xanthine dehydrogenase, as discussed in Chapter 3,

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involves at least two biological pathways N. crassa. It is essential for purine degradation

as a source of nitrogen and subject to nitrogen repression; it is also critical for recycling

purine bases in the purine salvage pathway. These findings imply the possible

interconnection between nitrogen assimilation circuits and the regulation of nucleotide

metabolic / biosynthesis pathways.

In a living organism, thousands of genes and their products function in a

complicated manner that create and sustain life. Traditional methods in molecular

biology usually work on a single gene at a time. This approach generally has limited

throughput, and the whole picture of gene function is difficult to obtain. In the past few

years, the development of DNA microarray technology makes monitoring the whole

genome on a single chip possible. So researchers can gain a better picture of the

interactions among thousands of genes. S. cerevisiae was the first eukaryotic organism to have its entire genome sequenced. Approximately 6,000 genes which encode identified and unknown proteins have been predicted. Yeast was also the first organism whose entire genome was represented on a DNA microarray which has even become commercially available. A recent study utilizing DNA microarray to access the global transcriptional profile in S. cerevisiae cultures limited for variant nutrients discovered a large number of transcripts were significantly changed by alternating growth condition.

A total of 225 transcripts were found with significantly altered level (higher or lower) when the carbon (glucose) supply became limited. In a nitrogen-limited growth condition, 66 transcripts were changed. Of there, 51 genes showed a significantly higher transcription level and 15 genes were expressed significantly lower in a manner specific

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to nitrogen limitation. Those increased in expression level included 25 structural genes

known to be involved in transporting and utilizing nitrogen compounds, plus five genes involved in cellular differentiation. The functions of the remaining 21 genes with a higher transcription level remain to be identified (Boer et al., 2003).

The study of global transcriptional profiles appears to be an efficient way to compare the expression level of thousands of genes simultaneously and to enable a quick

screening of specific genes involved in a particular biological condition. With the near

completion of the N. crassa genome project, all the genes predicted (10,082, vs. 6,000 of

S. cerevisiae) are already being printed into microarrays which will be available to workers in the field. A goal of using DNA microarray in metabolic studies is to discover new phenomena which are still unknown in current knowledge, such as the interconnections between different regulatory circuits.

Protein-DNA binding and protein-protein interaction are two major elements involved in transcriptional regulation. When proteins are used as probes, DNA microarrays can be used to identify genomic locations of transcription factors’ DNA binding sites, thus can identify the genes they might regulate. However, studies of protein-protein interactions will still require conventional approaches such as pull-down assays, immunoblotting, or the two-hybrid system. Mutagenesis procedures are also needed to create mutant strains. Another limitation of DNA microarray is that one obtains gene expression data only at the transcriptional level, but not at the translational or post-translational level, which can be very important in studying protein expression.

Furthermore, comparative gene expression studies using DNA microarrays will not be

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able to identify transcription factors whose expression levels are not changed when the conditions change. From the global transcriptional profile obtained with S. cerevisiae under nitrogen-limited growth, the genes identified with an increased transcription level are mostly structural genes, such as dal-1, dal-2, dal-3, dal-4, and dal-5 needed for transporting and metabolizing allantoin. The transcripts of the regulatory proteins that control their expression, gln-3 and dal-81, however, were not among the genes identified by this approach (Boer et al., 2003).

The availability of the new molecular techniques and the accessibility to the wealth of genetic information give researchers many options to solve questions. Some may prefer to utilize computer programs in attempts to identify patterns and connections that remain unknown to date. Meanwhile, more creative laboratory work is also needed to understand the complex network of metabolic regulation. The combination of both approaches is likely to rapidly advance our understanding of the metabolic regulatory systems that govern a broad array of important biological processes.

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