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IDENTIFICATION OF DOTHISTROMIN 

BIOSYNTHETIC PATHWAY GENES 

A thesis presented in partial fulfilment of 

the requirements for the degree of 

Masters of Science in Molecular Genetics 

at 

Massey University, Palmerston North 

New Zealand 

Carmel Jane Gillman 

1996 



ll 

ABSTRACT 

Dothistromin is a polyketide-derived toxic secondary metabolite produced by the 

filamentous fungus Dothistroma pini which causes the disease Dothistroma needle blight 

in Pinus radiata. Dothistromin is considered to be an important component in the disease 

process, although its exact function is yet to be identified. By isolating and identifying 

genes involved in dothistromin biosynthesis, and subsequently obtaining mutants 

blocked or altered in the synthesis of dothistromin, the role of this toxin in pathogenicity 

will be able to be assessed. Dothistromin is structurally related to the mycotoxins, 

aflatoxin (AF) from Aspergillus parasiticus and A. flavus, and sterigmatocystin (ST) 

from A. nidulans. Three intermediates in the ST and AF biosynthetic pathways 

(averantin, averufin, and versicolorin B) are thought to also be intermediates dothistromin 

biosynthesis. Due to these similarities, cloned AF pathway genes were used as 

heterologous probes in Southern hybridisation analysis to provide a direct method for 

identifying dothistromin biosynthetic genes. 

A fragment of the A. parasiticus nor-I gene, encoding a reductase involved in the 

conversion of norsolorinic acid (NA) to averantin (A VN) in the AF biosynthetic pathway, _ 

was used as a probe to detect a region of sequence similarity to D. pini genomic DNA. A 

D. pini genomic library was then constructed and screened, resulting in clone ACGN2. 

However, Southern hybridisation analysis suggested that this clone did not contain a 

homologue of the nor-I gene from A parasitic us. 

A fragment of the Aspergillus parasiticus ver-1 gene, encoding a reductase involved in 

the conversion of versicolorin A (VA) to ST in the AF biosynthetic pathway, was also 

used as a probe to detect a region of sequence similarity to D. pini genomic DNA. The 

D. pini genomic library was then screened. Two clones, ACGVl and ACGV2, were 

isolated and Southern hybridisation analysis confirmed that these clones contained 

sequences hybridising to the A parasiticus ver-1 gene fragment. Fragments of these 

clones which hybridised were then sequenced and compared to the GenBank database. 

The amino acid coding sequence of a 0.8 kb Sall region from clone ACGVl exhibited a 

high degree of similarity with the A. nidulans verA and A parasiticus ver-1 genes, 

involved in the ST and AF biosynthetic pathways, and the Magnaporthe grisea ThnR, 

and Colletotrichum lagenarium Thr 1 genes, involved in melanin biosynthesis. This data 

suggested a ver-1 homologue is present in the D. pini genome. Limited sequence 

analysis of a 2.1 kb region from clone ACGV2 suggested that a second independent copy 

of a ver-1-like gene may also be present in the genome. 



ill 

ACKNOWLEDGEMENTS 

I feel very overwhelmed and honoured when I think of all the people I would like to 

thank for their contribution to my project and to helping me through recent really difficult 

times. Firstly, I would like to thank my supervisor, Rosie Bradshaw. Her never ending 

faith in me, encouragement, guidance, and kindness kept me going, and enabled me to 

reach a goal that I wasn't sure I could reach. Thank you so much to Tania and Paul, 

without whom none of this would have been possible. I couldn't have won the battle 

without such wonderful, dedicated, patient, and loving friends. Thank you also to my 

parents for their love, strength, and financial support. Thank you to my brother and 

sister for their support. Because my project was done in two parts there are lots of 

people to thank for getting me started, and other people to thank for re-teaching me and 

building up my confidence again. Thank you to all of you. Thank you to all the people I 

did fourth year with, for making such a difficult year more bearable. Thank you Dianne 

and Karyn for getting me started on my lab work. Thank you to Carolyn for her always 

useful advice, and for saving lots of my sequencing gels. Thanks also to Mike and Rich 

for helpful tips. Thanks to Austen for his friendship. Thank you to Tash for keeping my 

project going while I was away. Thank you to Anita for always being really positive, 

especially for making the lab a fun place to be in. Special thanks must also go to Linda 

for her friendship, and her patience in helping me belong again in the lab. Thank you to 

Brendon for reminding me how to run a sequencing gel, and to Paul for teaching me 

sequencing the first time around. Thanks to Bran for late night company in the lab. 

Thank you to David for drawing my chemical structures. Thanks again to Tania, this 

time for helping me draw my thesis figures. Thank you to all the Friday night staff club 

regulars for keeping me sane and refreshed. Thank you to all the other past and present 

people in the MGU I haven't mentioned. Thank you to all my friends outside of the 

department, especially Craig and Maree for their support from afar, and Justin for always 

being there if I needed him. I would also like to thank my flatmates for keeping me in 

touch with reality over the last few months, especially Tony "I love birds, eh" Roeven for 

his reassurance and tolerance. rm sure fve forgotten heaps of people who also played a 

really valuable part in getting me where I am today, so even if you're not on this list I 

would like to thank you. Thanks must also go to Molecular Genetics Unit for providing 

the facilities and financial assistance that enabled me to undertake this project, especially 

Barry for getting our lab a freezer. In addition, thank you to John Linz (Michigan State 

University) for providing the Asperillus parasiticus nor-I and ver-1 gene clones. 



TABLE OF CONTENTS 

ABSTRACT 

ACKNOWLEDGEMENTS 

TABLE OF CONTENTS 

LIST OFT ABLES 

LIST OF FIGURES 

1.0 INTRODUCTION 

1.1 General Features 

1. 2 Infection 

1. 3 Chemical Control 

1.4 Resistant Strains 

1.5 Dothistromin Toxin 

1.6 Inactivation of Dothistromin 

1. 7 Aflatoxin Biosynthesis 

1.8 Gene Cloning Strategies in Aspergillus Species 

1. 9 Organisation and Arrangement of AF/ST Biosynthetic 
Pathway Genes 

1.10 Aims and Objectives 

2.0 MATERIALS AND METHODS 

2 . 1 Bacterial and Fungal Strains, 'A Clones and Vectors 

2.2 Media 

2. 2. 1 Fungal Media 

2.2.1.1 D. pini Media (DM) Broth 

2.2.1.2 D. pini Media (DM) Agar 

2.2.1.3 Malt Extract Agar (MEA) 

2.2.1.4 Malt Yeast Glucose (MYG) Agar 

11 

ill 

IV 

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xii 

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4 

5 

6 

10 

13 

14 

18 

20 

22 

22 

22 

22 

22 

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22 

23 



2.2.1.5 Yeast Peptone Glycerol (YPG) Agar 23 

2.2.1.6 Potato Dextrose Agar (PDA) 23 

2.2.1.7 Nutrient Malt Yeast (NMY) Agar 23 

2.2 .1.8 Minimal Medium (MM) Agar 23 

2.2.1.9 Nutrient Yeast (NY) Broth 23 

2.2.1.10 Nutrient Malt Yeast (NMY) Broth 23 

2.2.2 Bacterial Media 26 

2.2.2.1 Luria-Bertaini (LB) Media 26 

2 .2 .2.2 TB Top Agar 26 

2.2.2 .3 NZCYM 26 

2.3 Growth and Maintenance of Cultures 26 

2.3.1 Fungal Cultures 26 

2.3.2 Bacterial Cultures 27 

2.4 Buffers and Solutions 27 

2.4.1 TEG 27 

2.4.2 Tris-Equilibrated Phenol 27 

2.4.3 TE Buffer 28 

2.4.4 DNase free RNase 28 

2.4.5 10 x TAE Buffer 28 

2.4.6 10 x Gel Loading Dye 28 

2.4.7 20 x SSC 28 

2.4.8 TES (10/1/100) 28 

2.4.9 50 x Denhardt' s 29 

2.4.10 SM Buffer 29 

2.4.11 Acrylamide Mix 29 

2.4.12 10 x TBE Sequencing Buffer 29 



2.5 DNA Preparations 29 

2.5.1 Alkaline Lysis E. coli Plasmid Preparation 29 

2.5.2 Large Scale D. pini Genomic DNA Preparation 30 

2.5.3 Preparation of Genomic DNA for D. pini Library Construction 31 

2.5.4 Mini-prep of A Phage DNA 32 

2.5 .5 Extraction of DNA from Seaplaque Agarose 33 

2.5.5.1 Bio 101 Geneclean kit 33 

2.5.5.2 Gibco BRL GlassMax DNA Isolation Spin Cartridge System kit 33 

2.5 .6 Purification of DNA 34 

2.6 DNA Manipulations 34 

2.6. l Restriction Enzyme Digests 34 

2 .6.1.1 Plasmid Digests 34 

2 .6 .1.2 Genomic Digests 35 

2.6.2 Agarose Gel Electrophoresis 35 

2.6.2.1 Minigels 35 

2.6.2.2 Overnight Gels 35 

2.6.3 Determination of Fragment Sizes 36 

2.6.4 Determination of DNA Concentration 36 

2 .6.4.1 Concentration Standards 36 

2.6.4.2 Spectrophotometric Method 36 

2.6.4.3 Fluorometric Method 37 

2.7 Subcloning 37 

2.7.1 Preparation of Insert DNA 38 

2.7.2 Linearisation and CAP-Treatment of Vector DNA 38 

2.7.3 Ligation 38 

2.8 Transformation of E. coli 39 

2.8.1 Calcium Chloride Transformation 39 



2.8.1.1 Preparation of CaCh Competent Cells 39 

2.8.1.2 Transformation 39 

2.8.2 Transformation of E. coli by Electroporation 39 

2.8.2 .1 Preparation of Electro-Competent E. coli Cells 39 

2.8.2.2 Electroporation 40 

2.9 DNA Hybridisations 40 

2.9.1 Southern Blotting 40 

2.9 .2 Random-Primer Labelling of Probes 41 

2.9 .3 Separation of Unincorporated Nucelotides by Minispin Column 
Chromatography 42 

2.9.4 Hybridisation of Probe DNA to Southern Blots 42 

2.9.5 Autoradiography of Southern Blots 43 

2.9.6 Stripping Filters 43 

2.10 Genomic Library Construction 43 

2.10 .1 Establishing Conditions for Partial Digestion of High Molecular 
Weight Genomic DNA 43 

2.10 .2 Large Scale Preparation of Partially Digested DNA 44 

2.10.3 Partial Fill-in Reaction for Genomic DNA 44 

2.10.4 Ligation of Insert to Vector Arms 45 

2.10.4.1 Klenow/Ligation Controls 45 

2.10.4.2 Determination of Optimum Ligation Conditions 46 

2.10.5 Packaging of Ligated DNA and Titration of Recombinant Phage 46 

2.10.5 .1 Packaging of Ligated DNA 46 

2.10.5.2 Titration of Packaged Phage on LB Plates 46 

2.10.6 Large Scale Packaging of Ligated DNA 47 

2.10.7 Amplification of the Library 48 

2.11 Library Screening by Plaque Hybridisation 49 

2.11.1 Plating Phage 'A 49 



2.11.2 Filter Lifts 49 

2.11.3 Hybridisation of Phage "A DNA [a-32P]dCTP Labelled DNA so 

2.12 DNA Sequencing so 
2.12 .1 Preparation of DNA for Sequencing so 

2.12. 1.1 Preparation of Single Stranded M13 Template DNA so 
2.12.1.2 Preparation of Template DNA for AmpliCycle Sequencing 51 

2.12.2 Sequenase Version 2.0 Protocol 51 

2.12.3 AmpliCycle Sequencing Protocol 52 

2.12.4 Polyacrylamide Gel Electrophoresis (PAGE) of Sequencing 
Reactions 52 

3.0 RESULTS 56 

3.1 Determining Optimal Mycelial Growth Conditions in Culture 56 

3.1.1 Determining Optimal Solid Media for Mycelial Growth 56 

3.1.1.1 Temperature Effect on Mycelial Growth 56 

3.1.1.2 Media Effect on Mycelial Growth 56 

3.1.1.3 Effect of Inoculation Method and Incubation Period on Mycelial 
Growth 61 

3 . 1.2 Quantitation of Mycelial Growth in Liquid Media 61 

3.2 Southern Hybridisations 64 

3 .2.1 Detection of a D. pini Region Heterologous to the A. parasiticus 
nor-I Gene 64 

3.2.2 Detection of a D. pini Region Heterologous to the A. parasiticus 
ver-1 Gene 64 

3.3 Library Construction 68 

3.4 Isolation of A Clones Hybridising to the A. parasiticus ver-1 Gene 74 

3.4.1 Library Screening 74 

3.4.2 Restriction Digestion of "A Clones and Southern Hybridisation 74 

3.5 Further Characterisation of Clone "ACGV 1 80 



3.5.1 Mapping Clone ACGV 1 Further 80 

3.5.2 Subcloning of a ACGVl Region Required for Sequencing 85 

3.5 .3 Sequence Analysis of Clone ACGVl 85 

3.5.4 Sequence Identification 85 

3.5.5 Sequence Comparison 90 

3.5.6 Comparison of Intron Positions 90 

3.5.7 Comparison of GC Content 90 

3.5.8 Comparison of Codon Usage 93 

3.6 Further Characterisation of Clone ACGV2 93 

3.6.1 Further Hybridisation Analysis of Clone ACGV2 93 

3.6.2 Subcloning of a ACGV2 Region Required for Sequencing 93 

3.6.3 Sequence Analysis of Clone ACGV2 93 

3.6.4 Sequence Identification 97 

3.6.5 Comparison Between D. pini Sequences 97 

3.6.6 Comparison of GC Content 97 

3.7 Isolation and Characterisation of A Clone Hybridising to the 
A. parasiticus nor- I Gene 97 

3 .7.1 Library Screening 97 

3.7.2 Restriction Digestion of Clone '"ACGN2 and Southern Hybridisation 100 

4.0 DISCUSSION 104 

4.1 Identification of Clone '"ACGVl 104 

4.2 Role of the ver-1-like Genes 104 

4.2.1 The Role of the Aspergillus ver-1 and ver-A Genes 104 

4.2.2 The Role of the ThnR and Thr 1 Genes 106 

4.3 Duplication of the ver-1 Gene 107 



4.4 Identification of Clone )..CGV2 108 

4.5 Identification of Clone ACGN2 110 

4 .6 Potential Uses of the D. pini ver-1 Gene 111 

4 .6.1 Cloning of Other Pathway Genes 111 

4 .6 .2 Gene Disruptions 113 

4.6.3 Identification of Molecular Mechanisms which Regulate 
Pathway Genes 114 

5 .0 SUMMARY AND CONCLUSIONS 116 

APPENDIX 1.0 118 

APPENDIX 2.0 122 

REFERENCES 124 



LIST OF TABLES 

Table 1 Fungal and Bacterial strains, A. Clones and Vectors 24 

Table 2 Primers used in Sequencing Reactions 53 

Table 3 Optimisation of Media and Method of Inoculation 62 

Table 4 Quantitation of Mycelial Growth with Malt Extract Concentration 63 

Table 5 Titres of Small Scale, Large Scale, and Amplified Libraries 75 

Table 6 Data from Restriction Mapping of Clone t..CGVl 81 

Table 7 Data from Southern Hybridisation Analysis of Clone "A.CGV2 82 

Table 8 Codon Bias Table 94 

Table 9 Data from Southern Hybridisation Analysis of Clone "A.CGN2 103 



LIST OF FIGURES 

Fig. 1 Structures of sterigmtocystin, aflatoxin B 1, and dothistromin 8 

Fig. 2 Comparison of the aflatoxin biosynthetic pathway with the 

proposed dothistromin biosynthetic pathway 15 

Fig. 3A-F Demonstration of media differences on mycelial growth and 
appearance 57 

Fig. 4A-C Southern blot of D. pini genomic DNA probed with nor-I 65 

Fig. 5A-C Southern blot of D. pini genomic DNA probed with ver-1 69 

Fig. 6A-B Profiles of partial Mbol digestions of genomic DNA from D. pini 72 

Fig. 7A-B Mapping the position of ver-1 on clone A.CGVl 76 

Fig. 8A-B Mapping the position of ver-1 on clone )..CGV2 78 

Fig. 9A-B Further mapping analysis of clone )..CGV 1 83 

Fig. lOA-B Restriction map of clone )..CGV 1 from a D. pini genomic library 
that hybridised to Aspergillus parasiticus ver-1 86 

Fig. 11 Partial sequence of the putative D. pini ver-1 gene from clone 
)..CGVl 88 

Fig. 12 Comparison of D. pini sequence to other amino acid sequences 91 

Fig. 13 Southern blot of 1CGV2 probed with the D. pini ver-1 fragment 95 

Fig. 14 Partial sequence of a ver-1 hybridising fragment from clone 
)..CGV2 98 

Fig. 15A-B Southern blot of A.CGN2 probed with nor-1 101 



1 

1.0 INTRODUCTION 

1.1 General Features 

The filamentous fungus Dothistroma pini is a major pathogen of Pinus radiata and other 

pine species. It causes Dothistroma needle blight, a necrosis associated with red lesions 

or bands on the needles, often followed by premature needle cast (initially at the base of 

the crown) and consequent reduction of photosynthesis and wood yield. Sometimes this 

is followed by tree death (Gallagher 1971, Franich et al. 1982, Gadgil 1984 ). 

D. pini is of the order Dothideales in the Ascornycotina class. It is the anamorphic 

(asexual) form of Mycosphaerella pini. Differences in pathogenicity between the two 

forms have not been reported in any of the wide number of pine species that they infect 

(Evans 1984). D. pini is a necrotrophic pathogen which is believed to kill plant tissue 

and then live saprophytically (Peterson and Walla 1978). 

D. pini has caused serious defoliation in plantations of P. radiata, which is by far the 

most important susceptible tree species from an economic viewpoint (Phillips and 

Burdekin 1982). The disease is widely distributed throughout the world, having been 

recorded in Europe, South and East Africa, North and South America, India, Russia, and 

Australasia. D. pini was first identified in New Zealand in the central North Island in 

1964. It is now found in all of the North Island except the Northern tip and Great Barrier 

Island. In the South Island it is found in Nelson, Marlborough, North of the Wairou 

river, Westland, Southland, and Otago (Gadgil, 1984). It is of commercial concern in 

New Zealand in that it affects the health and vigour of extensively grown exotic pine 

species P. radiata, P. ponderosa and P. nigra, which are unfortunately all highly 

susceptible to the disease (Elliot et al. 1989). Because of the important role that forestry 

plays in the New Zealand economy, the disease is of economic significance (Gallagher 

1971). 

Infection appears as chlorosis and necrosis of needles on the main stern and base of lower 

branches. Under favourable conditions, and on a susceptible host, defoliation may be so 

severe that only the needles at the extremities of the branches remain. Needle infections 

are first evident as yellow flecks which extend to become bands around the needle. As 

necrosis develops, these bands take on a characteristic red tinge, this has led to the 

commonly applied name 'red band' (Phillips and Burdekin 1982). These band-like 

lesions bear 1-12 black strornata ( asexual fruiting bodies). The strornata vary in size 



2 

from 300-750 x 150-400 µm. Conidia from these fruiting bodies are then released into a 

film of water on the needle surface following rainfall (Gadgil 1984). Rain splash 

dispersal of conidia usually occurs within a single tree, although wider distribution may 

occur when mist clouds are present. The perfect stage of D. pini has been identified as 

Mycosphaerella pini on a range of pine hosts including P. radiata on Vancouver Island 

(Phillips and Burdekin 1982). Sexual wind dispersed spores are also formed in 

stromata, but these have never been observed in New Zealand (P. Gadgil, pers. comm.) 

and are never seen in culture (D. Morrison, pers. comm.). Conidia are multicellular, and 

it is unknown whether each cell is derived from the same origin. Multiple germ tubes, up 

to one per compartment, produced from these conidia, penetrate the needles through 

stomata. This is followed by both inter- and intra-cellular hyphal growth, a process 

taking three days or longer, depending on temperature and humidity. Within the needle, 

disruption of the mesophyll tissue occurs well in advance of the developing hyphae as a 

result of the pathogen toxin, dothistromin. Macroscopic symptoms do not appear on the 

needles for 5-10 weeks. This relatively slow rate of growth is also a feature of the 

fungus in culture (Gadgil 1967, Phillips and Burdekin 1982). 

1.2 Infection 

Though P. radiata is susceptible to the pathogen when young, in general mature trees 

older than 15-20 years show little infection suggesting increased resistance. Several 

factors have been proposed to be involved in this resistance. The stomata on mature-tree 

needles have a smaller pore (10-15 µm) than the young-tree needles (15-20 µm). 

Stomata of young susceptible trees are open pores, the guard cells and subsidiary cells of 

which have an epidermis covered with fine rodlet or microtubular wax. Contact of 

hyphae with the guard cell wax apparently causes them to expand to form an 

appressorium, a specific infection structure, prior to a hyphal peg penetrating between the 

guard cells. However, the majority of stomata on needles from mature Dothistroma 

needle blight-resistant trees were shown to be occluded with resinous material which 

frequently closes the pore between the overarching stomata! lips. Such material could 

present a mechanical barrier to ingress of hyphae, or possibly mask chemotrophic or 

chemotactic stimuli experienced by the hyphae during stages of stomata! penetration 

(Franich et al. 1977, Franich et al. 1983). 

The chemical nature of the occluding material may also be important. Detailed analysis of 

needle epicuticular wax from young and old P. radiata trees has shown quantitative 

differences in the acidic fraction of the wax, from young trees it consists mainly of 

dehydroabeitic acid, where as from mature trees it comprises mainly a complex mixture 



3 

of oxygenated resin acid derivatives (Franich et al. 1978). An in vitro test on D. pini 

showed these oxygenated resin acid derivatives inhibited both conidia germination and 

mycelium growth (Franich et al. 1982). An in vivo test of artificial inoculation showed 

plants treated with acetone (which depletes epicuticular and stomata! pore fatty and resin 

acids) had a mean infection level about twice that of the control (Shain and Franich 

1981). These experiments suggest that the presence of oxidised resin acids at the needle 

surface around, or occluding stomata could therefore be a preinfection chemical 

fungistasis factor partly responsible for the observed D. pini resistance of mature P. 

radiata trees. Once stomata! penetration and hyphal colonisation of mesophyll has 

occurred, the extent of tissue damage and the rate of fruiting body formation appears 

dependent on other resistance factors, among these being sensitivity of needle tissue to 

the dothistromin (Franich et al. 1983). 

Attempts have been made to correlate differences in monoterpene profiles (Franich et al. 

1982) and pH buffering capacities of foliage extracts (Franich and Wells 1977) from 

young and mature trees with differences in susceptibility to blight. A mixture of volatile 

compounds (consisting mainly of monoterpene hydrocarbons) from P. radiata foliage 

populations of young Dothistroma needle blight-susceptible and mature-resistant trees 

was shown to stimulate germination of D. pini spores and mycelial growth at specific 

concentrations. D. pini was found to be a pathogenic fungus well-adapted to growing in 

a monoterpene flux at the surface of, and within the pine needle. Differences in needle 

monoterpene hydrocarbon composition did not bear any simple relationship to mature­

tree resistance. Nor did the high buffer capacity of mature-tree needle homogenates 

appear to be directly related to Dothistroma needle blight resistance. 

Rainfall and other environmental factors have been observed to influence the disease 

intensity. Experiments on leaf wetness period and infection by D. pini suggest that if 

hydrated conidia are deposited on the surface of a susceptible plant host, germination and 

penetration will occur regardless of the length of wetness period that follows deposition, 

provided that suitable temperatures prevail. However, the severity of infection depends 

on the length of the dry period which follows deposition of conidia - the longer the dry 

period, the lower the severity (Gadgil 1977). Germination of conidia does not vary 

greatly with temperature but stromata appear sooner with higher temperatures. Warm 

temperatures ( optimally 16-18°C) combined with prolonged periods of high humidity (> 

96%) or the existence of free water films on needle surfaces have been observed to 

favour serious disease outbreaks (Sheridan and Yen 1970, Gadgil 1974). Infection of 

P. radiata requires 100 conidia/mm2, with even mature trees susceptible to infection if the 

inoculum is high enough. In addition, soil deficiencies (in boron or sulphur) have been 

associated with increased susceptibility to D. pini (Ades and Simpson 1989). 



4 

1. 3 Chemical Control 

It has been predicted that forestry will provide 25% of New Zealand's exports by the year 

2010, P. radiata currently comprises 93% of our 1,330,000 hectare commercial forest 

plantations (L. Bulman, pers. comm.) . Although P. radiata in New Zealand has few 

diseases, Dothistroma needle blight (the most significant disease) has been estimated to 

cost between $6.1 million (New 1989) and $20 million per year (P. Carter, pers. 

comm.). From this cost, 10% is due to chemical control, and 90% is due to wood loss. 

In the absence of management of Dothistroma needle blight, wood volume increment loss 

is directly proportional to the disease severity, so control of the disease is important. 

Control of Dothistroma needle blight in New Zealand is based upon the aerial application 

of copper fungicides to P. radiata plantations during the susceptible ages of 2-15 years. 

Stands are sprayed when stand infection levels are estimated at 25% or greater. This has 

been successfully carried out for more than two decades. Spraying every three to four 

years reduces the mean disease severity thus keeping the disease in check (Shain and 

Franich 1981, Dick 1989). 

Copper fungicide (as 50% cuprous oxide, Cu2O) can react with aqueous-exudates on P. 

radiata needles, and to a lesser extent, with D. pini metabolites, to form free or 

complexed Cu2+ in aqueous solution at concentrations sufficient to inhibit the 

germination of D. pini conidia. The interaction of Cu2O and geothermal H2S to produce 

CuS and subsequently CuSO4 can contribute to solubilising the fungicide. Low Cu2+ 

concentrations effectively reduce germ-tube growth and inhibit the production of 

secondary conidia as well as stimulating dothistromin biosynthesis. It is the combination 

of solubilisation and redistribution of Cu2+, or its complexes, and their ready uptake by 

D. pini conidia, which can explain the good control of Dothistroma needle blight by 

copper fungicides (Franich 1988). 

Although copper-based fungicide treatment is effective, and the development of more 

efficient application methods have lead to a reduction in treatment costs (from over $60/ha 

to under $15/ha in the twenty years to 1988), it is still expensive, costing $1.6 million per 

year to spray the 35% of New Zealand's forests which are affected by D. pini, as well as 

a further $4.5 million residual growth loss per year (L. Bulman, pers. comm., New 

1989). As a consequence of this expense, a supplementary or long term alternative to 

copper fungicide spraying, particularly on high disease risk sites which require multiple 

spraying to achieve control, is obviously desirable (Shain and Franich 1981, Franich et 

al. 1988). 



5 

1.4 Resistant Strains 

Early observations indicated that certain D. pini infected trees possess naturally increased 

resistance to Dothistroma needle blight. Thus it was thought that a good method of 

combating the disease would lie in the production of Dothistroma needle blight-resistant 

P. radiata somatic seedling stock with which to replenish milled forest (Gallagher 1971). 

The tree breeding programme at the New Zealand Forest Research Institute (NZ FRI) is 

producing a Dothistroma needle blight-resistant (DR) breed of P. radiata for growth in 

high risk sites. Over the last ten years breeding population parents have been screened 

and selected for increased Dothistroma needle blight resistance and improved growth and 

form (GF) (FRI 1987). The best DR seedlots are expected to reduce mean stand 

infection by at least 15%. The effects of spraying and resistance are expected to be 

additive. This resistance, however, will only be effective for as long as D. pini maintains 

its current levels of virulence (Carson and Carson 1991). Given that the pathogen has a 

far shorter life cycle than its host, it is likely that strains may evolve which are capable of 

overcoming current plant resistance mechanisms. 

In order to obtain reduction in D. pini infection that is economically and genetically 

meaningful it is necessary to simultaneously improve DR and GF. However, selection 

with heavy emphasis on Dothistroma needle blight resistance reduces the gain in growth 

rate on non-Dothistroma needle blight sites. So separate breeds are therefore used on 

sites with and without a D. pini problem. The nature of Dothistroma needle blight 

resistance remains unknown, although it has been suggested to be a complex trait 

involving at least three different resistance mechanisms (Carson and Carson 1991). 

The New Zealand P. radiata population is derived from three localities on the central coast 

of mainland California in North America and from two islands near Mexico. Despite this 

small natural range, considerable variation between provenances has been demonstrated 

(Ades and Simpson 1991). For effective selection of disease resistance and other traits it 

is favourable to have a narrow genetic variability base to capture high genetic gains. 

Current selection, aimed at the eventual clonal propagation of superior breeds, is focusing 

on improving growth rate, stem form, and wood properties (principally wood density), 

as well as disease resistance (Carson and Carson 1991). Thus facilitating the production 

of a greater wood volume and more clearwood (less branching), resulting in the need for 

minimal pruning and allowing uniform milling. However, a reduction in genetic 

variation means an increase in biological risk factors. If a more virulent D. pini strain 

evolved, a monoclonal host population could be entirely destroyed. Thus, maintaining 



6 

genetic diversity is recognised as being very important. A range of parent material from 

diverse genetic backgrounds is being used in the breeding programme to ensure that 

separate batches of somatic seedlings are genetically variable. 

Although the tree breeding programme is proving successful, difficulties remain in 

effectively and economically controlling Dothistroma needle blight. DR trees will only 

control the disease, not eliminate it. Investigation into the molecular biology of the D. 

pini disease process may provide a valuable approach to reduce or eliminate the infection. 

1.5 Dothistromin Toxin 

The red pigment in the Dothistroma needle blight lesions is due to the presence of the 

mycotoxin dothistromin, which is found in high concentrations in D. pini infected P. 

radiata needles (Gallagher 1971). The toxin has also been isolated from several 

Cercospora species and Mycosphaerella laricinia (Stoessl et al. 1990) as well as from 

axenic cultures of D. pini. The capacity to induce Dothistroma needle blight symptoms 

artificially with purified dothistromin has been demonstrated, thus strongly supporting 

the hypothesis that dothistromin plays a significant role in pathogenesis and should be 

considered a toxin (Shain and Franich 1981 ). The exact function of dothistromin in the 

disease process is yet to be identified. It may be a pathogenicity factor enabling the 

fungus to breach the physical and chemical barriers presented by the tree and thus infect 

tree tissues or it may be a virulence factor acting as a specific elicitor of plant defence 

responses. 

Extensive needle death in the presence of dothistromin is not thought to be due to the 

toxin itself, but due to benzoic acid synthesised and accumulated in cells adjacent to those 

initially killed by the toxin. Benzoic acid is found in a dark green region adjacent to the 

necrotic region of the infected needles where no hyphae are present and is bound to lignin 

polymers which are also present in disproportionately high amounts. Benzoic acid is 

highly toxic to D. pini (Gadgil 1967). This suggests that one purpose of the benzoic acid 

response is to restrict hyphal extension within the needle, leading to the proposal that it is 

a phytoalexin. Needles which respond by producing long lesions also create within the 

plant tissue a highly fungistatic environment. In the needles dothistromin is metabolised 

or phytolytically degraded to CO2 and oxalic acid, a mechanism thought to involve 

peroxidase catalysed oxidation of the toxin by hydrogen peroxide (Franich et al. 1986). 

Despite these considerations, dothistromin is considered to be an important component in 

the disease process. The comparatively rapid production of dothistromin in culture, i.e., 

6-10 days (Gallagher and Hodges 1972, Harvey et al. 1976) suggests that it is not a 



7 

staling product of old cultures. Furthermore, histological studies (Gadgil 1967) which 

demonstrated that host tissue was killed in advance of hyphal penetration, suggest the 

diffusion of a toxin from hyphae to uninfected tissue. Another point of evidence 

favouring the toxic role of dothistrornin, is that in a limited trial of clonal material of 

susceptible age, susceptibility was correlated with sensitivity to dothistrornin (Franich et 

al. 1977). However, in other limited tests a correlation between decreased sensitivity to 

dothistrornin and mature-tree resistance was not obtained (Franich et al. 1983). This 

could reflect a continuing sensitivity of mature trees to dothistrornin from the juvenile 

stage and their later acquisition of other mechanisms which retard needle penetration. A 

capacity to induce lesions artificially with dothistromin (Shain and Franich 1981) 

suggested a relationship between short lesion length and D. pini resistance. 

Dothistromin is the major metabolic by-product of D. pini cultures (Harvey et al. 1976, 

Danks and Hodges 1974). It has been shown using chemical, spectroscopic (Bassett et 

al. 1970, Gallagher and Hodges 1972), and crystallographic evidence (Bear et al. 1972, 

Assante et al. 1977) to be a difuroanthraquinone which is fused to the same tetrahydro-2-

hydro-bisfuran ring system as the mycotoxins , aflatoxin (AF) from Aspergillus 

parasiticus and Aspergillus flavus and sterigmatocystin (ST) from Aspergillus nidulans 

(Fig. 1, Bassett et al. 1970, Shaw 1975, Shaw et al. 1978). It is the furan ring structural 

feature of AF and ST (which are difuranocourain secondary metabolites) that is 

considered to be responsible for their toxicity (Elliot et al. 1989, Harvey et al. 1976). In 

view of this, Shaw et al. ( 1978) performed nuclear magnetic resonance (NMR) and 

mass-spectrophotometric analysis of the reaction products of D. pini mycelia grown with 

[13C]-labelled acetate. The resulting labelling pattern strongly suggested dothistrornin to 

be the product of a biosynthetic pathway involving at least one acetate-polymalonate 

intermediate, therefore showing similarities to AF. Dothistrornin has been identified as 

the phytotoxic metabolite in some Cercospora species (Stoessl 1984). Subsequent work 

with Cercospora arachidicola (Stoessl and Stothers 1984) has identified several other 

anthraquinones. Three of these compounds (averantin, averufin, and versicolorin B) are 

intermediates in the ST and AF biosynthetic pathways in Aspergillus species, and are 

likely to also be intermediates in dothistrornin biosynthesis (Fig. 2). 

On the strength of the remarkable structural similarity between dothistromin and AF, and 

because forest workers might be exposed to high levels of dothistrornin during the course 

of their work, mutagenic studies were initiated with dothistromin to confirm its proposed 

role as a toxin. At growth inhibitory concentrations, dothistromin strongly inhibits 

incorporation of [3H] uridine into RNA of Chlorella pyrenoidosa and Bacillus 

megaterium (Harvey et al. 1976). The similarity between the effects of dothistromin and 



Fig. 1 Structures of sterigmatocystin, aflatoxin Bl, and 

dothistromin 



9 

OH 

0 0 

Sterigmatocystin 

0 0 

0 0 

Aflatoxin B 1 

OH 0 

0 0 OH 

OH 0 OH 
OH 

Dothistromin 



10 

actinomycin D, which is known to inhibit the transcriptional process (Reich et al. 1967), 

supports the view that this may be a primary site of action of the toxin in inhibiting 

microbial growth. The inhibition of RNA synthesis by dothistromin is not surprising in 

view of its close structural similarity to AF. AF inhibits RNA synthesis in liver cells 

(Clifford and Rees 1966), and it has been shown to bind to DNA and thereby impair 

RNA polymerase-mediated transcription (Neely et al. 1970). AF is considered to be 

responsible for hepatotoxicity and potential human carcinogenicity (Ames et al. 1987). 

This information initiated a further series of mutagenicity studies of dothistromin 

including field sampling, environmental monitoring and epidemiological research which 

were coordinated by both the New Zealand Department of Health and the Dothistromin 

Advisory Working Group. Dothistromin was tested for mutagenicity in a wide variety of 

in vitro bioassays, most which were positive, including chromosome damage in human 

peripheral blood lymphocyte cultures (Elliot et al. 1989, Stoessl et al. 1990). In a mouse 

in vivo mutagenicity assay dothistromin appeared to be just as genotoxic as AF, causing a 

significant increase in the number of abnormal erythrocytes as a result of damage to the 

spindle apparatus or chromosome damage (Elliot et al. 1989). However, in a separate in 

vitro study, metaphase chromosome damage in human peripheral blood lymphocytes 

required high doses of dothistromin (Ferguson et al. 1986). This is in contrast to AF 

where aberrations were seen at low dose levels. These studies suggest that although AF 

and dothistromin have similar toxic properties, the toxicity of dothistromin is weaker. 

1. 6 Inactivation of Dothistromin 

Copper fungicide treatment is only a limited control mechanism, and the resistant varieties 

are only successful as long as the pathogen retains its present virulence levels. For this 

reason different approaches to Dothistroma needle blight control are being investigated 

focusing on the mode of action of dothistromin. 

One method of analysis of the role of dothistromin in the plant/fungal interaction is 

immunoassay. Immunoassays have been developed for other small molecules such as 

aflatoxins (Chu 1991). Three years ago at HortResearch (Palmerston North), the 

production of monoclonal antibodies (MAbs) to dothistromin was reported (Jones et al. 

1993). They utilised dothistromin-carrier protein conjugates to prepare these MAbs for 

use in the development of an ELISA for dothistromin which has the required sensitivity 

and specificity to monitor development of the toxin in D. pini cultures and pine leaf 

lesions caused by D. pini. In in vitro experiments so far the antibody competes quite 

well for dothistromin binding, but preincubation of the antibody with dothistromin prior 

to challenging the plant cells is required (P. Reynolds, pers. comm.). As an alternative 



11 

strategy, they have also obtained single-chain antibodies to the bifuran ring of 

dothistromin. Anti-idiotypic antibodies are currently being made against these with a 

view to isolating dothistromin binding proteins (W. Jones, pers. comm.). They aim to 

produce transgenic plants which express the anti-dothistromin antibody, thus inactivating 

the dothistromin toxin if D. pini infects them and hence eliminating the need for spraying 

by copper fungicides. The fundamental assumption underlying this approach is that 

dothistromin is the primary cause of disease symptoms. 

In the past five years a number of fungal genes have been isolated which were anticipated 

to have a function in pathogenicity (VanEtten et al. 1994). The roles of many of these 

genes in pathogenicity have been evaluated by the construction of mutants that lack a 

functional wild type gene through transformation-mediated gene disruption or UV 

mutagenesis. For example, in the Dutch elm disease (DED) pathogen 0phiostoma ulmi 

and the fungal soybean pathogen, Cercospora kikuchii. 

0. ulmi produces a low molecular weight polypeptide toxin called cerato-ulmin (CU) 

which has been implicated as an important factor in virulence (Takai 1974). CU is an 

extracellular hydrophobin and is thought to act by accumulating at, and plugging 

intercellular openings in, the xylem (Russo et al. 1982), or by direct interaction with host 

parenchyma cells resulting in enhanced respiration and electrolyte loss (Richards and 

Takai 1984). The evidence that CU plays a key role in DED pathogenesis has come 

largely from physiological studies. Purified CU induces both internal and external 

symptoms similar to those of DED, when applied to elm saplings (Richards and Takai 

1984). CU has been detected by immunocytochemistry and scanning electron 

microscopy only in aggressive (virulent) strains (Svircev et al. 1988), and isolate 

aggressiveness of the pathogen correlates with levels of CU production in vitro (Takai 

1974). However, classical genetics gave only a weak correlation between CU expression 

and virulence towards the host elm. To directly test the role of CU in DED, Bowden et 

al. ( 1994) recently cloned and characterised the cu gene from a highly virulent isolate of 

0. ulmi. More recently, Bowden et al. (1996) generated a CU-minus mutant by 

transformation mediated gene disruption of the cu gene. This disruption mutant produced 

no detectable CU m.RNA or detectable CU protein, but in greenhouse trials it retained full 

pathogenicity. These unexpected findings suggest that CU is not a primary pathogenicity 

factor in the development of DED symptoms. The biological role of CU has yet to be 

determined. It has been suggested (Bowden et al. 1996) that CU may be involved in 

surface hydrophobicity of aerial hyphae and conidiospores. The validity of this 

hypothesis is undergoing experimentation. 



12 

C. kikuchii produces the necrosis inducing polyketide toxin cercosporin. Cercosporin is 

a photosensitising toxin which requires light for both synthesis (Fajola 1978, Daub 1982, 

Ehrenshaft et al. 1991) and toxin activity (Daub 1982). Upon exposure to light, 

cercosporin interacts with molecular oxygen to produce highly toxic singlet oxygen 

which has been shown to cause membrane damage and cell death by the peroxidation of 

plant membrane lipids (Daub and Briggs 1983, Daub and Hangarter 1983). It is 

postulated that both tissue colonisation and nutrient acquisition by the fungus are 

facilitated by the action of cercosporin. Treatment of plant tissue with pure cercosporin 

reproduces the ultra-structural changes that are both consistent with the known mode of 

action of cercosporin and similar to the disease symptoms caused by the pathogen itself 

(Bails and Payne 1971). Also, cercosporin has been isolated from the necrotic lesions of 

several infected plant hosts (Fajola 1978, Venkataramani 1967). Although this evidence 

suggests that cercosporin plays an important role in diseases caused by Cercospora 

species, until recently, little was known about the actual mechanisms of pathogenicity. 

To contribute to a better understanding, Upchurch et al. (1991) isolated UV-induced 

cercosporin blocked mutants. These cercosporin-blocked mutants were used to inoculate 

soybeans which were shown to be non-pathogenic. This provided direct evidence that 

cercosporin is a crucial pathogenicity factor (Upchurch et al. 1991). 

The ability to obtain mutants altered or blocked in the synthesis of toxins (such as CU 

and cercosporin in 0. ulmi and C. kikuchii, respectively) offers considerable potential for 

use in elucidating the molecular basis of pathogenicity in D. pini. Although, indirect 

evidence has implicated the importance of dothistromin in Dothistroma needle blight, the 

true role of the toxin as a pathogenicity or virulence factor has yet to be determined. The 

multiseptate and multinucleate nature of D. pini conidia make it difficult to employ 

conventional UV mutagenesis for the isolation of a loss-of-function mutation, so 

dothistromin-mutants will be most easily isolated by disrupting the gene. Evaluation of 

the pathogenicity of dothistromin-minus D. pini strains could then be performed. This is 

vital information for the HortResearch programme, since if the toxin-minus mutants are 

still pathogenic there will be no resistance conferred by the production of anti­

dothistromin antibodies in the host trees. Moreover, if dothistromin is a virulence factor 

then, potentially, in the absence of toxin, the severity of the disease may increase due to 

the lack of a hypersensitive-type defence response in the tree. The effect of dothistromin 

in the disease process may become apparent only after dothistromin biosynthesis is better 

understood. 



13 

1 . 7 Aflatoxin Biosynthesis 

The polyketide-derived secondary metabolites, aflatoxin (AF) and sterigmatocystin (ST), 

are among the most toxic, mutagenic, and carcinogenic natural products known. AFs are 

produced only by certain Aspergillus parasiticus, A. flavus, and A. nomius strains, while 

ST, the penultimate intermediate in the AF pathway is synthesised as an end product by 

numerous ascomycetes and deuteromycetes including A. nidulans (Brown et al. 1996). 

These ubiquitous fungi are capable of infecting a wide variety of crops such as com, 

peanuts, and cottonseed, which, under the proper environmental conditions, can become 

contaminated with this potent mycotoxin (Cary et al.1996, Trail et al. 1995a). Because 

of their potent carcinogenic effects on laboratory animals including rats, ducks, and 

monkeys, aflatoxins are considered to be a potential threat to human health and are an 

economic problem in many areas of the world. Due to the difficulty in effectively and 

economically controlling aflatoxin contamination of food and feed by traditional 

agricultural methods (i.e. irrigation, application of fungicides or insecticides, and use of 

resistant crop varieties), recent efforts in several laboratories have focused on developing 

an in depth understanding of the molecular biology of the aflatoxin biosynthetic pathway. 

An understanding of the aflatoxin biosynthetic pathway may result in the identification of 

strategies to inhibit aflatoxin contamination of plant-derived products at the pre-harvest 

level. These strategies are focused on two main areas: (i) genetically modified crops to 

reduce fungal growth or inhibit aflatoxin biosynthesis (long-term approach); and (ii) 

utilisation of biological control organisms to competitively exclude the toxigenic fungus 

from infecting the crop (short-term approach). A detailed understanding of the aflatoxin 

biosynthetic pathway at the molecular level will aid in the pursuit of these approaches of 

control. 

The ST/ AF pathway was elucidated through the isolation and characterisation of A. 

flavus and A. parasiticus mutants blocked in AF production, radiolabelled-precursor 

feeding experiments, enzyme inhibitor studies, and biochemical characterisation of 

enzymatic activities. AF/ST biosynthesis is proposed to begin with the condensation of 

acetyl coenzyme A and malonyl coenzyme A via polyketide synthetase (PKS) to form the 

decaketide noranthrone. Alternatively, a six-carbon fatty acid, hexanoate, is first 

synthesised by a fatty acid synthetase and then extended by a PKS to generate 

noranthrone. Noranthrone is oxidised to norsolorinic acid (NA), which is converted to 

aflatoxin B 1 (AFB 1) through a series of pathway intermediates, including averantin 

(AVN), averufanin (AVNN), averufin (AVF), versiconal hemiacetal acetate (VHA), 

versiconal (VAL), versicolorin B (VB), versicolorin A (VA), demethylsterigmatocystin, 

sterigmatocystin (ST), 0-methylsterigmatocystin (OMST), and AFB 1. The generally 



14 

accepted pathway is illustrated in Fig. 2. As many as 17 different enzymes are proposed 

to be involved in aflatoxin biosynthesis (Trail et al. 1995a, Mahanti et al. 1996). 

1.8 Gene Cloning Strategies in Aspergillus species 

Aflatoxin-blocked mutants and purified enzymes have been used to clone several genes 

involved in the aflatoxin biosynthetic pathway. The cloning of these genes has been the 

key to increasing understanding of the molecular biology of the pathway. Cloned genes 

are useful probes for elucidating the molecular mechanisms that regulate the timing and 

expression of these genes. Two different strategies have been successfully utilised in the 

cloning of several genes encoding enzymes involved in AF biosynthesis in A. parasiticus 

and A. flavus and ST biosynthesis in A. nidulans. These genes include pksA, which 

codes for a polyketide synthase (Chang et al. 1995b, Trail et al. 1995b), nor-I, which 

codes for a reductase that reversibly converts NA to averantin (Chang et al. 1992, Trail et 

al. 1994), ver-I, which is involved in the conversion of VA to ST (Keller eta!. 1994, 

Skory et al. 1992), and omtA, which codes for an S-adenosylmethionine-dependent O­

methyltransferase that converts ST to OMST and dihydrosterigmatocystin to dihydro-O­

methylsterigmatocystin (Yabe et al. 1989, Yu et al. 1992). In addition to these structural 

genes, a regulatory gene, aflR, that codes for a regulatory factor (AFLR protein) has been 

cloned and was shown to be involved in the activation of pathway gene transcription 

(Chang et al. 1993, Payne et al. 1993). Also, a putative fatty acid synthase gene, umv8, 

potentially involved in polyketide backbone synthesis, and a gene, aad, homologous to 

aryl-alcohol dehydrogenase which may be involved in an intermediate step of AF 

biosynthesis have also been cloned (Yu et al. 1995). For introduction of DNA into the 

fungus, transformation systems were developed for A. parasiticus (Skory et al. 1990) 

and A flavus (Woloshuk et al. 1989). 

The nor-I (Chang et al. 1992) and ver-I (Skory et al. 1992) genes were cloned by 

complementation of aflatoxin blocked mutants which accumulate the brightly coloured 

pathway intermediates NA (brick-red) and VA (yellow), respectively. Complementation 

was achieved by introduction of a cosrnid DNA library using genomic DNA from a wild 

type aflatoxin-producing strain of A parasiticus. The functionally homologous verA 

gene of A. nidulans was isolated by hybridisation of ver-1 to an A. nidulans genomic 

DNA library (Keller et al. 1994). The predicted amino acid sequences of the ver-I and 

verA gene products are nearly identical (Keller et al. 1994), illustrating the high degree of 

identity between aflatoxin biosynthetic genes among these Aspergillus species. The 

predicted amino acid sequences of nor-I, ver-I, and verA contain an NAD(P)H binding 



Fig. 2 Comparison of the aflatoxin biosynthetic pathway with the 

proposed dothistromin biosynthetic pathway 

AEL
Pencil



Aflatoxin Bl 
Biosynthetic Pathway 

Polyketide 
jj, 

Norsolorinic acid (NA) 
jj, nor-I 

Averantin (AVN) 
jj, 

Averufanin (AVNN) 
jj, 

A verufin (A VF) 
jj, 

V ersiconal hemiacetal 
acetate (VHA) 

jj, 
V ersiconal (VAL) 

jj, 
Versicolorin B (VB) 

jj, 
Versicolorin A (VA) 

jj, ver-1 
Demethylsterigmatocystin 

jj, 
Sterigmatocystin (ST) 

.[J, 

0-Methlysterigmatocystin 
(OMST) 

.[J, 

Aflatoxin B 1 (AFB 1) 

16 

Proposed Dothistromin 
Biosynthetic Pathway 

Polyketide 
jj, 

jj, 
Averantin (AVN) 

jj, 

jj, 
Averufin (AVF) 

jj, 

jj, 
Versicolorin B (VB) 

jj, 

jj, 
Dothistromin 



17 

motif near the amino terminus and show significant identity (33% for ver-1/verA, 23% 

for nor-I) to several NADPH- and NADH-dependent reductase/dehydrogenase enzymes 

found in other polyketide biosynthetic pathways. Each sequence also contains a short­

chain alcohol dehydrogenase motif (Trail et al. 1994). 

To confirm the role of these genes in AF biosynthesis, gene disruption was conducted in 

toxigenic strains of A. parasiticus (nor-I ,Trail et al. 1994; ver-1, Trail et al. 1995b) and 

A. nidulans (ver-A, Keller et al. 1994). Disruption of the ver-A gene resulted in loss of 

detectable ST and accumulation of VA by A. nidulans, confirming its role in conversion 

of VA to ST. Similarly, disruption of ver-1 blocked the AF pathway, resulting in VA 

accumulation. Disruption of nor-I resulted in accumulation of large quantities of 

norsolorinic acid (NA). However, disrupted nor-I strains retained their ability to 

produce low levels of aflatoxin, supporting the hypothesis that there are one or more 

alternative routes (or enzymatic activities) in the AF pathway to synthesise averufin from 

NA (Yabe et al. 1993). 

The umv8 gene was cloned by complementation of an aflatoxin blocked mutant, umv8, 

derived by UV mutagenesis (Mahanti et al. 1994 ). Metabolite conversion studies 

confinned that umv8 has two blocks in the pathway, one block at nor-I and the other one 

prior to nor-I. Nucleotide sequence analysis of this gene revealed that the predicted 

protein contains a high degree of similarity (67%) and identity (48%) to the enoyl­

reductase and malonyl/palmityl transferase domains in the p subunit of yeast fatty acid 

synthase (FAS 1, Kottig et al. 1991) from Saccharomyces cerevisiae (Mahan ti et al. 1996, 

Trail et al. 1995a). umv8 was therefore thought to encode an FAS activity necessary for 

synthesis of the hexanoate starter. Consequently, its name was changed to fas-I. It is 

possible that synthesis of the hexanoate starter requires two FAS subunits (a and p; 
encoded by unique genes) analogous to those of yeast. Gene disruption combined with 

feeding studies will allow this hypothesis to be tested. 

A second approach for isolating genes, reverse genetics, relied on the purified pathway 

enzymes discussed above. Where purification has been possible, production of 

antibodies to the enzyme, and isolation of the gene from a cDNA expression library in E. 

coli, can be accomplished. This procedure was utilised to clone the omt-1 gene from A. 

parasiticus encoding the O-methyltransferase activity responsible for conversion of ST to 

O-sterigmatocystin (Yu et al. 1993). The predicted amino acid sequence derived from the 

cloned cDNA contained a motif found in other S-adenosylmethionine-methyl-dependent 

methyltransferases. More recently, Cary et al. (1996) reported the isolation of a full­

length cDNA from A. parasiticus harbouring a gene designated norA. The norA cDNA 



18 

clone was isolated with monoclonal antibodies (MAbs) raised against a purified A. 

parasiticus enzyme demonstating norsolorinic acid reductase (NOR) activity. They also 

identified and sequenced the norA homologue in A flavus (Cary et al. 1996). So far, 

these genes are the only pathway genes cloned by the reverse genetics approach. 

However, this approach should be successful in cloning several other genes encoding the 

purified pathway enzymes. 

Another molecular genetic approach for gene cloning, differential screening, has been 

used by Feng et al. ( 1992) in an attempt to isolate genes whose pattern of expression 

coincides with aflatoxin production in A parasiticus. This method is not based on 

specific knowledge of the function of the gene product, as in the two previous methods, 

and can therefore be advantageous when the timing of induction of gene expression is 

known but pure enzymes or blocked pathway mutants are not available. With this 

technique Woloshuk and Payne ( 1994) were successful in isolating an alcohol 

dehydrogenase gene, adhI, from A. flavus that was induced under growth conditions 

conducive to aflatoxin biosynthesis. However, to date, this method has failed to identify 

conclusively any genes directly involved in the aflatoxin biosynthetic pathway. 

1.9 Organisation and Arrangement of the AF/ST Pathway 
Genes 

Parasexual analysis using A. flavus and A parasiticus suggested that the genes involved 

in AF biosynthesis were linked (Papa 1978). Attempts to demonstrate linkage of nor-I 

and ver-I genes in A parasiticus by parasexual analyses, however, gave conflicting 

results (Bradshaw et al. 1983, Lennox et al. 1983, Papa 1984). Molecular and genetic 

analyses have now provided proof that many of the genes involved in AF biosynthesis in 

A. parasiticus and A. flavus are physically clustered on one chromosome. During the 

cloning and characterisation of the nor-I and ver-1 genes from A. parasiticus, one 

cosmid, NorA, was identified that hybridised to probes of both genes (Skory et al. 

1992). This tentative evidence for linkage was later confirmed by physical mapping of 

the corresponding region in the fungal genome in A. parasiticus (Trail et al. 1995b). 

aflR, umv8, and omt-I were recently mapped to this cluster and to a similar cluster of AF 

genes in A flavus (Yu et al. 1995, Trail et al. 1995b). 

Since as many as 17 different enzymes are thought to be required to complete AF 

biosynthesis it was hypothesised that the cosmid NorA (and the corresponding region in 

A. flavus) encoded several other pathway enzymes. To determine the size, location, and 

pattern of expression of other genes in the cluster, a transcriptional map of the 35 kb 



19 

genomic DNA insert in cosmid NorA was completed (Trail et al. 1995b). Twelve unique 

RNA transcripts localised to this cluster. Eight of these transcripts, previously 

unidentified, showed a pattern of expression similar to that of nor-I and ver-1, 

suggesting that the genes encoding them are also involved in AF biosynthesis. To 

directly test this hypothesis, gene-I (tentatively named because of its position at the far 

left end of the cluster), encoding one of the eight transcripts, was disrupted in an VA­

accumulating mutant of A. parasiticus. Thin-layer chromatography revealed that gene- I 

disruptant clones no longer accumulated VA. Southern hybridisation analysis of the 

disruptant clones confirmed that gene-I is directly involved in AF biosynthesis. 

Nucleotide sequence analysis of two regions within gene-I showed a high degree of 

identity and similarity with the P-ketoacyl-synthase and acyltransferase functional 

domains of the wA gene product in A. nidulans (which encodes a PKS involved in 

conidial pigment production, Mayorga and Timberlake 1992) and other polyketide 

synthases (Trail et al. 1995b ). It is possible that this putative aflatoxin PKS is involved 

in extending the hexanoate starter unit synthesised by the umv8 gene product. 

Recently, it has been shown that the A. nidulans ST pathway is conserved at the 

functional, regulatory, and physical levels with the AF pathway in A. flavus and A. 

parasiticus (Keller et al. 1994, Keller et al. 1995, Yu et al. 1995). For example, ver-A, a 

homolog of the A. parasiticus ver-1 gene is required for the same bioconversion in A. 

nidulans (Keller et al. 1994, Skory et al. 1992). In addition, the forced expression of the 

A. flavus AF regulatory gene, aflR, in A. nidulans induces expression of the ve r-A 

transcript, indicating that regulation of the ST/AF pathway is functionally conserved 

(Chang et al. 1993, Woloshuk et al. 1994). Moreover, a putative PKS required for ST 

biosynthesis is located within approximately 48 kb of ver-A in A. nidulans, which is 

similar to the grouping of homologous genes in A. parasiticus and A. flavus (Yu et al. 

1995, Keller et al. 1994, Yu and Leonard 1995). More recently, Brown et al. (1996) 

presented the entire sequence of a 60 kb region in the A. nidulans genome and found it to 

contain many, if not all, of the proposed genes needed for ST biosynthesis. This was 

based on three observations: (i) A total of eight of the genes, stcA (previously pksST, 

Yu et al. 1995), stcS (previously verB, Keller et al. 1995), stcU (previously verA, Keller 

et al. 1994), aflR (Yu and Leonard 1995), stcl, stcK, stcL, and stcN (Brown et al. 1996) 

have been shown to be necessary for ST biosynthesis. (ii) All 25 -transcripts 

corresponding to the proposed genes are coordinately regulated. (iii) The disruption of 

aflR, a putative pathway-specific regulator, resulted in loss or greatly reduced 

accumulation of all 25 transcripts (Yu et al. 1995). Functional assignment of the 

remaining cluster genes will require gene disruption followed by structural 

characterisation of any accumulating materials. It is possible that these studies will result 

in a revised ST/AF pathway. 



20 

Due to this increasing body of evidence that gene sequences are highly conserved among 

A. parasiticus, A. flavus, and A nidulans it is probable that cloned AF and ST pathway 

genes will provide a direct method for identifying dothistromin biosynthetic genes via 

their use as heterologous probes in Southern hybridisation analysis. 

1.10 Aims and Objectives 

The primary focus of current research is to obtain mutants blocked or altered in the 

synthesis of dothistromin. This will allow the application of molecular biology 

approaches to the study of dothistromin synthesis and regulation, and the ability to more 

directly assess the role of dothistromin in the pathogenicity of D. pini. Biochemical and 

molecular genetic studies will allow the isolation and identification of genes involved in 

dothistromin biosynthesis and will contribute to a better understanding of the structure, 

function, and organisation of the dothistromin biosynthetic pathway genes, as well as 

elucidation of the molecular control mechanisms that regulate dothistromin production 

(i.e. regulatory genes) . This study will focus on the isolation and cloning of genes 

required for dothistromin biosynthesis. 

Clones of aflatoxin biosynthetic genes from A parasitic us will be used as hybridisation 

probes in the hope that they will share sufficient sequence identity to D. pini dothistromin 

biosynthetic genes to permit their detection. Genes involved in dothistromin biosynthesis 

can then be isolated and cloned by screening a D. pini genomic library. 

The eventual characterisation of several genes will allow testing of the fundamental 

assumption that pathogenesis is primarily dothistromin-mediated, by producing isolates 

of D. pini which no longer produce the toxin and assessing if they are capable of 

invoking the benzoic acid response from the plant. Hence we can ascertain the precise 

roles of the fungal mycelium and the toxin in the disease process. A transformation 

system has recently been developed for D. pini (Bidlake 1996) using a positive selection 

system based on the E. coli hygromycin resistance gene (hph). This will enable targeted 

disruption of wild type dothistromin biosynthetic genes. 

The production of genetically stable atoxigenic strains of D. pini which, by disruption of 

several pathway genes (or a complete cluster), are rendered completely disabled in 

dothistromin production, could then be used for competitive exclusion of dothistromin­

producing strains in the field. Biocompetition of this type has proven to be quite 



21 

successful in laboratory and field tests with mutant aflatoxin strains (Skory et al. 1990, 

Skory et al. 1992). Furthermore, an understanding of the molecular control mechanisms 

involved in dothistromin biosynthesis may lead to the development of agents or 

genetically engineered plants that inhibit toxin production. 



22 

2.0 MATERIALS AND METHODS 

2 .1 Bacterial and Fungal Strains, A Clones and Vectors 

The bacterial and fungal strains, A. clones, and plasmids used in this study are listed in 

Table 1. 

2.2 Media 

All media was prepared using MilliQ water. After preparation the media was sterilised by 

autoclaving at 121 °c and 15 psi for 20 min. Liquid media was cooled to room 

temperature before addition of antibiotic(s) and inoculation. Solid media was cooled to 

approximately 50°C prior to antibiotic addition and pouring. Uninoculated plates were 

stored at 4°C. 

2.2.1 Fungal media 

2.2.1.1 D. pini Media (DM) Broth 

DM broth contained (g/1): malt extract (Oxoid), 50.0 (5%); and nutrient broth (Oxoid), 

28.0. When stated, malt extract content was altered to (g/1): either, 30.0 (3%) or 10.0 

(1%). 

2. 2 .1. 2 D. pini Media (DM) Agar 

DM agar contained (g/1): malt extract (Oxoid), 50.0; nutrient agar (Oxoid), 28.0; glucose 

(BDH), 20.0 (if required) 

2. 2 .1. 3 Malt Extract Agar (MEA) 

MEA containined (g/1): malt extract (Oxoid), 30.0, peptone (Oxoid), 5.0; agar (Davis), 

20.0; glucose (BDH), 20.0; uracil (Sigma), 2.2 (if required) 



23 

2.2.1.4 Malt Yeast Glucose (MYG) Agar 

MYG contained (g/1): malt extract (Oxoid), 5.0; yeast extract (Oxoid), 2.5; agar (Davis) 

20.0; glucose (BDH), 20.0; CuSO45H2O, trace. 

2.2.1.5 Yeast Peptone Glycerol (YPG) Agar 

YPG contained (g/1): yeast extract (Oxoid), 10.0; peptone (Oxoid), 20.0; agar (Davis), 

20.0; and (ml/1): glycerol (BDH), 30.0. 

2.2.1.6 Potato Dextrose Agar (PDA) 

PDA contained (g/1): potato dextrose broth (Difeo), 24.0; agar (Davis) 20.0; glucose 

(BDH), 20.0 (if required). 

2. 2 .1. 7 Nutrient Malt Yeast (NMY) Agar 

NMY agar contained (g/1): nutrient agar (Oxoid), 28; malt extract (Oxoid), 10.0; yeast 

extract (Oxoid), 10.0; glucose (BDH), 20.0 (if required). 

2. 2. 1. 8 Minimal Medium (MM) Agar 

MM agar contained (g/1): NaNO3 (BDH), 6.0; MgSO4.7H2O (BDH), 0.52; KCl (BDH), 

0.52; KH2PO4 (BDH), 1.52; FeSO4. 7H2O (BDH), trace; ZnSO4.7H2O (BDH), trace; 

glucose (BDH), 20.0; agar (Davis), 20.0. 

2.2.1.9 Nutrient Yeast (NY) Broth 

NY broth contained (g/1): nutrient broth (Oxoid), 28.0; yeast extract (Oxoid), 10.0. 

2. 2 .1.10 Nutrient Malt Yeast (NMY) Broth 

NMY broth contained (g/1): nutrient broth (Oxoid), 28.0; malt extract (Oxoid), 50.0; 

yeast extract (Oxoid), 10.0. 



Table 1 Fungal and Bacterial Strains, A Clones and Vectors 

Strain, A Clone or 

Plasmid 

Fungal Strains 

Aspergillus nidulans 

R21 

Dothistroma pini 

Dpl 

Bacterial Strains 

Escherichia coli 

KW251 

XLl-Blue 

A. Clones 

A.CGVl and )..CGV2 

)..CGNl 

Relevant Characteristics 

pabaAl yAl 

wild type strain (forest isolate, 

Long Mile Road, Rotorua) 

p- supE44 supF58 galK2 gaIT22 

metB 1 hsdR2 mcrB 1 mcrA-

argA81:TnlO recD1014 

supE44 hsdRl 7 rec Al endAl 

gyrA46 thi relAl lac- F fproAB+ 

lacUM15 TnlO (tetf)] 

A.GEM-12 clones containing 

D. pini genomic DNA hybridising 

to the A parasiticus ver-1 gene 

A.GEM-12 clone containing 

D. pini genomic DNA hybridising 

to the A. paraciticus nor-I gene 

Source or 

Reference 

Waldron and 

Roberts 1973 

P. Debnam 1993 

Murray et al. 

1977 

Bullock et al. 

1987 

This study 

This study 

24 



Table 1 (Continued) 

Vectors 

pNA17 

pBVer-1 

pUCl 18 

AGEM-12 

pCGl 

pCG2 

pUC19 containing a 1.7 kb 

SphVBgffi fragment containing 

the A. parasiticus nor-I gene 

pBR322 containing a 2.35 kb 

ClaVHindlll fragment containing 

the A. parasiticus ver-1 gene 

3.2 kb ampr 

lambda genomic cloning vector 

Chang et al. 1992 

Skory et al. 1992 

Messing 1983 

Frischauf et al. 

1983 

pUCl 18 containing a 0.8 kb SalI This study 

fragment from A.CGV 1 

M 13mp 18 containing a 2.1 kb This study 

BamHVSalI fragment from A.CGV2 

25 



26 

2.2.2 Bacterial Media 

2.2.2.1 Luria-Bertaini (LB) Media 

LB media contained (g/1): tryptone (Difeo), 10.0 ; NaCl, 5.0; yeast extract (Oxoid), 5.0. 

The pH was adjusted to 7.0 prior to autoclaving. For solid media, agar (Davis) was 

added to 15.0 g/1. 

When required LB was supplemented after autoclaving to give final concentrations of: 

ampicillin, 100 µg/ml from a stock solution of 100 mg/ml; tetracycline, 10 µg/ml from a 

stock solution of 10 mg/ml in methanol; isopropylthio-P-D-galactoside (IPTG), 30 mg/ml 

and 5-bromo-4-chloro-3-indolyl-P-D-galactoside in dimethylformamide (X-gal), 60 

µg/ml . 

2.2.2.2 TB Top Agar 

TB top agar contained (g/1): tryptone (Difeo), 10.0 ; NaCl, 5.0; agar (Davis), 8.0. This 

was cooled to 45-50°C following autoclaving and supplemented with 10 rnM MgSO4. 

2.2.2.3 NZCYM 

NZCYM contained (g/1): NZ amine, 10.0; NaCl, 5.0; Casamino acids (Difeo), 1.0; yeast 

extract, 5.0; MgSO4.7H20 , 2.0. NaOH was added until the pH was 7.5. 

2.3 Growth and Maintenance of Cultures 

2.3.1 Fungal Cultures 

A (8.0 mm x 8.0 mm) chunk of D. pini mycelia (cut with a scalpel blade) was ground in 

1ml of sterile MilliQ water using a plastic grinder in an eppendorf tube. From this, 200 

µl was spread onto DM plates with sterile cellophane discs which were sealed with 

parafilm. D. pini fungal cultures were grown at 20°C for 7 days, and then stored at 4°C 

for up to 6 months before subculturing. When stated, two other methods of inoculation 

were also used: ground up mycelia were streaked onto agar plates, or 3-4 pieces of 

mycelia (approximately 3 mm x 3 mm) were placed on the agar plate. DM broth cultures 

were inoculated in the same way with 1 ml of inoculum/100 ml of DM broth in a 1 litre 

siliconised flask. These were grown at 20°C with gentle shaking ( 100 rpm) for up to 7 



27 

days. Mycelial growth was quantitated as follows . The entire contents of each broth 

culture was vacuum filtered through pre-dried and -weighed Whatman no. 1 filter paper, 

using a Buchner funnel, and the filtrate discarded. The mycelial extracts were washed, 

then dried in a hot air oven at 80°C for 2 hr, weighed, and expressed as mycelial dry 

weight (g/100 ml) . 

Note: the inoculum size is not strictly quantitative due to varying sizes (5.0 mm-10.0 mm 

x 5.0 mm-10.0 mm) of mycelia chunks being unavoidably cut depending on the 

morphology and thickness of the mycelial material. 

2.3.2 Bacterial Cultures 

E. coli cultures were maintained on LB plates supplemented with the appropriate selective 

antibiotics. Cultures were grown at 37°C, sealed with parafilm, and then stored at 4°C. 

They were regularly restreaked onto fresh LB plates. 

2.4 Buffers and Solutions 

2.4.1 TEG 

TEG contained: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), and 10 mM EDT A. 

2.4.2 Tris-Equilibrated Phenol 

Tris-equilibrated phenol was prepared by melting solid phenol at 50°C. An equal volume 

of 100 mM Tris-HCI (pH 8.0) was added at room temperature and stirred for 15 min, 

then the phases left to separate for 15-30 mins. The aqueous phase was then decanted, 

and the phenolic phase retained and repeatedly washed with 100 mM Tris-HCI until the 

pH reached 8.0. After equilibration the phenolic phase was retained and an equal volume 

of 100 mM Tris-HCl (pH 8.0) was added. Hydroxyquinoline was added to a final 

concentration on 0.1 % (w/v). The equilibrated phenol was stored in a brown bottle at 

4°c. 



28 

2.4.3 TE Buffer 

Tris EDTA buffer (10 mM Tris-HCVlmM Na2EDTA (10/1) or 10 mM Tris-HCV0.l mM 

N a2EDT A(l 0/0.1)) was prepared to the required concentration from 1 M Tris-HCl (pH 

7.5) and 0.5 M Na2EDTA (pH 8.5) stock solutions. 

2.4.4 DNase free RNase 

DNase free RNase contained: 10 mg/ml pancreatic RNase A, 10 mM Tris-HCl (pH 7.5), 

and 15 mM NaCl. This was heated to 100°C for 15 min to inactivate the DNase, allowed 

to cool slowly to room temperature, checked for residual DNase activity, dispensed into 

aliquots, and stored at -20°C. 

2.4.5 10 x TAE Buffer 

10 x Tris acetate EDTA buffer contained: 400 mM Tris, 11.4 mVl glacial acetic acid, and 

20 mM EDT A. The pH was adjusted to 8.5. 

2.4.6 10 x Gel Loading Dye 

10 x gel loading dye contained 50% (w/v) glycerol, 1 x T AE buffer (see Section 2.4.5), 

12% (w/v) urea and 0.4% (w/v) bromophenol blue. 

2.4.7 20 x SSC 

20 x standard saline citrate contained (g/1): NaCl, 175.4 g; and trisodium citrate, 88.2 g 

to give final concentrations of 3 M NaCl and 0.3 M sodium citrate. The pH was adjusted 

to 7.0. 

2.4.8 TES (10/1/100) 

TES buffer (10/1/100) contained 10 mM Tris-HCl (pH 8.5), lmM Na2EDTA (pH 7.5) 

and 100 mM NaCL 



29 

2.4.9 50 x Denhardt's 

Denhardt's solution (50 x) contained (g/1): Ficol, 10; PVP, 10; and BSA, 10. This was 

made up to 1 litre with sterile MilliQ water, filter sterilised, and stored in 5 ml aliquots at 

-20°C. 

2.4.10 SM Buffer 

SM buffer contained (g/1): NaCl, 5.8; MgSO4.7H2O, 2; and 50 ml/1 1 M Tris-HCl (pH 

7.5). 

2. 4 .11 Acrylamide mix 

Acrylamide mix contained (g/1): urea, 288; acrylamide, 34.2; and bis-acrylamide, 1.8. 

This mix was made up to about 500 ml and deionised with 1 g of Amberlite MB-3 

(Sigma), then filtered through a sintered glass funnel (porosity 1 ), 60 ml of 10 x TBE 

sequencing buffer (see Section 2.4.12) was then added and the volume made up to 600 

ml with MilliQ water. This was stored at 4°C. 

2. 4 .12 10 x TBE Sequencing Buffer 

10 x TBE sequencing buffer contained (g/1): Tris, 162.0; Na2EDTA, 9.2; and boric acid, 

27.5. The pH was adjusted to 8.8. For running sequencing gels this buffer was diluted 

10 x with MilliQ water to give final 1 x concentrations of 134 mM Tris, 2.5 mM 

Na2EDTA and 45 mM boric acid. 

2.5 DNA Preparations 

2.5.1 Alkaline Lysis E. coli Plasmid Preparation 

This protocol is based on a procedure described by Sambrook et al. ( 1989) which is a 

modification of the methods of Birnboim and Doly (1979) and Ish-Horowicz and Burke 

(1981 ). A 2 ml volume of LB (Section 2.2.2.1) supplemented with ampicillin was 

inoculated with a single bacterial colony, and incubated overnight on a shaker at 37°C. 

From this overnight culture, 1.5 ml was transferred into an eppendorf tube, and the cells 

pelleted by centrifugation for 1 min. The media was removed by aspiration, leaving the 

pellet as dry as possible. The cells were resuspended by vortexing in I 00 µl of TEG 



30 

(Section 2.4.1). This mixture was kept at room temperature for 5 min. A 200 µl aliquot 

of a freshly prepared solution of 0.2 M NaOH and 1 % SDS was added, and mixing was 

carried out by several rapid inversions. This was stored on ice for 5 min. Then 150 µl 

of ice cold 5 M potassium acetate was added, and the mixture was vortexed gently for 10 

seconds, then stored on ice for 5 min. The mixture was centrifuged for 5 min, and the 

supernatant was transferred to a clean tube. An equal volume of Tris-equilibrated 

phenol/chloroform was added, and the mixture vortexed and stored at room temperature 

for 2 min. The phases were separated by centrifugation for 5 min. The aqueous phase 

was transferred to a clean tube, and 2 volumes of 100% ethanol was added. The mixture 

was vortexed and stored at room temperature for 2 min. The DNA was collected by a 5 

min centrifugation, and the ethanol was drained from the tube. The pellet was washed 

with 70% ethanol, centrifuged for 5 min, and the ethanol drained from the tube. The 

pellet was dried, then resuspended in 25 µl of TE buffer (Section 2.4.3). RNase (10 

mg/ml, Section 2.4.4) was incorporated in restriction digests at 0.5 µg/µl 

2.5.2 Large Scale D. pini Genomic DNA Preparation 

DNA was extracted from D. pini using a modification of the method described by Raeder 

and Broda (1985). D. pini was grown on DM agar (Section 2.2.1.2) plates overlaid with 

sterile cellophane discs. Mycelia from four plates were removed from the cellophane 

discs, and freeze-dried. This was ground to a powder in liquid N2 using a mortar and 

pestle, and suspended in 4 ml of extraction buffer (200 mM Tris-HCl (pH 8.0), 250 mM 

NaCl, 25 mM EDT A, 0.5% (w/v) SDS) per 100 mg of dry weight mycelium which was 

vortexed thoroughly. For each 4 ml of extraction buffer, 2.8 ml of phenol equilibrated 

with extraction buffer was added and mixed thoroughly. To this, 1.2 ml of 

chloroform:isoamyl alcohol (24: 1) was added, then mixed, and centrifuged at 17,300 g 

(12,000 rpm, SS34) for 40 min at 4°C. The supernatant was transferred to a clean corex 

tube and re-extracted with phenol/chloroform in the same proportions, then centrifuged 

for 15 min. DNase free RNase (10 mg/ml, Section 2.4.4) was added to the supernatant 

at 100 µg/ml, and incubated at 37°C for 30 min. Phenol/chloroform (1:1) was added and 

the mixture was centrifuged 15 min. An equal volume of chloroform was then added to 

the aqueous phase which was centrifuged for 20 min. The DNA was precipitated by 

adding 0.54 volume of isopropanol to the aqueous phase and centrifuged for 5 min. The 

supernatant was then discarded. The pellet was washed with 70% ethanol, centrifuged as 

before and the supernatant discarded. The DNA was vacuum dried and resuspended in 

100 µl TE buffer 10/1 (Section 2.4.3), and the concentration of the DNA determined 

(Section 2.6.4). 



31 

2.5.3 Preparation of Genomic DNA for D. pini Library Construction 

DNA was extracted from D. pini using a modification of a method designed to obtain 

high molecular weight DNA from plant tissue (Ausubel et al. 1990). The basis of this 

method is that fungal cells are lysed by the detergent Sarkosyl and the lysates digested 

with Proteinase K. After clearing the insoluble debris form the lysate the nucleic acids 

are precipitated and the DNA purified on a caesium chloride gradient. 

Fresh fungal tissue (10-50 g) was harvested, then frozen with liquid nitrogen and ground 

to a fine powder in a mortar and pestle. The frozen powder was transferred to 2 x 250 ml 

Nalgene centrifuge bottles and extraction buffer (100 mM Tris-HCI, pH 8.0, 100 mM 

EDTA, 250 mM NaCl, 100 µg/ml Proteinase K) at 20 ml/g was immediately added. An 

appropriate volume of Sarkosyl (10% (w/v) N-lauryl sarcosine) was added to achieve a 

final concentration of 1 %. The mixture was incubated for 2 h at 55°C, and the lysate 

centrifuged for 10 min at 5,860 x g (6,000 rpm, GSA) at 4°C, to pellet the debris. To 

the supernatant, 0.6 volume of isopropanol was added, mixed gently, then placed at 

-20°C for 30 min. The DNA was then precipitated by centrifugation for 15 min at 

10,400 x g (8,000 rpm, GSA) and the supernatant discarded. The pellet was 

resuspended in 9 ml of TE buffer (Section 2.4.3), then 9.7 g of solid caesium chloride 

was added and mixed gently until dissolved. The lysates were incubated on ice for 30 

min, then centrifuged for 10 min at 7,500 x g at 4°C. A 0.5 ml volume of ethidium 

bromide ( 10 mg/ml) was added and the lysates incubated on ice for 30 min, then 

centrifuged for 10 min at 7,500 x g at 4°C. The supernatant was transferred to two 5 ml 

quick-seal ultracentrifuge tubes which were balanced and well sealed, then centrifuged 

overnight at 300,000 x g (60,000 rpm, Sorvall Ultracentrifuge) at 20°C. Using a large­

bore needle (15-G) and syringe the DNA band was collected. To do this a hole was first 

punched into the top of the tube using the needle, and the DNA band was removed by 

inserting the collecting needle/syringe through the tube wall directly below the band. By 

repeatedly extracting the collected DNA with CsCl-saturated isopropanol the ethidium 

bromide was removed. Two volumes of water and 6 volumes of ethanol was added to 

the DNA solution and mixed, then centrifuged for 10 min at 7,710 x g (8,000 rpm, 

SS34) at 4°C. The DNA pellet was resuspended in TE buffer, reprecipitated by adding 

0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol, then incubated at 

-20°C for 30 min and centrifuged briefly. The DNA pellet was air dried, resuspended in 

500 µl of TE buffer, and the concentration of the DNA was determined (Section 2.6.4). 



32 

2.5.4 Mini-prep of A Phage DNA 

This method is a modification of the Liquid Lysate method of phage preparation 

(Sambrook et al. 1989) which was originally described by Leder et al. (1967). A 50 ml 

volume of LB (Section 2.2.2.1) supplemented with 0.2% maltose and 10 mM 

MgSO4.7H2O was inoculated with a single colony of KW251 and shaken at 37°C 

overnight. Volumes of 100 µl of titred eluted phage (diluted to give 106-107 phage/100 

µl) and 100 µl of the overnight bacterial culture were combined and incubated at 37°C for 

30 min. The phage mixture was transferred to 50 ml of NZCYM (Section 2.2.2.3) in 

500 ml flasks and shaken vigorously at 37°C for 6-8 h until lysis occurred. The culture 

was harvested immediately upon clearing. A few drops of chloroform were added and 

the culture shaken for a further 15 min to lyse any remaining cells, then transferred to 

sterile 250 ml Nalgene bottles and centrifuged for 10 min at 16,300 g (10,000 rpm, 

GSA) to pellet the debris. The lysate was transferred to a fresh tube and stored at 4°C. 

DNase free RNase (10 mg/ml, Section 2.4.4) and DNase (10 mg/ml, Sigma) were added 

to the liquid lysate to give final concentrations of 10 µg/ml, and incubated for 1 h at 

37°C. NaCl and PEG 6000 were added to give final concentrations of 0.5 Mand 10% 

w/v, respectively, and dissolved using a magnetic stirrer, then precipitated on ice for two 

hours. The phage were pelleted by centrifugation at 4,920 g (5,500 rpm, GSA) for 10 

min at 4°C, the supernatant discarded and the bottles left upside down for 20 min to 

drain. The pellet was resuspended in 1 ml of SM buffer (Section 2.4.10), transferred to 

an eppendorf, then microcentrifuged at 12,000 rpm for 10 min. The supernatant was 

transferred to a fresh tube and Proteinase K was added (final concentration 0.1 mg/ml) 

and incubated at 37°C for 30 min. The phage suspension was extracted twice with an 

equal volume of phenol/chloroform, vortexed for 20 min and centrifuged at 16,300 x g 

for 5 min. An equal volume of chloroform was then added, vortexed for 10 min and 

centrifuged for 5 min. The aqueous phase was transferred to a clean tube and 2 volumes 

of ethanol+ 0.3 M ammonium acetate were added, the mixture centrifuged for 10 min, 

and the supernatant was discarded. The DNA pellet was washed with 70% ethanol and 

centrifuged for 5 min, the supernatant discarded, then recentrifuged briefly to remove 

excess liquid. The pellet was air dried and resuspended in TE buffer 10/0.1 (Section 

2.4.3) for 20 min at 65°C, then centrifuged to remove debris. The supernatant was 

transferred to a fresh eppendorf and stored at 4°C. 



33 

2.5.5 Extraction of DNA from Seaplaque Agarose 

Restriction enzyme digested DNA (Section 2.6.1.1) and a size marker used to determine 

the molecular weight of the DNA fragments (Section 2.6.3) were size fractionated by 

electrophoresis (Section 2.6.2) on a 1 % Seaplaque agarose (Biorad) gel (150 x 200 x 8 

mm or 140 x 110 x 8 mm) in 1 x T AE buffer (Section 2.4.5) at 34 V for 16-20 h. The 

gel was stained for 30 min in ethidium bromide solution, and destained in MilliQ water. 

The DNA fragments to be isolated were visualised under U.V. light, photographed, and 

the appropriate band was excised from the gel with a sterile scalpel blade. The excess 

agarose was trimmed away, then the DNA containing the band was placed in a 1.5 ml 

eppendorf tube. To extract the DNA from the agarose, one of the following two kit 

based methods below were used, according to the manufacturers instructions. 

2. 5. 5 .1 Bio 101 Geneclean kit 

The Bio 101 Inc. Geneclean DNA purification process (Labsupply Pierce) is based on a 

procedure described by Vogelstein and Gillespie (1979). To the excised DNA band 2.5 to 

3 volumes of Nal stock solution was added and incubated for 5 min at 50°C to melt the 

agarose. The GLASSMILK was vortexed vigorously for a min, then 5 µl was added to 

the Nal/DNA solution, mixed, and incubated on ice for 5 min to allow binding of the 

DNA to the silica matrix, mixing every 1-2 min to ensure that the GLASSMILK stayed in 

suspension. The GLASSMILK/DNA complex was microcentrifuged for 5 seconds, and 

the supernatant removed and set aside until the DNA recovery was confirmed. 500 µl of 

ice cold NEW wash (7 ml NEW stock concentrate diluted with 140 ml sterile MilliQ 

water and 155 ml 100% ethanol) was added to the GLASSMILK/DNA pellet, and the 

pellet resuspended by pipetting up and down. This solution was microcentrifuged for 5 

seconds and the supernatant was discarded. The wash procedure was repeated twice. 

Following removal of the supernatant from the third wash, the tube was spun for 10 

seconds and the liquid remnants removed. The GLASSMILK/DNA pellet was then 

resuspended in 20 µl of TE buffer, incubated at 50°C for 5 min, then centrifuged for 30 

seconds, and the supernatant containing the eluted DNA was placed in a new tube. 

2. 5. 5. 2 Gibco BRL GlassMAX DNA Isolation Spin Cartridge System kit 

The GlassMAX Spin Cartridge purification process (Life Technologies) is based on a 

procedure described by Vogelstein and Gillespie (1979). A stock of wash buffer was 

prepared by transferring 2.5 ml of Wash Buffer Concentrate to a labelled 200 ml screw­

cap container, adding 45 ml of distilled water and 52.5 ml of absolute ethanol, then 



34 

mixing thoroughly. This was stored at 4°C. The excised DNA band was weighed in a 

preweighed eppendorf tube, and 0.45 ml of Binding Solution per 0.1 g of agarose gel 

was added and incubated at 50°C for 5-7 min until the agarose was fully melted. Up to 

550 µl of DNA/Binding Solution mixture was added to the GlassMAX Spin Cartridge. 

Volumes greater than 550 µl required more than one loading. The cartridge was 

centrifuged at 13,000 x g for 20 seconds and the tube emptied. The solution was saved 

until recovery of the DNA was confirmed. A 0.4 ml volume of cold wash buffer was 

added to the spin cartridge and centrifuged at 13,000 x g for 20 seconds, and the tube 

emptied. This step was repeated twice. After removal of the final wash buffer, the tube 

was centrifuged at 13,000 x g for 1 min. The spin cartridge was inserted into a fresh 

Sample Recovery Tube and 20 µl of TE preheated to 65°C was added, then the spin 

cartridge centrifuged at 13,000 x g for 20 seconds to elute the DNA. 

2.5.6 Purification of DNA 

This method was based on that of Sambrook et al. ( 1989). A known volume of a 

solution of DNA was transferred to a centrifuge tube. One volume of Tris-equilibrated 

phenol was added to the DNA solution and vortexed thoroughly. This was stored on ice 

for 5 min, and then microcentrifuged for 5 min. The aqueous phase was transferred to a 

clean eppendorf tube. This process was repeated, first with an equal volume of 

phenol:chloroform (1: 1 v/v), and then with an equal volume of chloroform. Either, 2.5 

volumes of 95% ethanol with 0.1 volumes of 3 M sodium acetate was added, or 0.6 

volumes of isopropanol was added. This mixture was vortexed thoroughly, and stored at 

-70°C for 30 min or left at -20°C for at least 2 h to precipitate the DNA. The DNA was 

pelleted by microcentrifugation for 15 min, and washed twice with an equal volume of ice 

cold 70% ethanol. After centrifugation for 1 min, the ethanol was drained off, and the 

DNA pellet was vacuum dried, then resuspended in TE buffer. 

2. 6 DNA Manipulations 

2. 6. 1 Restriction Enzyme Digests 

2. 6. 1.1 Plasmid Digests 

Plasmid DNA (typically 50-250 ng) was digested in a total volume of 10-25 µl. 

Commercially prepared buffer (lx) was specifically matched to the appropriate restriction 

enzyme to give the salt concentration recommended by the manufacturer. When a higher 



35 

salt concentration was required for addition of an enzyme to a double digest, 1 M NaCl 

was used to adjust the salt concentration accordingly. A 0.5 µl volume of restriction 

enzyme ( 10 u/µl) was used per 10 µl reaction volume. Digestion was generally 

performed for 2 h at 37°C. An aliquot of the digest was then run on a minigel (Section 

2.6.2.1) to check that the digestion was complete. 

2. 6 .1. 2 Genomic Digests 

Genomic DNA (typically 3-10 µg) was digested in a 30-60 µl reaction volume. 

Commercially-prepared buffer (lx) was specifically matched to the appropriate restriction 

enzyme to give the salt concentration recommended by the manufacturer. A 0.5 µl 

volume of restriction enzyme (10 u/ul) was used per 10 µl digest volume, along with 100 

µg/ml acetylated BSA (Promega) and 500 µg/ml DNase free RNase (Section 2.4.4). 

Digestion was performed overnight at 37°C. An aliquot of the digest was then run on a 

minigel (Section 2.6.2.1) to check that the digestion was complete. If incomplete 

digestion was observed, a second quantity of enzyme was added, and the mixture was 

incubated further at 37°C. 

2. 6. 2 Agarose Gel Electrophoresis 

2.6. 2 .1 Mini gels 

Agarose was dissolved in 1 x TAE buffer (Section 2.4.5) to give a 0.7%-1.0% (w/v) gel, 

cooled, and poured into a Horizon 58 gel apparatus (83 x 57 x 4 mm). When set, the gel 

was covered with 1 x T AE buffer. Gel loading dye (Section 2.4.6) was added to an 

aliquot of digested DNA to give a final concentration of 10%, then this mixture was 

loaded onto the gel. The DNA was size fractionated at 80 V for 30 to 60 min, stained in 

0.01 % ethidium bromide solution for 10 min, destained in MilliQ water, visualised on a 

UV transilluminator and photographed using Polaroid film 667. 

2. 6. 2. 2 Overnight Gels 

Agarose was dissolved in 1 x TAE buffer (Section 2.4.5) to give a 0.6%-1.0% (w/v) 

gel, cooled and poured into a Biorad DNA sub-cell (150 x 200 x 8 mm) or Horizon 

11.14 ( 140 x 110 x 8 mm) gel apparatus. Digested DNA (including 0.1 volume of gel 

loading dye) was size fractionated in 1 x TAE electrophoresis buffer at 34 V for 16-20 h. 

Gels were stained in 0.01 % ethidium bromide solution for 30 min, and destained in 



36 

MilliQ water. They were visualised on a UV transillurninator and photographed with a 

ruler alongside using Polaroid film 667 or 665. 

2.6.3 Determination of Fragment Sizes 

Samples of HindilI/EcoRI-digested A DNA and/or HindIIl-digested A DNA or BRL 1 kb 

ladder standard markers were run alongside DNA samples on agarose gels. The relative 

mobility (mm) of each fragment was measured as the distance migrated from the centre of 

the well to the centre of the band. The molecular weight was then calculated by 

interpolation from a plot of the distance migrated in the same gel by suitable size 

(molecular weight) markers, against the logarithm of the molecular weight (kb or bp) of 

the size markers, or by use of the computer program Cricket Graph, or the molecular 

weight tool box of the IS-1000 Digital Imaging System. 

2.6.4 Determination of DNA Concentration 

DNA was quantified by three methods, by intensity of ethidium bromide fluorescence in 

a gel for samples of very low concentration, spectrophotometrically for pure solutions of 

high concentrations, or fluorometrically for impure samples of high or low concentration. 

2. 6. 4. 1 Concentration Standards 

EcoRI/HindIII-digested A DNA (200 ng), and linearised pBR322 concentration standards 

representing 2.5 ng/5µ1, 5.0 ng/5µ1, 10.0 ng/5µ1, and 20 ng/5µ1 were run on 1 % agarose 

rninigels (Section 2.6.2.1) alongside a known volume of the DNA sample. The 

concentration of the DNA sample was estimated by visual comparison with the 

concentration standards. 

2. 6. 4. 2 Spectrophotometric Method 

To 995 µl of TE buffer, 5µ1 of DNA was added, and mixed gently. The absorbance of 

the solutions in quartz cuvettes with a 1 cm light path was determined at both 260 nm and 

280 nm using the Shimadzu automatic spectrophotometer zeroed with TE buffer for a 

blank. The reading at 260 nm allowed calculation of the concentration of nucleic acid 

present in the sample since an OD of 1 corresponds to approximately 50 µg/ml of double 

stranded DNA. The ratio of readings at 260 nm and 280 nm (OD26o/OD2so) was used as 

an estimate of the DNA purity. Pure DNA has an OD26o/OD2so value of 1.8-2.0 



37 

2. 6. 4. 3 Fluorometric Method 

For impure DNA samples, or pure samples of low concentration, DNA was quantitated 

on a Hoefer Scientific TKO 100 Fluorometer. This method was suitable for quantitating 

down to 10 ng/µl and only 2 µl of sample was needed. The fluorometer was turned on at 

least 15 min before use. 10 ml of 10 x TNE buffer stock solution (100 mM Tris-HCI, 

10 mM EDTA, and 1.0 mM NaCl, pH 7.4) was diluted with 100 µl of Hoescht 33258 

dye (1 mg/ml) and 90 ml of sterile MilliQ water to give a 1 x TNE working solution 

(Working Dye Solution B). The TKO 105 glass fluorometry cuvette was filled with 2 ml 

of Working Dye Solution B. The sides of the cuvette were wiped clean and the cuvette 

was placed in the sample chamber - always in the same orientation ("G" imprint to the 

front). The fluorometer was adjusted to zero with the scale knob at 50% sensitivity (i.e. 

5 clockwise turns of the knob from the counter clockwise position). A 2 µl aliquot of the 

reference standard calf thymus DNA (1 mg/ml in 10 mM Tris-HCl, 50 mM EDT A, pH 

8.0) was delivered into the 2 ml of dye solution in the cuvette and mixed by pipetting up 

and down without introducing bubbles into the solution. The cuvette chamber was 

closed and the "scale" knob adjusted until the display read "1000" indicating 1000 ng/ml. 

This was repeated twice more, or until the reference standard read "1000" reproducibly. 

The cuvette was emptied and drained thoroughly before samples. DNA samples of D. 

pini were measured in the same manner in units of ng/ml. Each sample was blanked by 

adjusting the "zero" control knob each time 2 ml of Working Dye Solution B was loaded. 

The "scale" control knob was not adjusted. 

2. 7 Subcloning 

The process of subcloning typically involved recovery of DNA fragments from agarose 

gels (Section 2.5.5), ligation of DNA fragments into a suitable host vector (Section 

2.7.3) , transformation of ligation mixtures into a suitable E. coli host (Section 2.8.2), 

and then screening for recombinants by gel electrophoresis (Section 2.2.6.2.1) of 

diagnostic restriction digests (Section 2.6.1.1) of plasmid DNA isolated from 

transformants by the alkaline lysis method (Section 2.5.1). Blue/white selection (a.-

complementation) was employed to screen for putative recombinants in a suitable E.coli 

background (strain XLl-Blue, Table 1). Transformed cells were then plated onto 

medium containing ampicillin, IPTG and X-gal (Section 2.2.2.1) and white colonies 

were screened for recombinants as outlined above. 



38 

2.7.1 Preparation of Insert DNA 

DNA was digested with the appropriate restriction enzyme(s) (Section 2.6.1.1) and 

electrophoresed through a Seaplaque agarose gel (Section 2.6.2) to separate DNA 

fragments. The fragment(s) of interest were excised from the gel and then purified 

(Section 2.5.5). 

2.7.2 Linearisation and CAP-Treatment of Vector DNA 

Approximately 5.0 µg of vector DNA was digested to completion by the appropriate 

restriction endonuclease (Section 2.6.1.1), dephosphorylated by addition of 1.0 µl of calf 

alkaline phosphatase (CAP, Boehringer 1 U/µl) and the mixture incubated for 30 min at 

37°C. The reaction was terminated by the addition of 5 rnM EDTA, 0.5% (w/v) SDS, 

and 50 µg/rnl Proteinase K (final concentrations), mixed by inversion, and incubated for 

1 h at 37°C. A phenol/chloroform extraction (Section 2.5.6) and ethanol precipitation 

(Section 2.5.6) were then performed and the precipitated DNA resuspended in TE (10/1) 

at a concentration of 200 ng/µl. This method was based on that of Sambrook et al. 

(1989). 

2.7.3 Ligation 

Ligation mixtures contained 2.0 µl of the manufacturers (New England Biolabs) 5 x 

ligation buffer, a 2-3 times molar excess of insert:vector (at least 20 ng of DNA insert and 

at least 20 ng of vector DNA), 1 µl of a 10-fold dilution (40 units) of T4 DNA ligase 

(New England Biolabs), and MilliQ water to 10 µl. Ligation mixtures were incubated 

overnight at 4°C. To check that ligation had occurred, a 1.0 µl aliquot of the ligation mix 

was removed prior to addition of T4 DNA ligase and the sample was examined on an 

agarose gel (Section 2.6.2.1) alongside a 1.0 µl sample removed after addition of T4 

DNA ligase and overnight ligation. This method was based on a modification of the 

method proposed by Dugaiczyk et al. (1975). 



39 

2.8 Transformation of E. coli 

2.8.1 Calcium Chloride Transformation 

This procedure from Sambrook et al. (1989) is a variation of that developed by Cohen et 

al. (1972) 

2. 8 .1.1 Preparation of CaCI2 Competent Cells 

A 50 ml volume of LB broth (Section 2.2.2.1) in a 250 ml flask was inoculated with the 

desired E. coli strain and grown at 37°C on a shaking platform at 225 rpm to mid log 

phase (OD6oo 0.45-0.60, about 2 h). The cells were chilled on ice for 10 min then 

harvested by centrifugation at 3,000 rpm for 5 min at 4°C. The cells were resuspended 

in 0.5 volume of ice cold 100 mM CaCl2 chilled on ice for 10 min, then pelleted by 

centrifugation and resuspended in 0.05 volume of 100 mM CaCh. The cells were kept 

on ice and used within 48 h, or 0.3 volume of 50% glycerol/100 mM CaCh was added 

and the mixture dispensed into 200 µl aliquots, frozen on dry ice, and stored at -80°C. 

2. 8 .1. 2 Transformation 

Competent cells (200 µl, stored on ice for 30 min before use) were mixed with 1-2 µl of 

DNA (Section 2.5 .1) and left on ice for 30 min. Following heat shock at 42°C for 2 

min, 5 ml of prewarmed (37°C) LB broth was added and the cells incubated for 30 min at 

37°C. The cells were then centrifuged at 270 x g (1,500 rpm, SS34) for 5 min, the 

supernatant decanted and the cells resuspended in about 100 µl LB broth (amount 

remaining in tube). The cells were then spread onto a fresh LB + amp plate and 

incubated at 37°C for 16 h. Ampicillin resistant colonies were picked and grown for 

DNA extraction. 

2.8.2 Transformation of E. coli by Electroporation 

This method from Sambrook et al. ( 1989) was based on a procedure developed by 

Dower et al. (1988). 

2. 8. 2. 1 Preparation of Electro-Competent E. coli Cells 

One litre of LB broth was inoculated ( 1/100) with the desired E. coli strain and grown at 

37°C with vigorous shaking to mid-log phase (OD6oo 0.5-1.0, about 3 h). The cells 



40 

were chilled on ice for 20 min then harvested by centrifugation at 4,000 g for 10 min (all 

centrifugations were at 4°C). The cells were washed sequentially (by resuspension, 

centrifugation at 4,000 x g to pellet, then draining supernatant) in ice cold water (1 litre 

followed by 500 ml) and then ice cold 10% glycerol (20 ml then finally resuspended in 4 

ml). Cell suspensions were stored at -70°C in 200 µl aliquots. 

2. 8. 2. 2 Electroporation 

Electrocompetent E. coli cells (Section 2.8.2.1) were thawed gently at room temperature, 

divided into 40 µl aliquots in ice cold microcentrifuge tubes, 1-2 µl of DNA added 

(generally ligations from Section 2.7.3), mixed and the DNA/cell mixture left on ice for 1 

min. The Biorad Gene Pulser Transfection Apparatus was set to 25 µF and 2.5 V and 

the pulse controller to 200 Q resistance, in parallel with the sample chamber. The 

mixture of DNA and cells was transferred to an ice-cold 0.2 ml cuvette, shaken to the 

bottom, then pulsed at the above settings and the time constant checked. When a time 

constant of 4-5 mseconds was obtained the cells were immediately resuspended in 1 ml 

of LB medium (Section 2.2.2.1) and incubated at 37°C for 1 h to aid recovery of 

transformed E. coli. A positive (typically 2 ng of pUCl 18) and negative (water only) 

control was always employed. Cells were plated at suitable dilutions onto selective LB 

plates (Section 2.2.2.1). 

Transformants were screened via informative restriction digests (Section 2.6.1.1) of 

plasmid DNA isolated by alkaline lysis (Section 2.5.1) followed by gel electrophoresis 

(Section 2.6.2.1). 

2.9 DNA Hybridisations 

2.9.1 Southern Blotting 

DNA from overnight electrophoresis gels (Section 2.6.2.2) was transferred to Hybond-N 

nylon filters (Amersham) by a modification of the method described by Southern (1975). 

The DNA was denatured by gently shaking the gel in denaturing solution (0.5 M NaOH/ 

1.5 M NaCl) for 2 x 10 min. This was followed by shaking in neutralising solution (1.5 

M NaCl/0.5 M Tris-HCl, pH 7.2) for 2 x 15 min. The gel was equilibrated in 20 x SSC 

(Section 2.4.7) for 15 min, then the blotting apparatus assembled. Four thin pieces of 

sponge larger in size than the gel to be blotted, were placed in a plastic container and 

saturated with 20