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CONSTRUCTION OF A NOVEL FUNGAL GUS EXPRESSION PLASMID, 

AND ITS EVALUATION IN ASPERGILLUS NIDULANS. 

A Thesis Presented in Partial Fulfilment of the 

Requirements for the Degree of 

Master of Science in Genetics 

at 

Massey University, Palmerston North 

Aotearoa I New Zealand 

Tania Louise McGowan 

1996 



ii 

ABSTRACT 

A GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site 

for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. 

A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpd.A 

promoter (glyceradehyde-3-phosphate dehydrogenase) into the multi-cloning site of 

pFunGus for the assessment of the plasmid's transformation and expression properties 

in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified 

by analytical restriction digests and by its property of GUS expression in A. nidulans. 

The plasmid was integrated into the A. nidulans genome via cotransformation with the 

phleomycin resistance plasmid, pAN8-1. Transformation frequencies of between 3 and 

250 transformants per µg of pAN8-1 DNA were obtained. Initial screening for 

cotransformation yielded no pFG-gpd transformants. Attempts to improve 

cotransformation frequencies by optimisation of cotransformation conditions were 

unsuccessful. However, large scale screenings of transformants lead to cotransformants 

being isolated at a very low cotransformation frequency. Approximately 0.45% of 

pAN8-1 transformants possessed the GUS phenotype. 

The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. 

Six out of the eight transformants had a single copy integration. Of the remaining two 

transform.ants, one had three copies integrated at separate locations, one of which was 

disrupted, and the other had four copies integrated as tandem repeats, one of which was 

disrupted. All the transforming DNA appeared to be integrated ectopically. 

The physiology of the transformants was assessed by dry weight increase, colony 

extension and total protein content. These showed that the transformants biology was 

not significantly compromised by the transforming DNA. 

Finally, high levels of GUS expression were observed in all pFG-gpd transformants and 

the GUS expression per copy of the GUS expression cassette integrated into the genome 

was constant. These results showed that the transformed gene copy number determined 

the levels of gene activity rather than the position of integration in the genome. 



lll 

Overall these results demonstrate the potential application of the versatile GUS 

expression plasmid, pFunGus for reporter gene studies in filamentous fungi. 



lV 

ACKNOWLEDGMENTS 

Firstly, I would like to thank my supervisor, Jan Schmid, for his guidance throughout, 

I'm sure we have both learnt a lot from the experience. Also, to Barry Scott whose 

interest and encouragement was invaluble. Many thanks to Carolyn Young who 

provided much required assistance with the cloning work, as well as much, much more. 

And to Diane Bird who aided with invaluable discussions and ideas on the 

transformations. 

Thanks to the many staff and students of the Department of Microbiology and Genetics, 

whose friendly smiling faces in the corridor always provided me with a pick-me-up 

when I most needed it. In particular I would like to thank Karyn for being a great lab 

mate (I missed you when you were gone), and more recently Scott. To Mike, Kate, 

Linda, Richard, Carolyn, David, Miranda, Anita, Bennett, Brendon and more that I've 

forgotten, thanks for being mates. 

Special thanks go to Mum, Dad, Nana, Carl and Bryce who provided constant love and 

support (and computer consultants), even through the frustration of wondering whether I 

would ever leave varsity. 

To all the flatmates that have been and gone, especially Steve, Lisa, Dave and Austen, 

you guys made me laugh. A special, special thanks to Austen, you've put up with living 

with me, working with me and you're still a wonderful, wonderful friend! To the 

'friendly folk' at MUSA, thanks for a great year (even if it meant this thesis was longer 

in the making), and kept fighting the good fight, and to the rabble rousers all over 

Aotearoa, keep up the struggle. To special friends, Ruth, Andrew, Nicki the gift of 

friendship is great and I don't take it lightly, thanks! Finally, to Paul and Carmel, your 

love and energy sustained me throughout. 

Thanks are also due to AGMARDT, for their generous financial assistance, as well as 

Lottery Science and Massey University Graduate Research Fund for their financial 

support of this project. 



TABLE OF CONTENTS. 

ABSTRACT II 

ACKNOWLEDGMENTS iv 

TABLE OF CONTENTS. V 

LIST OF FIGURES. X 

LIST OF TABLES XI 

1. INTRODUCTION. 1 

1.1 THE BIOLOGICAL IMPORTANCE OF FUNGI ............ ....... .... .................... ...... . 1 

1.2 THE BIOLOGY AND GENETICS OF ASPERGILLUS NIDUI.ANS . ................... 1 

1.3 TRANSFORMATION IN FILAMENTOUS FUNGI. .................................... .. ...... 5 

1.3.1 Overview ............................................................................................................ 5 

1.3.2 Components of Fungal Expression Vectors used in Transformations ............... 5 

1.3.3 DNA Uptake ....................................................................................................... 9 

1.3.4 Fate of Transforming DNA: Integration Events .............................................. 10 

1.3.5 Fate of Transforming DNA: Transient Transformants ................................... 13 

1.3.6 Position Effects and Gene Copy Number in Determining Levels of 

Transformed Gene Expression ........................................................................ 14 

1.3.7 Cotransfonnation ............................................................ .............. ........ ........... 15 

1.4 THE USE OF REPORTER GENES IN THE STUDY OF FUNGAL 

PROCESSES ............................................................................................................... 16 

1.4.1 Overview of Methods to Study Fungal Cell Processes .................................... 16 

1.4.2 The ~-glucuronidase (gusA) Reporter Gene System ........................................ 18 

1.4.3 GUS Expression Plasmids ............................................................................... 19 

1.5 AIMS AND OBJECTIVES OF THIS STUDY ..................................................... 20 

2. MATERIALS AND METHODS. 21 

2.1 FUNGAL STRAINS, BACTERIAL STRAINS AND PLASMIDS ...................... 21 

2.2 GROWTH MEDIA ................................................................................................ 21 

2.2.1 Liquid Media .................................................................................................... 21 



2.2.2 Solid Media ... .. .... ................. ... .......... ..... ..................... ........ .. ... ......... .............. . 23 

2.2.3 Media Antibiotics and Supplements ...... ......... .... .. ........ .... ............................... 23 

2.3 MAINTENANCE OF CULTURES ............... .... ....... ...... ... ..... .... ...... ............. ....... 24 

2.4 CO:MMON BUFFERS AND SOLUTIONS .......... .......... .. ....... .. .. ... ..... ...... ...... ..... 24 

2.4.1 TE buffer ... ............ ....... .. .................. ... .......... ..... ........... .... .. ........... .... ......... ..... 24 

2.4.2 1 x TBE Buffer ... ........ ... .......... .................... .. ......... ...... ........ ........... .......... ....... 24 

2.4.3 1 x T AE Buffer ........ ......... ... ............. .................................... ............ ............... 24 

2.4.4 Tris-Equilibrated Phenol. .. ........................ ....... ...... .. ..... ... ................ ............ .. .. 25 

2.4.5 DNase free RNaseA ....... .......... ..................................... ..... .. ................... ....... .. 25 

2.4.6 Lysis Buffer ......... ................. ......... .. ........ ... ........ ........ ... ........... ............ .... ........ 25 

2.4.7 Gel Loading Buffer (10 x) .... .. ...... ................... ....... ........ .......... ............ ... ......... 25 

2.4.8 OM Buffer. ... ................ .. ... .. .............. ..... .... ...... ............... .. .. ... .... ....... .......... ..... 25 

2.4.9 STC Buffer. ... ... .. .. .......................... ...... .... ........ ... ...... ..... ... ..... ........ .. ... .... ...... ... 25 

2.4.10 40% PEG Solution ................... .............. .................... .......... .............. ..... ....... 25 

2.4.11 GUS Extraction Buffer. ...... ......................... .. .... .. ...... ................. .... ............... . 26 

2.4.12 10 x SSPE ....................................... ................. .... ..................................... ..... 26 

2.4.13 lOOOx Ethidium Bromide Stock (for staining of agarose gels) ....... ...... ......... 26 

2.5 DNA PREPARATIONS ......... ................................. ................................. ............. 26 

2.5.1 Phenol/Chloroform Purification of DNA. ......... ................ ............ ................... 26 

2.5.2 Concentration of DNA by Ethanol Precipitation ........... .................................. 26 

2.5.3 Boiling DNA Mini-preparation ............ .......................... ......... ....... ..... ............. 27 

2.5.4 Large Scale Plasmid Preparation with Purification by PEG Precipitation ....... 27 

2.5.5 Large Scale Plasmid Preparation with Purification by CsCl/EtBr 

Centrifugation .............. .... .... ......... ................... ....... .. ....... ... ......... .............. ...... 28 

2.5.6 Mini Plasmid Preparation for Checking Presence of Insert DNA .................... 29 

2.5.7 Mini-preparation for Isolation of DNA from Fungal Cultures .. ........ ............... 30 

2.5.8 Extraction of DNA Fragments from Agarose Gel. ...................... ...... ......... .... . 30 

2.6 DETERMINATION OF DNA CONCENTRATION ............................. ............... 31 

2.6.1 Determination of DNA concentration by spectrophotometric assay ................ 31 

2.6.2 Determination of DNA concentration by fluorometric assay ............ ............... 31 

2.7 DNA MANIPlJI..ATIONS .......... ........ ........... ... ........................... .......................... 32 

2.7.1 Restriction Enzyme Digests ................... ..... ........................ ............................. 32 



2.7.2 Agarose-gel Electrophoresis of DNA. ................................................. .... ... .... .33 

2.7.3 Determination of Fragment Sizes .................... ........ ........... .. ....... ..................... 33 

2.7.4 Repairing 3' or 5' Overhanging DNA Ends to Generate Bunt Ends using the 

Klenow Fragment. ..................................... ...................................................... 34 

2.7.5 Dephosporalytion of Digested DNA 5' Ends ........................ ................... ... .... .34 

2. 7 .6 Ligation Reactions .......................................................................................... .34 

2.8 TRANSFORMATION OF E. COLi . ............................... .......... ................. .......... .35 

2.8.1 Transformation using Calcium Chloride ......................................................... .35 

2.8.2 Transformation using Electroporation .................... ......................................... 36 

2.9 COTRANSFORMATION OF A. NIDULANS . ....................... .. ........................... .37 

2.9.1 Preparation of Spore Suspensions from A. nidulans . ....................................... 37 

2.9 .2 Preparation of Fungal Protoplasts ............... ....... .................... ... ......... ....... ....... 38 

2.9.3 Cotransformation of A nidulans (Method l) ...... .. ................ ...... ....... ............. .39 

2.9.4 Cotransformation of A nidulans (Method 2) ........................... .... ................... . 39 

2.9.5 Selection of Cotransformants ........................................... .......... .. .. ...... .... ....... .40 

2.9.6 Single Spore Purification of Transformants ...................... ......... .................. .. . .40 

2.10 MEASUREMENT OF TRANSIENT EXPRESSION OF TRANSFORMED 

DNA ............... .......... ....... .......... .............................................. ......... .. .. ........... .......... ... 40 

2.11 DNA HYBRIDISATIONS ........ ......... ...... .......... ..................... ............................ 41 

2.11.1 Southern Blotting ........................................................................................... 41 

2.11.2 Radioactive (32P) Labelling of DNA Probe ................................................... .42 

2.11.3 Southern Hybridisation ... ..... ................... ...... ...... .... ........................ ...... ......... 43 

2.11.4 Autoradiography of Southern Blots ........ ............... ............ ............................ 44 

2.12 GROWfH RATE DETERMINATION OF FUNGAL TRANSFORMANTS ... 44 

2.12.1 Colony Growth ..................... ........... ............... ................. ... ........ .................... 44 

2.12.2 Dry Weight Determination ........................................ ... ...... ............. ............... 44 

2.13 MEASUREMENT OF GUS EXPRESSION ....................................................... 45 

2.13.1 Protein Quantification ................... ...... ............................... ... ......................... 45 

2.13.2 Quantification of GUS Expression . ......................................... ......... ............ .46 

2 .14 STATISTICAL ANALYSIS ................................................................................ 47 

2.14.1 Linear Regression, Correlation Coefficient, and Exponential Regression ..... 47 

2.14.2 Z-test. ............................................................................................................. 48 



3. RESULTS. 49 

3.1 CONSTRUCTION OF A GUS EXPRESSION PLASMID, PFUNGUS ..... ........ .49 

3.1.1 Preparation of DNA Fragments Required for pFunGus Construction ............ .49 

3.1.2 Ligation of DNA Fragments for pFunGus Construction ............................ ...... 53 

3 .1.3 E. coli transformation of Ligated Fragments and Preliminary Analyses of 

Transformants .................................................................................................. 53 

3 .1.4 Identification and Confirmation of Potential pFunGus Containing E. coli 

Transformants .................................................................................................. 54 

3.2 CONSTRUCTION OF PFG-GPD, A PROMOTER CONTAINING 

DERIVATIVE OF PFUNGUS ......... ........................................................................... 58 

3.2.1 Preparation of DNA Fragments Required for pFG-gpd Construction ............. 58 

3.2.2 Ligation of DNA Fragments for pFG-gpd Construction .................................. 60 

3.2.3 E. coli Transformation of Ligated Plasmid and Preliminary Analysis of 

Transformants . ......................... .............. ..... ... .... .................. .. ..... .......... .......... . 61 

3.2.4 Identification and Confirmation of Potential pFG-gpd Containing E. coli 

Transformants .............................................. ... ................................................. 61 

3.3 TRANSIENT EXPRESSION OF THE PFG-GPD PLASMID . ............................ 67 

3.3.1 Transient GUS Expression Levels in A. nidulans Transformants .. .... ............. . 67 

3.4 COTRANSFORMATIONS OF A. NIDUI.ANS . ................................................... 70 

3 .4.1 Preparation of Transforming DNA for all Cotransformations ................... ... ... 70 

3.4.2 Initial Cotransformations of A. nidulans R21 .................................................. 71 

3.4.3 Attempt at Optimisation of Cotransformation Procedure using pNOM102 . ... 75 

3.4.4 Large Scale Cotransformations of A. nidulans Yielding Cotransformants with 

pFG-gpd + pAN8- l .......................................................................................... 77 

3.4.5 Selection and Purification of Cotransformants . ..................................... .......... 79 

3.5 ANALYSIS OF PFG-GPD A. NIDUI.ANS TRANSFORMANTS ........................ 80 

3.5.1 Analysis of Integration of pFG-gpd into the A. nidulans Genome ................... 80 

3.5.2 Physiological Characteristics of A. nidulans Transformants ........................... 89 

3.5.3 Analysis of Total Protein Levels in Transformants at Mid Log Phase ............ 92 

3.5.4 Analysis of GUS Expression in pFG-gpd Transformants . .............................. . 96 

4. DISCUSSION. 99 

4.1 OVERVIEW .......................................................................................................... 99 



4.2 EVALUATION OF COTRANSFORMATION ..... .. ........................... .. ................ 99 

4.2.1 Possible Reasons for Low Levels of Cotransformation Frequencies of pFG-

gpd .... ............. ........... .. .. .. ........................... .. .. ... ........ ... ......... ..... ............. ......... 99 

4.2.2 Possible Explanations for Higher Cotransformation Frequencies Observed 

with pNOM102 than with pFG-gpd ...... .............. ........ .... .... ................... ...... . 103 

4.3 EVALUATION OF INTEGRATION EVENTS IN TRANSFORMANTS ........ 104 

4.3.1 A Predominance of Single Copy Integrations with Intact GUS Expression 

Cassettes Observed in pFG-gpd Transformants ............... .......... ............ ....... 104 

4.3.2 Analysis of Ratios Between Type I/ II/ III Integration Events Show a 

Predominance of Type II (Ectopic) Integration Events ........... ................. ..... 105 

4.3.3 Physiological Response of A. nidulans to the Presence of pFunGus 

Derivatives ............................... .. ... ............ ......... ... ............................. ..... ....... 106 

4.3.4 Correlation Between GUS Expression Levels and Transformed Gene Copy 

Number. ......... ................................ .... ..... ....................................................... 106 

4.4 SUMMARY AND FUTURE PROSPECTS ....... ................................................ 108 



LIST OF FIGURES. 

Figure 1 Life cycle of Aspergillus nidulans, (reproduced from Pontecorvo, 1953) ......... .3 

Figure 2 Schematic representation of types of expression cassettes .......................... ..... ... 8 

Figure 3 Diagram of the intergration events observed in fungal transformation ............. 11 

Figure 4 Summary of pFunGus Construction ...... .. ....... .... .... .......... ... .............................. 50 

Figure 5. Agarose gel showing the profile of bands produced for analysis of the potential 

pFunGus clone ... .... .... .... .. ..... .......... ....... ... .................... ....... .... ... ................. .. .... 54 

Figure 6 Restriction map of pFunGus .... .. .. ..... ....... ... ............ .. ............... .. ... .... ... .......... .... 59 

Figure 7 Agarose gel showing the profile of bands produced for analysis of the potential 

pFG-gpd clones ............ ........ ..... .. ...... ...... .... .... ...... ............. ...... ..... ....... .. .. .. ... ..... 62 

Figure 8 Restriction map of pFG-gpd ... .. ......... .... .......... ..... ... ........ ...... .... .... ... .. ..... .... .. .... 66 

Figure 9 Agarose gels showing the profile of bands produced by digesting A nidulans 

DNA to demonstrate consistency in the DNA concentration and degree of 

digestion between lanes ................. .... .... .......... .... .. .... ................. .. ...... .... ..... .. .... 82 

Figure 10 Profiles of the integration of the GUS cassette in pFG-gpd transformants . ... . 84 

Figure 11 Diagrammatic representation of the types of integrations observed .......... .... . 87 

Figure 12 Graphs of colony extension versus time ...... ... ... .............. .......... ....... ............... 90 

Figure 13 Growth of untransformed and transformed A. nidulans in liquid media defined 

as dry weight. ...... ......... .................. ........................ ............................................ 93 



LIST OF TABLES 

Table 1 Plasmids and Strains .......................................................................................... 22 

Table 2. Summary of band sizes produced from digests for the analysis of the potential 

pFunGus clone ... ................................................ ........ .. ... ............... .. ..... ..... ........ 57 

Table 3 Summary of band sizes produced from digests for the analysis of the potential 

pFG-gpd clones .......................... ........................................................................ 64 

Table 4 GUS enzyme specific activity in transformed protoplastsa .. .... ...................... .... 69 

Table 5 Results of initial cotransformation experiments ........... ....... .... ....... ..... ... ............ 73 

Table 6 Summary of cotransformations with pNOM102 and pAN8-1 ........................... 76 

Table 7 Summary of results for the final cotransformation experiments ........................ 78 

Table 8 Summary of the types of integrations ................................................................. 86 

Table 9 Growth rates of transformants determined by dry weight versus time ..... ..... ..... 94 

Table 10 Protein concentrations in GUS extracts of transformants in mid-log phase of 

growth ................................. : .............................................................................. 95 

Table 11 Specific activities of GUS Enzyme in pFG-gpd transformants showing the 

correlation with copy number. ................................ ........................................... 98 



1 

1. INTRODUCTION. 

1.1 THE BIOLOGICAL IMPORTANCE OF FUNGI. 

Fungi are eukaryotic organisms that have provided model systems for studying cell 

processes, as stated by Watson et al., 1987 in a text on molecular biology: "If we can 

solve the same problem equally well with yeast or with human cells, common sense tells 

us to stick with the simpler less expensive system". This thinking led to a wealth of 

research using fungi, and headed an understanding of basic processes in eukaryotic cells, 

in particular of meiosis and mitosis. New research developments have enabled studies 

in animal and plant systems to become increasingly viable, therefore research of fungi 

has turned to studying them in there own right as unique life forms, for their 

contribution to the ecosystem, and importance medically and economically (Wessels, 

1991). 

Fungi obtain organic nutrients by either infecting other living organisms as parasites, or by 

breaking down dead organic matter as saprophytes (Alexopoulos and Mims, 1979). They 

are responsible for the majority of known plant diseases as well as many diseases of 

human and animals (Wessels, 1991). They also provide the basis for many industrial 

processes, including commercial fermentations for organic acid production (e.g. citric 

acid) and in the food industry for the preparation of cheeses. The health industry depends 

on fungi for the production of drugs, for example corgmentrine and cortisone, vitamin 

preparations, and finally the manufacture of a number of antibiotics, e.g. penicillin and 

cephalosporin (reviewed by Davies, 1991; Martin, 1991). 

1.2 THE BIOLOGY AND GENETICS OF ASPERGILLUS NIDULANS. 

Aspergillus is a widely distributed genus that plays a major role in the natural world as 

well as in human processes, positively in industry for the production of secondary 

metabolites and negatively in causing disease in plants, animals and spoilage of foods 

(Martinelli, 1994). The species A. nidulans has been utilised as a model system for the 



2 

study of biological and genetic processes in filamentous fungi . Several features of A. 

nidulans make it suitable for study as a model organism (Pontecovo, 1953), and the 

following discussion will describe A. nidulans from this perspective. 

Aspergillus nidulans is a filamentous fungi which develops as hyphae which are 

collectively known as mycelia. Hyphae have cell walls enclosing a cytoplasmic mass 

which moves inside the tubes. As the cell wall is built at the tip the cytoplasmic mass 

moves forwards and ramifies into branches (Wessels, 1991). In A. nidulans hyphae are 

septate with multi-nucleate compartments. Undifferentiated hyphae may form structures 

that produce asexual spores that can then germinate and produce more hyphae. The 

asexual stage is prevalent in A. nidulans and produces spores, ( conidia) that contain a 

single haploid nucleus. The sexual stage can occur by the fusing of two hyphae, either 

from different or the same mycelia making A. nidulans is a homothallic species. Sexual 

recombination results in meiosis and the production of a multi-cellular structure, an ascus, 

containing four diploid ascospores. This defines A nidulans as a member of the fungal 

class, Ascomycetes (Martinelli, 1994). This life cycle is shown in Figure 1. 

Aspergillus nidulans has minimal nutritional requirements, and prefers to grow at 

temperatures around 37-40°C. At 37°C it grows relatively rapidly with a rate of hyphal 

extension of approximately 4µm per minute on complete or minimal media (Pontecorvo, 

1953). Conidia lend themselves to plating techniques used for yeast and bacteria. Rapid 

growth generally means conidia are available for harvest after 48 hours, and colonies are 

classifiable by morphology, colour of conidia, etc., thus allowing quick analysis of many 

generations. Tetrad analysis can be applied to the ascospores produced by the sexual stage 

and these ascospore have been utilised for the long term storage of strains (Pontecorvo, 

1953). 



Figure 1 Life cycle of Aspergillus nidulans, (reproduced from Pontecorvo, 1953). 

Zy.;;i;--Meiosis----.

0 
® ~Asc~s 

2, f] 8 :__: •:: ·. ..:<:: 

3 



4 

The genetics of filamentous fungi has been well studied, especially in A. nidulans. This 

species, relative to other fungi, has a small genome of approximately 20,000kb, about 7 

times the size of E. coli and 1 % the size of a human haploid genome. The genome is 

organised on 8 chromosomes per haploid genome (reviewed by Turner, 1993). Unique 

sequences make-up 97-98% of the total genome, with the remaining 2-3% repeated 

sequences being assigned to the ribosomal RNA genes. The 5' region of a typical gene 

carries sequences responsible for the binding of regulatory proteins and general 

transcription factors. Many genes are strongly regulated by activation and/or repression at 

the level of transcription (reviewed by Caddick and Turner, 1993). A feature of fungal 

genome organisation seen in A. nidulans is functionally related genes occurring in clusters. 

These contain two or more loci that code for related functions, examples of these are the 

loci involved in; alcohol utilisation (Lockington et al., 1985), penicillin biosynthesis, 

(McCabe et al., 1990), and nitrate utilisation (Johnston et al., 1990). Of the 82+ genes 

isolated by cloning, many have been sequenced permitting studies of their structure and 

function (reviewed by Matinelli et al., 1994 ). For these reasons A. nidulans provides a 

parallel system for the study of processes in more complex and less well defined fungi. 

Methods for transforming sequences into A. nidulans are well established (f ilbum et al., 

1983; Ballance et al., 1983; Yelton et al., 1984). The DNA is integrated into the genome 

and produces mitotically stable transformants. The multi-nucleate nature of hyphae means 

balanced heterkaroytes can be created (Pontecorvo, 1953). These contain a mixture of two 

parental nuclei that differ at one or more genetic loci, and provide the opportunity create 

and investigate lethal mutations. Transformation has provided an opportunity to study 

gene expression and regulation at the molecular level. This is discussed in more detail in 

Section 1.3. 

In summary A. nidulans provides a good model system for the study of biological and 

genetic phenomena in filamentous fungi. It is (i) rapidly growing, (ii) its genome 

organisation is relatively simple with little repeating DNA and (iii) regulation of gene 

expression predominantly occurring a the level of transcription, (iv) many control and 

structural genes have been cloned and sequenced, and (v) transformation systems have 

been established. 



5 

1.3 TRANSFORMATION IN FILAMENTOUS FUNGI. 

1.3.1 Overview. 

Transformation is an experimental method for introducing DNA sequences into 

genomes. It was first reported in filamentous fungi (Neurospora crassa) in 1973 

(Mishra and Tatum, 1973), and now is widely established in many types of fungi. 

Development of transformation systems has not only allowed the introduction of 

endogenous sequences into fungi, but the transfer of genes between fungal species and 

from prokaryotes, animal, and plants into fungi. This is termed 'heterologous 

expression', and predominantly these genes are transcribed, translated, and give 

functional products. Many gene products of bacteria, plant and animal origin have been 

produced under the control of fungal regulatory systems, and provide many applications 

(reviewed by Saunders et al., 1989; Davies, 1994). Also, regulatory systems are 

transferable between Ascomycetes. This shows conservation of regulatory mechanisms 

within this class of filamentous fungi (Austin et al., 1990; Couteaudier et al, 1993). 

There is extreme flexibility in the genes that can be expressed in A. nidulans (reviewed 

by Davies, 1994; Goosen et al., 1992). These include; vertebrate proteins for 

pharmaceuticals, other fungal proteins for industrial and research purposes, bacterial 

proteins for selection systems (Section 1.3.2.1) and insect proteins, as reporter genes for 

the study of gene expression (reporter genes are discussed in Section 1.3.7). 

1.3.2 Components of Fungal Expression Vectors used in Transformations. 

Vectors used for transformation in filamentous fungi contain, (i) the expression cassette 

containing the DNA sequence to be expressed attached to fungal regulatory elements, 

(ii) a bacterial origin of replication, (iii) bacterial selectable marker for cloning purposes 

e.g. ampicillin resistance, and may also contain (iv) a fungal selectable marker. (The 

latter can also be supplied on a separate vector though the process of cotransformation; 

Section 1.3.7) In the following section some of the vector components will be described 

(reviewed by Turner, 1994). 



6 

J.3.2.1 Selectable Markers in A. nidulans. 

Several types of selectable markers are available for fungi. These are either auxotrophic 

selectable markers, or dominant selectable markers (reviewed by Goosen et al., 1992; 

Turner, 1994). Auxotrophic markers require the complementation of a phenotype in a 

mutant strain. For example, an A. nidulans strain with a mutation in the orotidine-5' -

phosphate decarboxylase (pyrG) gene will only grow when media is supplemented with 

uracil or uridine. When transformed with the pyrG gene, transformants can be selected 

on media lacking uracil (Oakley et al., 1987). The drawbacks in using auxotropic 

markers are the requirement for the appropriate mutation in the recipient strain, and that 

the mutant strains are often compromised even when supplied with the required 

substrate (Turner, 1994 ). Therefore these transformants do not truly represent the wild 

type genetic background and it is often more desirable to select on the basis of a 

dominant selectable marker. 

Fungal antibiotic resistance markers have been difficult to obtain due to the variable 

sensitivity of filamentous fungi to antibiotics commonly used in yeast and higher 

eukaryotes (Goosen et al., 1992). Therefore dominant selectable markers have been 

created by fusing bacterial antibiotic genes to fungal expression signals. These hybrid­

dominant selectable markers have been constructed to confer resistance to hygromycin 

B (Punt et al.,1987), and phleomycin (Mattern et al, 1988) for example. Phleomycin 

resistance is becoming the dominant selectable marker of choice due to A. nidulans' 

sensitivity to it. Little or no growth is seen in the wild type strain at antibiotic 

concentrations of greater than lOµg per ml of growth medium (Punt et al., 1992). 

Phleomycin is a metalloglucopeptide antibiotic causing DNA strand scission, and the 

resistance gene from Streptomyces hygroscopicus produces a product that binds and 

inactivates the antibiotic (Malpartida et al., 1983). 

1.3.2.2 The Expression Cassette. 

A simple expression cassette contains; a fungal promoter ( or regulatory element), the 

coding region, and the fungal transcription stop sequence. For the expression of 

heterologous genes the expression cassette may also contain a sequence for a stabilising 



7 

protein, a signal sequence e.g. for secretion, or other processing sequences such as a 

cleavage sequence (Davies, 1994). The types of expression cassette constructs are 

shown in Figure 2. 

In A nidulans several promoters have been used in expression cassettes. The choice is 

generally based on whether a constitutive or inducible promoter is required. Most 

promoters used originate from genes involved in carbon-source utilisation pathways, as 

their expression tends to be high (reviewed by Davies, 1994). Constitutive promoters 

are generally more convenient because inducible conditions are not required and the 

product is obtained faster. Examples of constitutive promoters are glyceraldehyde-3-

phosphate dehydrogenase (gpd.A; Punt, 1990), and triose phosphate isomerase (tpi; 

McKnight et al., 1986). However, if the protein to be produced is not stable, is toxic to 

the cell, or the product needs to be produced at a certain developmental stage, then 

inducible promoters can be used. Under the control of these promoters gene expression 

is turned on by a chemical or environmental trigger. Commonly used examples of these 

are the promoters of acetamidase (amdS; Turnbull et al., 1989), and alcohol 

dehydrogenase I (a/cA;Gwynne et al., 1987). Finally, parts of promoters and regulatory 

elements have been used fqr the assessment of these elements in driving gene 

expression. For example, the promoter of the A nidulans isopenicillin N synthase gene 

has been dissected by this method (Perez-Esteban et al., 1993) 

All eukaroytic genes contain a conserved sequence in the region immediately 

surrounding the translational start codon, called the Kozak sequence (Kozak, 1987). 

This is involved in the recognition and binding of the gene expression machinery. If 

heterologous genes being expressed are not of eukaroytic origin then they are often 

modified to contain this sequence (Ballance, 1991 ). 



Figure 2 Schematic representation of types of expression cassettes. 

A. 

B. 

c. 

D. 

Fun al Promoter 

SPF 
Fun al Promoter 

SP 

Coding Region. 

Coding Region. 

Termination 
Sequence 

Termination 
Sequence 

===~========= 
CS 

8 

A and B are cassettes designed for the expression of intracellular gene products, while C 

and D are cassettes designed for secreted gene products (Section 1.3.2.2). SPF, 

stabilising protein fusion; SS, signal sequence for secretion; SP, secreted protein coding 

sequence; CS, endogenous site for enzymatic cleavage of unrequired sequences. This 

diagram presents constructs described by Davies (1994). 



9 

Finally, transcriptional terminators are required for the correct termination and 

processing of the mRN A product (Devchand and Gwynne, 1991 ). 

1.3.3 DNA Uptake. 

Transformation procedures are well established in A. nidulans (Tilbum et al., 1983; 

Ballance et al., 1983; Yelton et al., 1984). The most commonly used and most successful 

transformation techniques involve preparation of osmotically sensitive cells by the 

removal of the fungal wall by cell wall-digesting enzymes. These protoplasts are 

subsequently treated with CaC}i, polyethylene glycol (PEG), and exogenous DNA. 

After an incubation period the mixture is diluted and aliquots spread on stabilised 

selective media, either directly or in a soft agar overlay (methods reviewed by Goosen et 

al., 1992; Fincham, 1989). Filamentous fungi do not have non-chromosomal replicons 

with high copy numbers, such as those used in yeast and bacteria, therefore transformation 

relies on integration of the DNA into the genome (Davies, 1991). Three types of 

integration events are observed (Hinnen et al., 1978), and these are discussed in detail in 

Section 1.3.4. 

A model of DNA uptake during transformation is the 'competent cells' model that 

predicts that not all cells are capable of DNA uptake, but that those that are will take up 

multiple DNA molecules (Grotelueschen and Metzenberg, 1995). Evidence from 

cotransformation experiments (Section 1.3.4) heavily favours the 'competent cells' 

model as cotransformation with two separate vectors occurs at a high frequency (Austin 

and Tyler, 1990; Wemars et al., 1987). 

If only a certain number of cells are competent, what keeps the rest incompetent? 

Grotelueschen and Metzenberg (1995) hypothesised that this could be a feature of either 

permeability of the cell membrane or the characteristics of the nuclei. They showed that 

the requirement for competence is at the level of the nucleus. They then hypothesised 

that the limiting event in transformation is the availability of suitable sites for 

integration. The nuclei in a protoplast population divide asynchronously, and therefore 

there may be only a narrow window in the nuclear cycle in which the genome is 

available to be transformed. It was shown by Contour-Clamped Homogeneous Electric 



10 

Field (CHEF) gel electrophoresis and Southern hybridisation analysis that there are 

competent chromosome sites for ectopic integration (Grotelueschen and Metzenberg, 

1995). This followed hypothetical models proposed in mammalian and yeast cells that 

ectopic integration may occur at pre-existing breaks in the chromosome (Roth and 

Wilson, 1988; Schiestl et al., 1993). 

The hypothesis that the limiting event in transformation is DNA integration is 

corroborated by transient expression studies (Monke and Schafer, 1993). Research 

showed that the frequency of transformation resulting in transient expression levels in 

protoplasts far exceeded the frequency of stable transformation. Also that at least 99% 

of transformants lose the transforming DNA. This implies that the limiting factor in 

transformation is DNA integration rather than DNA uptake (Monke and Schafer, 1993). 

1.3.4 Fate of Transforming DNA: Integration Events. 

Stable transformation of A. nidulans requires the integration of the exogenous DNA into 

the genome. Three types of integration were first defined in yeast by Hinnen et al. 

( 1978), and those observed in filamentous fungi are very similar (reviewed by Goosen et 

al., 1992; summarised in Figure 3). Type I integration occurs at a homologous site 

resulting from a single crossover event between homologous sequences present in the 

plasmid and the genome. This results in a linked duplication of the sequences separated 

by the plasmid DNA. Type II integration occurs at a heterologous sites in the genome. 

It is called 'ectopic integration' and results from a single cross-over event between two 

non-homologous sequences. With both these types of integration the transformation can 

contain one or more copies arranged in a tandem array. Finally, type ill integration 

occurs a homologous site resulting in a double cross-over event between the 

homologous sequences. This is termed gene conversion or replacement and results in 

the plasmid sequence replacing the sequence in the genome. Methods have been 

developed using type ID integration for the deletion, mutation, and cloning of genes (e.g. 

Miller et al, 1985; Wemars et al, 1987). All these integration types have been shown to 

occur simultaneously in a single fungal transformant (Yelton et al., 1984). 



11 

Figure 3 Diagram of the intergration events observed in fungal transformation. 

Type I integration results in integration at a homologous site, type II results in ectopic 

integration and type III results in sequence replacment (Section 1.3.4). The diagram 

represents the types of integration events described by Hinnen et al, (1978). 



A. Type I integration 

Genomic DNA 

! 
Genomic DNA 

#- * * * ! * I 1* * * ! * , ... ... 

B. Type II integration 

//- * * * * * * ~ 
Genomic DNA Genomic DNA 

! 
#- * * * * * * 7}' I** ·..:1 ., ... I 

Genomic DNA Plasmid DNA 

C. Type m integration 

Genomic DNA 

! 
Genomic DNA 

/,.'L,'/~---------;~·=·:::J*F,....:*:.....:·t:~*'.::~*1-----------"""J// /r , r 1 1 // 

~ 7 

# 



13 

The frequencies with which the different types of integration will occur in a given 

transformant are difficult to predict as they vary depending on the composition of the 

vector, the nature of the selective marker, the form in which the vector is used (circular 

or linear), and the genetic background of the recipient strain. However one general rule 

is that the number of multi-copy transformants can be increased by increasing the total 

amount of DNA (reviewed by Goosen et al., 1992). 

1.3.5 Fate of Transforming DNA: Transient Transformants. 

In transformation experiments two types of transformants are commonly observed. The 

first type grow strongly and spore well on selective media, the second type present 

weaker growth and fail to grow when transferred to fresh selective media. The latter 

type has been classified as abortive transformants (Tilbum et al., 1983). It is 

hypothesised that the molecular basis of this is transient expression of the transformed 

vector. The vector is not integrated into the genome and is lost during cell division. 

The product of the transient expression may confer the transformed phenotype until it is 

titrated out by mitosis. Transient expression can be used to investigate gene expression 

without the need for stable integration. This method saves time, circumvents the 

problems of multi-copy integration and position effects (Section 1.3.6), and is useful for 

establishing transformation techniques and for rapid evaluation of expression signals 

(Menke and Schafer, 1993; Judelson et al., 1993). 

Stable expression requires integration into the genome and once this has occurred 

stability remains through many rounds of sub-culturing on non-selective media (Yelton 

et al, 1984). However meiotic instability has been observed in some filamentous fungi, 

and has been extensively studied in N. crassa. It has been shown to be due to gene 

inactivation by methylation, and is termed RJP - Repeat Induced Point Mutation (Selker, 

1990). RJP seems limited to a subset of filamentous Ascomycetes, and has not been 

reported in A. nidulans, although other forms meiotic instability, for example unequal 

recombination, may still occur (Goosen et al, 1992). 



14 

1.3.6 Position Effects and Gene Copy Number in Determining Levels of 

Transformed Gene Expression .. 

Once an expression cassette has integrated into the genome levels of transformed gene 

expression may still vary depending on the number of copies of the cassette introduced 

and the area of the genome that the cassette is introduced into (position effects; Turner, 

1994). There are two sources of position effects. Firstly, the transformed gene may 

integrate downstream of promoter elements. These elements may be positively or 

negatively regulated, and therefore will have an additive effect with the transformed 

regulatory elements to alter the expression of the transformed gene . Secondly, the 

region of the genome that the DNA integrates into may be transcriptionally active or 

inactive, and may vary in activity depending on the developmental stage or on external 

stimuli (Couteaudier et al., 1993). It has been clearly shown that position effects play a 

large role in the expression of transformed genes in animals and plants, while they play 

a very small role in bacteria (Martinelli, 1994). The significance of position effects 

seems to climb as the complexity of the genome increases and the genome contains 

larger amounts of repeated DNA sequences. 

In keeping with this is the observation that in fungi the strength of position effects is 

connected with the genome complexity. For example in the position effects are 

significant in the fungal class, Oomycetes, although to a lesser extent than plants and 

animals (Judelson et al., 1993). The genome of Oomycetes' is more complex than the 

genome of the Ascomycetes', also, A. nidulans has a relativity simple genome with only 

2-3% consisting of repeated sequences (Mao and Tyler, 1991; Section 1.2). Therefore 

compared to Oomycetes, animals and plants, position effects may be expected to play a 

less significant role in determining the levels of expression of a transformed gene. This 

has been demonstrated by a correlation between an increase in copy number and an 

increase in hygromycin resistance (Punt et al., 1987) in Aspergillus species. In the 

production of heterogeneous gene products, e.g. human interferon, high copy number 

also gave increased yields of the product (Gwynne et al., 1987). A comprehensive study 

of the activity of interferon a-2 under the control of alcA (alcohol dehydrogenase) 

expression signals was carried out for transformants containing different copy numbers 



15 

of the expression cassette. The importance of the copy number of the alcR (alcohol 

dehydrogenase regulatory gene) was also analysed. It was concluded that expression 

increased in a roughly linear fashion with the number of copies present up to a number 

of 7-10 copies. It was hypothesised that when the alcA copy number reached 10 copies 

the alcR product was titrated out. This was corroborated by experimental evidence 

showing that the multi-copy alcR transforrnants continued the linear relationship up to 

20 copies. Therefore, in this case copy number showed a linear relationship with yield 

(Davies, 1991). However, in another Ascomycetes, Fusarium oxysporum, it was 

concluded that position effects were more important than gene copy number. This was 

based on the comparison of two single copy transforrnants, one single copy transformant 

showed enzyme activity 80-fold higher than another single copy transformant. Plasmid 

rescue and sequence analysis indicated that the highly expressing single copy 

transforrnant had integrated downstream of a recognisable enhancer element 

(Couteaudier et al., 1993). Studies on the relative effects of copy number and position 

are complicated due to the difficulty in detennining the number of active copies of the 

cassette accurately. 

1.3. 7 Cotransformation. 

When a sequence is introduced into an organism, it may be a disadvantage to need to 

physically link the sequence to a selectable marker. However, introduction of a vector 

containing a non-selectable sequence can be achieved by dual transformation with a 

vector containing a selectable gene, without the need for prior covalent linkage of the 

two vectors. This process is called cotransformation, and it has been established in A. 

nidulans (Wemars et al., 1987). Cotransformation occurs since not all protoplasts in a 

preparation are available to be transformed, however a single protoplast that is 

competent can transform more than one plasmid simultaneously (Grotelueschen and 

Metzenberg, 1995; discussed in Section 1.3.2). 

This principle was first demonstrated by Wemars et al. (1987), A. nidulans was 

cotransformed with vectors containing the amdS gene and trpC gene. Each vector was 

initially selected for, and transformants screened for the presence of the second vector. 

Results showed that the cotransformation frequencies varied depending on which vector 



16 

was initially selected for; 95% of the amdS+ transformants were also trpc+, while only 

62% of the trpc+ transformants were amdS+. This was hypothesised to reflect a 

relatively large number of amdS genes becoming inactivated during the act of 

integration, since similar results were obtained with a plasmid containing both the genes 

together. This shows that the frequency of cotransformation is dependent on the nature 

of the selection system. It was also observed that cotransformation frequencies 

increased with increases in the total amount of cotransfonning vectors, and increases in 

the ratio of cotransfonning vector to transfonning vector ( containing the selectable 

marker). 

Since these initial experiments cotransformation has been extensively used to introduce 

a variety of genes for the production of heterologous gene products in industry, e.g. E. 

coli enterotoxin subunit B (Turnbull et al., 1989) and human interferon a-2 (Gwynne et 

al, 1987) and reviewed by Saunders et al., 1989. Also for the study of gene expression 

by the introduction of the reporter genes e.g. P-galactosidase (lacZ; Van Gorcom et al., 

1986) and p-glucuronidase (gusA; Roberts, et al., 1989) (discussed in Section 1.3.7). 

Most papers report cotransformation as a percentage of the transformants, of above 

60%. In a study by Roberts et al., (1989) it was reported that when cotransfonning with 

vector containing the gusA gene and the selectable marker argB, they reported 

cotransformation frequencies "up to 50%". 

1.4 THE USE OF REPORTER GENES IN THE STUDY OF FUNGAL 

PROCESSES. 

1.4.1 Overview of Methods to Study Fungal Cell Processes. 

Through microscopy, with or without chemical staining procedures, it is possible to see 

cells and the organelles within them, but this gives only limited information about the 

metabolic processes that are occurring. A classical path for the elucidation of fungal cell 

metabolic processes has used a combination of classical genetic techniques for the creation 

of mutants, and the characterisation of these mutants by biochemical analysis. In A 

nidulans, examples of metabolic processes that have been studied this way are; carbon 



u 

metabolism, alcohol metabolism, inorganic nitrogen assimilation, sulphur metabolism etc 

(reviewed in Martinelli and Kinghorn, 1994 ). A question not answered by this type of 

biochemical analysis is how these processes are spatially organised in the fungal cells. 

Recent staining techniques utilise fluorochromes, dyes that are absorbed by cell organelles 

or bind to specific residues on or inside the cells (reviewed by Butt et al., 1989). Upon 

binding to their target these produce a fluorescence that is detectable through a 

microscope. In mycology, this has revealed much information about cell processes, and 

the differentiation and growth of the hyphae. However, these dyes have disadvantages. 

Their cytological use is rather limited, they are only available for certain cell types under 

certain conditions, and it is not known what damage they or the cell fixing process 

required inflicts on the cell (Yuan and Heath, 1990). Recent molecular genetic techniques 

have been developed that allow the study of specific cell processes by both visual 

observation and biochemical assay. This involves the linking the promoter of a gene 

involved in a process of interest to a reporter gene (Bardonnet, 1988). 

A reporter gene is a gene whose product is easy to detect and assay. When fused to 

expression signals from a gene of interest, the reporter gene expression reflects the 

expression characteristics determined by those signals in the original gene (Jefferson, 

1987). There are several considerations when using reporter genes in fungi. For analysis 

of expression signals downstream from the promoter the reporter gene may need to be 

fused in frame with the protein-coding region of the gene of interest, and some processing 

will not occur as for the endogenous protein. The reporter gene product will not be 

transported in the same manner as the endogenous protein, although the fusing of a signal 

peptide may alleviate this. Because fungal vectors must integrate into the genome, the 

level of reporter gene activity may not only reflect the activity conferred by the expression 

signals, but also will be influenced by position effects and gene copy number (Section 

1.3.6). Another consideration is the stability of the reporter gene product. A stable gene 

product may be necessary for some applications, but for quantitative measurements, the 

levels of reporter gene activity may not represent the levels of promoter activity at that 

time but the accumulation of the reporter gene product over development. This problem 

can be circumvented by using inducible promoters (Section 1.3.2). 



18 

The first reporter gene used in filamentous fungi was the E. coli p-galactosidase (lacZ). 

This system is commonly used in bacteria and plants (reviewed by Silhavy and Beckwith, 

1985), and was experimented with in A nidulans (van Gorcom et al., 1985; 1986) . . In A 

nidulans, lacZ fusions have facilitated the molecular characterisation of promoters and 

their individual elements, such as the promoters of 3' -phosphoglycerate kinase (Streatfield 

et al., 1992) and isopenicillin-N-synthetase (Perez-Esteban et al., 1993). However, the 

sensitivity of this system is low due to the endogenous P-galactosidase enzyme activity 

that is present in many fungi (Roberts et al, 1989). 

1.4.2 The J3-glucuronidase (gusA) Reporter Gene System. 

Recently attention has focused on an alternative reporter gene, the E.coli uidA (or gusA) 

gene. Encoding p-glucuronidase, a hydrolase enzyme (Jefferson, 1987). It has been 

developed as a powerful tool for the assessment of gene activity in transgenic plants and 

for the development of molecular genetic analysis of plants. This system is effective in 

plants as there is no endogenous enzyme activity, and both histochemical and fluorometric 

substrates are available (Jefferson, 1987). These substrates are cleaved by the GUS 

enzyme to give a product that can be qualitatively or quantitatively detected. Another 

attractive feature of the gusA gene system is that its product is able to tolerate large amino­

terminal additions, therefore construction of translational fusions are feasible and provide 

a valuable method for assaying the behaviour of transit or signal peptides (Jefferson, 

1987). Also, with the appropriate signals the GUS enzyme can be translocated across 

chloroplast membranes with high efficiency indicating an ability to be translocated across 

all cell membranes (Jefferson, 1987). 

The use of gusA as a potential reporter gene in fungi was initially studied by Roberts et al. 

(1989). In this study, A. niger, A. nidulans, and Fulviafalva were transformed with the 

gusA gene driven by the A. nidulans promoter, gpdA. Histochemical staining of mycelia 

and assays of cell extracts with a fluorometric substrate showed that the gusA gene had 

been expressed. Unlike lacZ fusions in fungi, no significant endogenous GUS activity was 

observed. Since these initial studies gusA systems have been set up in other fungi, e.g. the 

plant pathogens Pseudocercosporella herpotrichoides (Bunkers, 1991), and Fusarium 



19 

oxysporum (Couteaudier et al., 1993). The gusA reporter gene system has been used in 

several applications. GUS activity was demonstrated in perennial ryegrass infected with 

gusA transformed Acremonium endophyte. This demonstrated the use of a surrogate host 

for the introduction of foreign genes into perennial ryegrass (Murray et al., 1992). 

Transient expression of transformed genes has been demonstrated in Cochliobolus 

heterosrophus using gusA (Monke and Schafer, 1993). The GUS reporter gene system 

has been used to demonstrate lack of conservation of gene expression machinery between 

the classes of fungi, Ascomycetes and Oomycetes (Judelson, 1992; Pieterse et al., 1995). 

Finally, gusA fusions to the ad.hi (alcohol dehydrogenase) and verl (aflatoxin biosynthesis 

gene) promoters of A. flavus have allowed the evaluation of transcriptional control and 

induction of aflatoxin production in situ (W oloshuk and Payne, 1994; Flaherty et al., 

1995). 

1.4.3 GUS Expression Plasmids. 

The components of GUS expression vectors are essentially the same as those for the 

expression of other heterologous genes (see Section 1.3.2). The GUS expression cassette 

is the most important component in the expression vector. It includes the gusA gene, 

transcriptional regulatory sequences, and sequences required for the termination and 

processing of the RNA transcript (Devchand and Gwynne, 1991). Because of the 

application GUS expression vectors to studying and comparing many promoter elements, 

a basic GUS vector without the expression sequences, and with a multi-cloning site for 

ease in cloning in these sequences can be initially constructed (this study). 

A popular promoter for driving gusA expression when systems are being developed is 

the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (e.g. Roberts et 

al., 1989; Bunkers, 1991). This is a gene from the glycolytic pathway, therefore it is 

strongly expressed in all cell types at all times in development. Other attractive features 

of this gene are that it is expressed in the cytosol, and that regulation is at the 

transcription level (Punt et al, 1988). This promoter has previously been used to drive 

the expression of lacZ (Punt et al, 1990), and other heterologous genes (e.g. phleomycin 

resistance; [Punt, 1992]). 



20 

1.5 AIMS AND OBJECTIVES OF THIS STUDY. 

The aim of this project was to develop a new fungal GUS expression vector with a 

multi-cloning site and to assess its properties relevant to uses in reporter gene studies. 

The steps to be taken in the project were: 

• Construction of the GUS expression vector containing a multi-cloning site, 

• Testing of the vector by insertion of the constitutive promoter, gpdA, 

• Evaluation of its performance in cotransformation using the dominant selectable 

marker, phleomycin resistance, 

• Testing the physiological impact of the presence of the vector DNA in 

transformation, 

• Assessing the relationship between transformed gene copy number and GUS 

expression levels in the transformants.