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EXPRESSION ANALYSIS OF AN AF LATOXIN NOR-1/UIDA REPORTER FUSION
IN ASPERGILLUS PARASITICUS

By

David L. Wilson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTERS OF SCIENCE

Department of Food Science and Human Nutrition

1999

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ABSTRACT

EXPRESSION ANALYSIS OF AN AF LATOXIN NOR-I/UIDA REPORTER FUSION
IN ASPERGILLUS PARASITICUS

By

David L. Wilson

Aflatoxins are carcinogenic contaminants of the agricultural environment that are
produced as a result of fungal infection of plant tissue. Reporter gene fusions are
powerful tools that can be used to monitor the interaction between plant and fungus, and
are being developed in our laboratory to aid in the analysis of aflatoxin production in
Aspergillus parasiticus. This study evaluates the expression of a B-glucuronidase (GUS)
reporter gene (uidA) which is fused to the nor-1 aflatoxin gene. Under aflatoxin -
inducing conditions, our data showed that the patterns of nor-1 and nor-1 /uidA transcript
and protein accumulation were similar. The data also demonstrated that the timing of
aflatoxin production and GUS activity were identical, while the pattern of aflatoxin
accumulation was consistent with the levels of GUS activity. Transformation
experiments were supportive of a position-dependent expression mechanism operating in
the aflatoxin gene cluster (Liang et al., 1997). Integration of the nor-I/uidA reporter
construct outside of the aflatoxin gene cluster resulted in a GUS‘ phenotype. In order to
avoid chromosomal position effects on reporter fusion expression, the 3' nor-1 region of
the chromosome was targeted for integration of the nor-1 luidA reporter construct.
Transformants with the reporter integrated at this site will be used in future reporter
expression studies. A qualitative GUS assay and a PCR assay were developed to screen

for transfonnants with this type of integration event.

This thesis is dedicated to my family. They provided constant support and the occasional

distraction.

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ACKNOWLEDGMENTS

I am grateful for the guidance of Dr. John Linz during the course of my research.
His support and critical analysis of my designs and results not only shaped this work but
also my development as a scientist. I would also like to thank Dr. James Pestka, Dr. Gale
Strasburg, and Dr. Mark Uebersax for their valuable time and expertise as part of my
guidance committee. I was fortunate to have all of these scholars as teachers during my
degree program and each provided a unique perspective of the relationship between the
food industry and food science. My friendships with Matt Rarick, Mike Miller, Tzong-
Shoon Wu, Ludamila Roze, Frances Trail, Shun-Hsin Liang, Nibedita Mahanti, Richard
Zhou, Li-Wei Lee, Ching-Hsun Chiou, Kristin Kneely, and Jaimie Oatley were important
for the every day insight and support crucial for success in scientific endeavors. To all I

owe a debt of friendship and the rights to hear my paradigm shitting ideas.

iv

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TABLE OF CONTENTS

LIST OF TABLES ................................... . .................. vi
LIST OF FIGURES .................................................... vii
LIST OF ABBREVIATIONS ............................................. ix
LITERATURE REVIEW ................................................. 1
Overview ........................................................ 1
Economic Impact .................................................. 2
Hypothesis ....................................................... 4
Reporter Fusions .................................................. 6
Fungal Fusions .................................................... 8
Beta-Glucuronidase ................................................ 9
GUS Reporter Constructs .......................................... 10
Position-Dependent Expression ...................................... 13
Gene Clusters .................................................... l4
SpoCl Cluster ................................................... 18
MATERIALS AND METHODS ........................................... 20
Strains, Plasmids and Culture Conditions ......... ' ..................... 20
Fungal Transformation ............................................. 23
Spore Preparation ................................................. 24
Fungal DNA Isolation and Southern Blotting ........................... 25
Fungal RNA Isolation and Northern Blot Analysis ....................... 26
Nucleic Acid Hybridization ......................................... 27
Qualitative and Quantitative GUS assays .............................. 27
Western Blot Analysis . . . ' .......................................... 28
Polymerase Chain Reaction Analysis ................................. 29
Aflatoxin Analysis ................................................ 30
RESULTS ............................................................ 32
Transformation and screening of A. parasiticus ......................... 32
Southern Hybridization Analysis ..................................... 36
PCR Analysis .................................................... 51
Time Course Analysis of NR-l/D8D3 ................................. 52
Time Course Analysis of NR-l/D8D3 Transcripts ....................... 53
Time Course Analysis of NR-l/DSD3 Proteins .......................... 53
Time Course Analysis of NR-l/D8D3 Protein Activity ................... 68
DISCUSSION ......................................................... 70
REFERENCES ........................................................ 77

 

 

Table

Table

 

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Table

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Tal

Ta

LIST OF TABLES

Table l. NR-l transformation frequencies from three separate experiments. ........ 32

Table 2. The ratios of GUS+ transformants from three separate transformation
experiments. The plasmids pAPGUSNNA and pAPGUSNNB were used to
transform NR-l. A qualitative GUS assay was used to screen niaD“ colonies
from these transformations, for their ability to express GUS activity on an
aflatoxin inducing media (YES agar). ................................. 33

Table 3A. Time course analysis of transforrnant D8D3. The dry weights of D8D3 and
control cultures were determined at 24, 48, and 72 h after inoculation in aflatoxin
inducing medium (YES broth). D8D3 was tested in triplicate while only single
cultures of the control strains were harvested for analysis. The data were reported
in mg of mycelia. ................................................. 52

Table 33. Time course analysis of transformant D8D3. The GUS activities from the
mycelia of D8D3 and control strains were determined at 24, 48, and 72 h. D8D3
was tested in triplicate while only single cultures of the control strains were
analyzed. The data were reported in nanomoles 4-methylumbelliferone/min/mg
of protein. ....................................................... 69

Table 3C. Time course analysis of transforrnant D8D3. The aflatoxin concentrations in
the filtrates of cultures of D8D3 and control strains were determined at 24, 48, and
72 h. D8D3 was tested in triplicate while only single cultures of the control
strains were analyzed. The data were reported in ng/ml as determined by direct
competitive ELISA. ............................................... 69

Table 4. GUS phenotypes of pAPGUSNNB transformants on media supportive (YES)
and non-supportive (CZ) of aflatoxin production. Qualitative GUS assays (see
Methods) were performed on fungal colonies afier 48 h of growth on both types of
media. Site of vector integration is indicated in parenthesis. ............... 70

Table 5. GUS activities in NR-l transformants. Mycelia from each of the strains were
harvested at 48 hrs after growth in aflatoxin inducing medium (YES broth). One
mg of protein from each sample was used to determine GUS activity. The data
were reported in nanomoles 4-methylumbelliferone/min/mg of protein. The GUS
activities for the transformants that are presumably genetically identical to D8D3
were reported in an experiment separate from the analysis that produced the GUS
activity data for the D8D4 and D8D6 strains. Transformant D8D6 has the GUS
reporter fusion located in the 5’ region of nor-1 whereas D8D4 has the reporter
integrated at niaD (Figure 5) ......................................... 70

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LIST OF FIGURES

Figure l. Aflatoxin gene cluster ............................................ 5
Figure 2. Construction of pAPGUSNNA and pAPGUSNNB reporter plasmids. ..... 21
Figure 3. Qualitative GUS assay of fungal colonies transformed with pAPGUSNNB.
............................................................... 34
Figure 4A. Schematics for Southern hybridization and PCR analyses of niaD+
transformants ..................................................... 37
Figure 4B. Schematics for Southern hybridization and PCR analyses of m'aD+
transformants ..................................................... 39

Figure 4C. Schematics for Southern hybridization analysis of niaD+ transformants. . . 41

Figure 5A. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants ..................................................... 43

Figure SB. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants ..................................................... 45

Figure 5C. Southern hybridization analysis of NR-l and NR- l/pAPGUSNNB
transformants ..................................................... 47

Figure 6. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants ..................................................... 49

Figure 7. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB GUS+
transformants ..................................................... 54

Figure 8. Southern hybridization analysis of NR—l and NR-l/pAPGUSNNB
transformants ..................................................... 56

Figure 9. PCR assay for identifying transformants with reporter constructs integrated
into the nor-l region of the NR-l chromosome. ......................... 58

Figure 10. Dry weight analysis of transformant D8D3 and control strains. ......... 60

Figure 11. Northern hybridization analysis of nor-1 and uidA transcripts in transformant
D8D3 and control strains. .......................................... 62

Figure 12. Western blot analysis of Nor-1 and GUS proteins in transformant D8D3 and
control strains. ................................................... 64

vii

Figuq

 

 

Figure 13. Analysis of GUS activity and aflatoxin production in transformant D8D3 and
control strains. ................................................... 66

viii

LIST OF ABBREVIATIONS

AF Aflatoxin

AF Bl Aflatoxin Bl

CZ Czapek—Dox

GAL B-galactosidase
GUS B-glucuronidase
SGC SpoCl gene cluster

YES Yeast Extract Sucrose

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LITERATURE REVIEW

Overview

Aflatoxins comprise a family of toxic and carcinogenic secondary metabolites
produced primarily by the filamentous fungi Aspergillusflavus and Aspergillus
parasiticus (Diener and Davis, 1969). The aflatoxins were first characterized in the early
1960’s as feed contaminants and the causative agents of Turkey—X-Disease, an illness that
decimated the domestic turkey population in the London region of England (Blount,
1961; Sargeant et al., 1961). Thin layer chromatographic analysis on suspect feed led to
the identification of the major aflatoxins (termed Aflatoxin B,, B2, G, and G2) based on
their Rf values and fluorescent color under ultraviolet light (Hartley et al., 1963;
Goldblatt, 1969; Pons and Goldblatt, 1969). Aflatoxin B, (AFB,) is considered the most
acutely toxic of the aflatoxins with lethal doses in test animals such as ducklings, rats,
hamsters, guinea pigs, rabbits, dogs and trout generally resulting in gross liver necrosis
(Wogan, 1966). Day old ducklings are among the most sensitive species with an LD50 of
approximately 0.5 mg/kg after 48 hours (Asao et al., 1963; Hartley et al., 1963; Wogan,
1966).

Animal studies have also determined AFB, to be extremely hepatocarcinogenic.
An early analysis on the carcinogenic effect of AFB, on ducklings, reported that eight of
eleven birds developed hepatic tumors after being fed rations containing 30 ppb AFB, for
fourteen months (Carnaghan et al., 1965). A similar study using rainbow trout showed
24 of 30 fish developed hepatomas after consuming a diet consisting of 7.9 ppb AFB, for
fifteen months (Sinnhuber et al., 1968). In one of many experiments with rats, 25 of 25

animals fed rations containing 15 ppb AFB, for up to 80 weeks developed hepatocellular

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carcinomas (Wogan and Newbeme, 1967).

The initiation of the carcinogenesis process requires mutations in genomic DNA,
and AFB, has mutagenic potential due to its in vivo oxidation to the highly reactive
AF B,-8,9-epoxide by hepatic cytochrome P450 (Eaton and Gallagher, 1994). This
epoxide can form adducts with the N7-guanine of DNA and consequently induce
mutations. Numerous studies in animals have demonstrated that AF B,-DNA adduct
formation and cancer risk are linearly proportional (Bechtel, 1989; Eaton and Gallagher,
1994). With regard to the human health threat, the available evidence on the
carcinogenicity of AFB, has been deemed sufficient by the International Agency for
Research on Cancer to classify AFB, as a human carcinogen (IARC, 1993).
Epidemiological analyses have indicated a role for the p53 tumor suppressor gene in
human hepatocellular carcinomas fiom regions of the world with dietary aflatoxin
exposure (Hsu et al., 1991; Bressac et al., 1991; Eaton and Gallagher, 1994). Codon-
specific GC->TA transversions and GC->CG transitions in the p53 gene of these
carcinoma cells have been identified, and the same types of mutations can be found in
human cells mutagenized with AFB, and the AFB, epoxide (Loechler, 1994; Aguilar et
al., 1993). The formation of adducts between the AFB, epoxide and this p53 mutational
hotspot region have also been demonstrated (Kobertz et al., 1997).
Economic Impact

A. flavus and A. parasiticus occupy a broad niche in the agricultural environment
and are capable of producing aflatoxins on commercially important substrates such as
corn, peanuts, cottonseed and treenuts (Diener etal., 1987 ; Ellis et al., 1991). When
drought-damaged or insect-damaged, these crops become susceptible to ftmgal

colonization, which in turn can lead to aflatoxin biosynthesis and crop contamination

 

 

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(Diener et al., 1987; Pier, 1992). The transportation and storage of these commodities at
inappropriate temperatures and moisture levels also provides opportunities for fungal
growth and toxin formation (Ellis et al., 1991; Pier, 1992).

Aflatoxin contamination of the food supply occurs in many regions of the world
(Jelinek et al., 1989) jeopardizing both human and animal health. Acute aflatoxin
poisoning can have a dramatic impact on animal production as illustrated by the high
mortality rate of Turkey-X-Disease (Blount, 1961). However, the more common
financial concern among the beef, poultry, swine and dairy industries is with chronic
aflatoxin exposure, which results in inferior feed conversion, growth suppression,
decreased reproductive potential, impaired immune responses and toxin carryover into
meat, milk and eggs (Pier, 1992; Shane, 1994).

Because of the global threat of aflatoxins to health and the economy, many
countries have regulatory limits on the amount of toxin permitted in food and feed. The
United States Food and Drug Administration has set an action level of 20 ppb total
aflatoxins in food. The allowable levels in different areas of the world range from zero
ppb to 500 ppb total aflatoxins (Gourama and Bullerman, 1995; Rustom, 1997).

The restrictions on aflatoxins in the food supply leads to an additional economic
burden on growers, who are unable to market contaminated commodities or are forced to
sell them at reduced prices. Detoxification methods, which include treatment with
ammonia, sodium bisulfite, calcium hydroxide or formaldehyde are options available to
help eliminate post-harvest contamination. A more recent approach to maintaining
animal performance while using contaminated feed is to include alumina, silica or
aluminosilicates as toxin binding materials. In theory, these binding compounds inhibit

toxin absorption in the intestinal tract, thus preventing AFB, metabolism by the animal

 

 

 

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(Piva et al., 1995). Despite their apparent effectiveness, the detoxification methods raise
concern over the safety, nutritional stability and ftmctionality of treated food and feed,
and ultimately offer only short term solutions while maintaining a long term financial
onus on producers.

Hypothesis

A major objective in the aflatoxin control effort is the prevention of crop
contamination prior to harvest. Two strategies based on the molecular biology of
aflatoxin biosynthesis have been developed to help achieve this goal. One approach is to
produce highly competitive, atoxigenic strains of Aspergillus, that when released into the
farming environment, can exclude toxigenic fungi from colonizing crops (Cotty, 1990;
Domer et al., 1992). The success of such a scheme would most likely depend on the
ability to genetically engineer, and monitor in the field, fungal strains lacking key
aflatoxin regulatory and/or anabolic genes. The second strategy involves the genetic
engineering of crops. This approach is targeted to the identification of either endogenous
plant genes or foreign genes which produce compounds that repress or inhibit aflatoxin
formation and/or fungal growth $urow et al., 1997). Manipulating these genes to be
constitutively expressed in plants or induced in response to external stimuli should help
reduce aflatoxin levels in food and feed. In both strategies, fiindarnental knowledge
about the biosynthesis of aflatoxin and its molecular regulation is requisite.

A proposed pathway for aflatoxin biosynthesis has been reported (Bhatnagar et
al., 1993), and the genes encoding proteins involved in aflatoxin production have been
found to be clustered in the genomes of A. flaws and A. parasiticus (Figure 1) (Y 11 et al.,
1995; Trail et al., 1995; Payne and Brown, 1998). Our laboratory has targeted the nor-1

and ver-IA genes of A. parasiticus, which express two distinct enzymes in the aflatoxin

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pathway, for regulatory studies. The nor-I gene encodes a reductase (NOR), catalyzing
the conversion of norsolorinic acid to averantin early in the pathway (Trail at al., 1994),
while ver-IA is believed to express 3 reductase involved in a metabolic step converting
versicolorin A to sterigrnatocystin late in the pathway (Skory et al., 1992). Translational
fusions of the nor-1 and ver-IA 5’ regions with the Escherichia coli beta-glucuronidase
reporter gene (uidA) (Jefferson et al., 1986) were constructed in order to help identify cis-
acting regions and trans-acting factors essential for the regulation of these genes. These
aflatoxin gene fusions should also be useful for monitoring aflatoxin biosynthesis in
response to environmental stimuli and as tools to evaluate compounds with a potential
aflatoxin inhibiting nature. This study focuses on the nor-1 gene, and is based on the
hypothesis that the expression of the nor-I/uidA reporter fusion is similar to the
expression of wild-type nor-1 gene. If the two genes can be shown to be regulated in a
similar manner, then the nor-1 luidA reporter construct can be used as a reliable indicator
of wild-type gene expression and aflatoxin biosynthesis.

Reporter Fusions

Reporter genes express stable enzymes for which sensitive and efficient assays
have been developed. By fusing the structural gene of a reporter enzyme to the promoter
region of a gene of interest, the regulation of a variety of genes can be studied with the
same reporter enzyme assays.

Early gene firsions were often characterized in strains of Escherichia coli where
transposition and deletion events brought expression of lactose metabolizing genes under
tryptophan operon control (Reznikoff et al., 1969; Miller et al., 1970; Bassford et al.,
1978). In these fusions, activity of beta-galactosidase (GAL), the product of the E. coli

lacZ gene, was used to indicate tryptophan promoter control over the lactose genes. GAL

 

 

 

 

 

 

 

 

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hydrolyzes lactose to its constituent monomers glucose and galactose, but maintains
specificity for substrates such as orthonitrophenyl-J-D-galactoside (ONPG) and 5-bromo—
4chloro-3indolyl-J-D-galactoside (XGAL) which substitute an indicator moiety for the
glucose. The cleavage of the ONPG and XGAL chromogenic substrates allows for
sensitive assays of GAL expression (Beckwith, 1978). The ease in assaying for GAL
spurred the development of in viva methods for fusing lac genes to other E. coli
promoters whose products were difficult to detect or unknown (Casadaban, 1976;
Casadaban and Cohen, 1979).

The ability to manipulate DNA in vitro, with endonuclease digestion and ligation
into plasmid vectors, increased the flexibility and efficiency for locating and examining
promoters of both procaryotes and eucaryotes. In one general scheme, the chromosomal
DNA of an organism was digested, and the fragments inserted into plasmid cloning sites
upstream of a gene conferring antibiotic resistance. The selectable antibiotic gene lacked
transcription control signals. Fusions containing transcription initiation signals could be
identified by transforming E. coli with recombinant plasmids and then selecting
transformants for antibiotic resistance (An and Friesen, 1979; Neve et al., 1979; West at
al., 1979). Vectors containing lac genes were utilized to isolate and characterize
promoters in a similar manner. After identifying promoter regions, assays for lacZ
expression could be used to measure gene regulation on different types of media and to
isolate mutations within a regulatory insert. Also, genes which express factors that act on
a promoter of interest, could be isolated by introducing cloned DNA in trans of IacZ
fusions, and then analyzing for the affect on GAL activity (Casadaban and Cohen, 1980).

The GAL enzyme has the additional reporter advantages of being able to tolerate

both large additions of amino acids to its amino terminal end (Muller-Hill and

7

Kania,1974), and/or the elimination of at least its first 27 amino acid codons (Welply et
al., 1980) without the loss of enzyme activity. By replacing the amino terminal portion
of GAL with the amino end of a protein of interest, a translational fusion can be
constructed to study both transcriptional and translational initiation signals (Casadaban et
al., 1980; Casadaban et al., 1983). Translational fiision expression results in the
production of hybrid proteins. With lacZ fusion technology it is possible to create a
series of hybrid proteins that can be useful in identifying amino terminal sequences
involved in protein localization (Bassford et al., 1978; Silhavy et al., 1977).
Fungal Fusions

The 5’ regions of the Saccharomyces cerevisiae orotidine-5’-monophosphate
decarboxylase (ura3) (Rose et al., 1981) and iso-l-cytochrome c (cycI) (Guarente and
Ptashne, 1981) genes were used to demonstrate the first fimgal expression of reporter
fusions. In both studies, translational lacZ fusions were created in vitro using vectors
with E. coli and yeast origins of replication and selectable markers. The absence of
endogenous GAL activity in S. cerevisiae allowed for assay of fusion expression without
background interference. Since ura3 is under uracil regulation and cycI is repressed by
glucose, expression of GAL was tested under appropriate media conditions to give data
supporting the assertion that the fusions used ura3 and cycI expression control signals.

As was the case with S. cerevisiae, the application of fusion technology in
filamentous fungi was preceded by the development of transformation methodologies.
Transformation experiments with the pioneering organisms Neurospora crassa (Case et
al., 1979) and Aspergillus nidulans (Tilbum et al., 1983; Yelton et al., 1984; Wemars et
al., 1985) showed that chromosomal integration of transforming plasmid sequences is

essentially as has been defined for yeast (Hinnen et al., 1978). Typically, the

 

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transforming DNA can be classified into one of three types of integrative events. Type I
is characterized by a complete plasmid integration at a homologous location in the
chromosome. Type II integration is identical to Type I with the exception that it occurs at
a heterologous chromosomal locus. Type III involves gene replacement of the selectable
marker with other vector sequences being excluded from integration. Studies with
integrative vectors containing A. nidulans trpC/IacZ (van Gorcom et al., 1985) and
gpd/IacZ (Kolar et al., 1988) fusions, established the feasibility of using reporter
constructs to analyze gene expression in filamentous fungi.

Beta-Glucuronidase

Beta-Glucuronidase (GUS) is the product of the uidA gene of E. coli, and
catalyzes the cleavage of B-O-glycosidic linkages to glucuronic acid. Detoxification
processes in vertebrates often produce natural glucuronides. , In these reactions, certain
waste products (aglycones) are conjugated to glucuronic acid and excreted. E. coli, as
part of the flora of the vertebrate gut, is uniquely able to metabolize these compounds
(Jefferson, 1989).

The development of GUS as a reporter enzyme was in response to concerns
regarding the high endogenous GAL activity in several biological systems. Alternative
enzyme markers, such as chlorarnphenicol acetyltransferase and firefly luciferase, were
relatively expensive, difficult to assay, and lacked applications for analyzing tissue
specific expression in higher eucaryotes (Jefferson et al., 1986; Jefferson et al., 1987).

The ~73 kilodalton GUS protein has several characteristics that make it an
excellent reporter enzyme. It is a stable protein that is resistant to thermal inactivation at
50°C, and has a broad pH optimum ranging from 5.0 to 7.5 (Jefferson et al., 1986). It can

also tolerate the addition of large amino terminal sequences without the loss of enzyme

activity and can traverse membranes when signal sequences are attached to its amino
terminus (Jefferson et al., 1986; Jefferson et al., 1987; Jefferson, 1989). A major
advantage of GUS as a reporter enzyme, is the absence of endogenous GUS activity in
many bacteria, fungi, and plants (Jefferson, 1989). With this lack of background
interference and the commercial availability of sensitive and specific chromogenic and
fluorogenic substrates, tissue and subcellular localization analyses can be performed with
relative efficiency (Jefferson et al., 1986; Jefferson et al., 1987).
GUS Reporter Constructs

Filamentous fungi are of economic interest because they can produce homologous
and heterologous proteins in industrial fermentations, they are successful plant
pathogens, and because they produce food contaminating mycotoxins (Bennett, 1997).
Research efforts into optimizing protein production in fermentations, and in
understanding plant-fungus interactions have often focused on the analysis of gene
expression in these fungi. Many laboratories have recognized the advantages of GUS
fusions as potential tools for gene expression studies. In one such application, GUS
expression vectors, lacking selectable markers, were successfully utilized in transient
expression assays. These assays were used to confirm DNA uptake and the firnctionality
of reporter constructs during development of a stable transformation system for the potato
and tomato pathogen Phytophthora infestans (J udelson and Michehnore, 1991). A rapid
transient GUS assay was also used to establish optimal transformation parameters, and to
identify fitnctional promoters in the corn pathogen Cochliobolus heterostrophus (Monke
and Schafer, 1993). The expression of GUS activity in stably transformed strains of C.
heterostrophus did not have an observable effect on their ability to infect the host.

The cotransforrnation of A. nidulans, A. niger and the tomato pathogen Fulvia

10

 

 

 

     

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fidva, with a glyceraldehyde 3-phosphate dehydrogenase (gpdyuidA fusion vector
resulted in about 50% of the selected colonies expressing detectable GUS activity
(Roberts et al., 1989). This study indicated that GUS expression in F. fidva did not have
a discernible effect on fungal pathogenicity and that the plant tissue harboring the
pathogen could be efficiently monitored by GUS assay. In similar cotransformation
studies with the same gpd/uidA fusion vector, 50% to 75% of selected F usarium
oxysporum colonies (Couteaudier et al., 1993), 69% of selected Claviceps pwpurea
colonies (Smit and Tudzynski, 1992), and 38% to 92% of selected Pseudocercosporella
herpotrichoides colonies (Bunkers, 1991) expressed GUS. Reporter activity in P.
hemotrichoides cotransforrnants appeared to have no effect on fungal growth rate,
morphology or pathogenicity; and C. purpurea cotransformants exhibited normal
sclerotial formation and expressed GUS activity during the parasitic cycle. Because GUS
expression in F. oxysporum cotransforrnants did not have an effect on their ability to
cause disease, it was utilized to monitor plant-pathogen interaction in competition studies
between pathogenic and nonpathogenic populations.

Bipolaris sorokiniana, a fungal pathogen of cereal crops, was also successfully
transformed with a gpd/uidA fusion vector. Transforrnants were able to infect plants and
express GUS activity prior to the development of obvious disease symptoms. It was
suggested that GUS activity could be used for quantification of the fungus in plant tissue
because of the high sensitivity of the fluorometric GUS assay, the lack of background
activity in plants, and the positive correlation between GUS activity and ergosterol
content (a frequently used indicator of fungal biomass) during infectious growth
(Liljeroth et al., 1993).

A ver-I/uidA fusion in A. flavus enabled the use of more reliable and sensitive

11

GUS assays to replace direct aflatoxin measurements in experiments designed to identify
aflatoxin inducing compounds in plant extracts (F laherty et al., 1995). A GUS reporter
construct that included the promoter region of the constitutively expressed beta-tubulin
gene, was used in A. flavus as a marker to indicate fungal colonization of aflatoxin
resistant and susceptible maize genotypes (Brown et al., 1995). GUS activity was also
used to demonstrate the regulation of uidA by the 5’ region of the Colletotrichum
gloeosporioides cap5 gene. Cap5 is involved in appressorium formation and GUS
expression in this study was found to be located exclusively in conidia forming
appressoria (Hwang and Kolattukundy, 1995).

In order to study the mechanisms of transcriptional induction of a B-amylase gene
(amyB) in Aspergillus oryzae, an amyB/uidA transcriptional fusion was constructed,
inserted into a vector with a selectable marker and transformed into an appropriate
auxotrophic A. oryzae mutant. Data fiom expression studies with B-amylase inducing
and non-inducing sugars, indicated that GUS activity was controlled by the amylase
promoter (Tada et al., 1991). Rhizopus niveus, because of its exceptional ability to
secrete a variety of enzymes, is potentially useful for the production of commercially
important proteins. The 5’ regions of R. niveus 3-phosphoglycerate kinase genes (pgk),
were used to construct pgk/uidA transcriptional fusions. The expression of GUS in R.
niveus transformants was then used to indicate the ability of these high activity promoters
to produce heterologous proteins (Takaya et al., 1994). In efforts to understand the
molecular regulation of penicillin biosynthesis, ipnA/lacZ and ach/uidA fusions were
both employed in the same A. nidulans strain. IpnA and ach encode two enzymes
involved in penicillin metabolism. GAL and GUS assays were able to be performed on

the same fungal extracts to identify trans-acting mutations that affected both ipnA and

12

ach expression (Brakhage and Den Brulle, 1995).
Position-Dependent Expression

The integration of transforming DNA in filamentous fungi can occur at
homologous or heterologous genomic sites, and the chromosomal environment can be a
factor in the expression of the integrating genes (Timberlake and Marshall, 1989). In a
study involving phosphoglycerate kinase/lacZ fusions in A. nidulans, transformants were
characterized based on fusion integration position in the chromosome (Streatfield er al.,
1992). Transforrnants with single fusion integrations at a homologous site (selectable
marker locus), and transformants with integrations at other undetermined sites (probably
heterologous loci), were identified and their GAL activities compared. All homologous
integrations maintained similar reporter activity levels. The heterologous integrations
gave relatively higher or lower activity levels, and in certain cases these transformants
completely lacked GAL activity. In other studies with Phytophthora infestans (J udelson
et al., 1993) and Aspergillus oryzae (Hata et al., 1992), chromosomal position was
believed to be a possible factor affecting expression of uidA reporter fusions. This
assumption was based on the differences in GUS expression among individual
transformants and the prevalence of non-homologous recombination in these
experiments. An analysis of F usarium oxysporum transformants containing single copies
of an A. nidulans @d/uidA reporter fusion, showed an approximately 80-fold difference
in GUS activity between certain isolates (Couteaudier et al., 1993). These transformants
were characterized by unique chromosomal positions of fusion integration. Subsequent
plasmid rescue experiments indicated that the putative elements responsible for higher
GUS activity could be isolated from the F usarium chromosome. In a study that

examined the effect of chromosomal position on the cloned glutamate dehydrogenase

13

gene (can) of Neurospora crassa (Kinsey and Rambosek, 1984), transformants with am
integration in a non-normal chromosome expressed only 5%-20% of the dehydrogenase
activity of the wild-type, while transformants with a normal am linkage relationship
produced wild-type activity levels. It was hypothesized that the lower am expression
levels were the result of the separation of an enhancer element from the am gene during
cloning, which was not effectively replaced by the chromosomal copy in non-normal
chromosome integrations. The phenomenon of position-dependent expression has been
recognized as a potential hindrance to promoter activity studies, and it has been suggested
that efforts be made in such studies to target gene integration to a specific locus in order
to avoid position effects on gene transcription (van Gorcom et al., 1986; Harner and
Timberlake, 1987; Tirnberlake and Marshall, 1989).
Gene Clusters

The clustering, or close linking of genes involved in the same metabolic pathway
appears to be an emerging theme in the filamentous fimgi, particularly when secondary
metabolic pathways and pathways expressing activity under a limited range of growth
conditions are examined (Keller and Hohn, 1997). Physical and transcriptional maps of
the proline catabolism gene cluster and the ethanol catabolism gene cluster in Aspergillus
nidulans have been determined (Hull et al., 1989; Fillinger and Felenbok, 1996). The
proline cluster comprises five inducible genes, ranging over 14.2 kb of chromosome, and
includes a gene expressing a positive acting, pathway regulator. A centrally located, cis-
acting region in this cluster regulates an adjacent gene and also appears to have an
enhancer-type effect on a non-adj acent cluster gene (Hull et al., 1989; Sophianopoulou et
al., 1993). Since proline can be used as both a carbon and nitrogen source, this pathway

is subject to regulation by the broad domain, carbon catabolite repressor CreA (Arst and

14

MacDonald, 1975), and the global nitrogen regulator AreA, which acts positively to
mediate nitrogen metabolite repression (Arst and Cove, 1973; Sophianopoulou et al.,
1993). The ethanol-utilization cluster consists of seven inducible genes, extending over
approximately 15 kb of DNA. It is positively regulated by a cluster gene expressing the
pathway-specific, trans-acting protein AlcR. This pathway is also controlled by carbon
catabolite repression mediated by CreA. The genes in the cluster have been categorized
based on their interactions with AlcR and CreA (F illinger and Felenbok, 1996).

A quinic acid (QA) catabolism gene cluster has been defined in both N. crassa
(Geever et al., 1989) and A. nidulans (Lamb et al., 1990). The 18 kb, QA-inducible
cluster in Neurospora contains five structural genes and two pathway specific regulators.
One of these regulators, a positive-acting protein, has been shown to bind the promoter
regions of the QA genes, while the second regulator protein is believed to repress the
pathway by binding and inhibiting the activator protein. Repressor activity is inhibited
by QA, and transcription of the cluster is repressed by glucose (Geever et al., 1989).
Similarly, the 17 kb Aspergillus cluster is characterized by seven genetic loci, which are
controlled by carbon catabolite repression and induced by QA. .The cluster includes two
regulatory genes that express a pathway activator protein and a repressor protein. Both
clusters have three pairs of divergently transcribed genes, however, the order of genes in
the two clusters is different. Southern hybridization data suggests that A. firmigatus and
Penicillium chrysogenum also contain a QA cluster (Lamb et al., 1990).

The three structural genes encoding the nitrate assimilation pathway in A.
nidulans are localized in a cluster (Johnstone et al., 1990). These genes are induced by
nitrate via a pathway-specific, positive-acting factor, and are also subject to the activity of

the AreA protein (Johnstone et al., 1990; Unkles et al., 1991). There are also data which

'15

indicate a regulatory role for the product of the niaD gene in the expression of the other
two structural genes in this cluster (Cove, 1979; Unkles et al., 1991). The nitrate
reductase and nitrite reductase genes in A. parasiticus, A. oryzae, A. niger, P.
chrysogenum, and Leptosphaeria maculans have also been found to be linked (Chang et
al., 1996).

The genes responsible for antibiotic production in procaryotes and eucaryotes are
commonly associated in clusters (Smith et al., 1990a; Hopwood et al., 1995). Two well-
characterized examples in the filamentous fungi are penicillin (PE) biosynthesis in A.
nidulans and P. chrysogenum (MacCabe et al., 1990; Smith et al., 1990a; Smith et al.,
1990b). In each of these examples, the identified linkage group consists of three
structural genes ranging over approximately 13 kb of chromosome (Smith et al., 1990a).
Promoters in the Penicillium cluster appear to be controlled by carbon catabolite
repression and nitrogen repression (F eng et al., 1994). The areA homologue in P.
chrysogenum has been designated are, and it has been suggested that the nre gene
product is the factor responsible for mediating nitrogen repression of PE biosynthesis in
this fimgus (Haas and Marzluf, 1995). Data from studies on A. nidulans have also
defined a role for the PacC protein in the transcriptional regulationof the PE cluster
(Espeso et al., 1993). PacC is a‘transcriptional activator and repressor, which is part of a
complex regulatory system, controlling gene expression in response to external pH
(Tilbum et al., 1995). The PE cluster in Aspergillus is also regulated by carbon source,
and it has been argued that this regulation is CreA-independent (Espeso et al., 1993).

Other fungal secondary metabolites that have their biosynthetic genes clustered
include the mycotoxins. The best characterized examples are the groups of genes

expressing sterigrnatocystin (ST) in A. nidulans (Brown et al., 1996); and aflatoxin (AF)

16

inA. flavus (Y u et al., 1995) and A. parasiticus (Trail at al., 1995; Yu et al., 1995). The
genes involved in AF biosynthesis are clustered in a 60 kb stretch of chromosome in both
of the AF producing species (Y u et al., 1995). Northern hybridization analysis in A.
parasiticus, has identified more transcripts expressed from the AF cluster than there are
known genes, indicating that the complete AF cluster is yet to be defined (Trail et al.,
1995). Examination of six AF genes in both A. flaws and A. parasiticus, has revealed a
highly conserved gene sequence and organization between the two clusters (Y u et al.,
1995). Despite this conservation, the AF profile in these two organisms is inconsistent,
and it has been suggested that the differences may be due to the subtleties in gene spacing
within the two clusters and/or the duplication of part of the gene group in A. parasiticus
(Liang et al., 1996; Yu et al., 1995). ST is an AF precursor in A. flavus and A.
parasiticus, but it is expressed as an end product in A. nidulans. The ST biosynthetic
genes in A. nidulans are located in a 60 kb linkage group, from which 25 transcripts are
coordinately expressed. All of the genes identified in AF metabolism are homologues of
ST cluster genes, however, there are differences in gene order and direction of
transcription between the AF and ST clusters (Brown et al., 1996). Both the AF (Payne
et al., 1993; Chang et al., 1993; Woloshuk et al., 1994) and ST (Yu et al., 1996) gene
clusters encode a homologous, pathway-specific regulator, that appears to act positively
in the transcriptional regulation of these toxins (Payne et al., 1993; Chang et al., 1993;
Chang et al., 1995; Yu et al., 1995).

Fumonisin, another mycotoxin, appears to have its biosynthetic genes clustered in
Gibberellafujilmroi (Desjardins et al., 1996); and a possible trichothecene gene cluster in
Fusarium sporotrichioides has been identified through mutant complementation studies

(I-Iohn et al., 1993). Alternaria alternata has three genes required for biosynthesis of

17

melanin clustered in a 30 kb region of chromosome, and the pattern of expression of these
genes has indicated coordinated regulation of this secondary metabolism cluster (Kimura
and Tsuge, 1993).

SpoCl Cluster

Secondary metabolism produces compounds that are generally considered to be
nonessential for growth. In a laboratory environment, secondary metabolites are usually
expressed after log growth of a culture, and primary metabolism provides the building
blocks used in their construction (Drew and Demain, 197 7; Demain, 1992). The
functions of many secondary metabolites are unknown, and this has contributed to their
classification as secondary in importance, when considering the entire metabolic
activities of an organism (Drew and Demain, 197 7; Bennett and Christensen, 1983).
Several possible functions of secondary metabolic activity have been proposed and these
include roles in cellular differentiation, reproduction, and protection fiom competition or
predation (Davies, 1992; Vining, 1992).

The SpoCl gene cluster (SGC) was first isolated in experiments performed to
identify genes involved in A. nidulans sporulation (Zimmermann et al., 1980). This
study provided evidence that developmentally regulated genes were nonrandomly
dispersed in the Aspergillus genome, and many genes of this type were subsequently
shown to be clustered (Orr and Timberlake, 1982). Characterization of the original 13.3
kb SGC fiagment, confirmed that the expression of transcripts from this chromosomal
region were temporally coordinated and that transcript accumulation was specific to
conidia (Timberlake and Barnard, 1981). Further analysis of the cluster provided a
transcript map that extended over 38 kb of DNA and included 19 coding regions that

were preferentially expressed during conidiation. In addition, the cluster was found to be

18

flanked by 1.1 kb direct repeats (Gwynne et al., 1984; Miller et al., 1987).

To investigate the possibility that the clustering of these spore-specific genes
served a regulatory purpose, transformation experiments were performed in which a gene
fiom the SGC was integrated at ectopic chromosomal locations, and conversely a
constitutively expressed gene was integrated into the cluster. The expression of these
genes was then monitored in hyphae and conidia (Miller et al., 1987). The results
indicated that chromosomal position was a factor in cell-specific expression of the cluster
gene examined. In these experiments the wild-type strain did not show expression of the
cluster gene in hyphae, while derivative strains, with the cluster gene placed at
presumably random heterologous locations, expressed this gene in hyphae. The
conclusion by the authors that a property of the SGC negatively regulates transcription of
cluster genes in hyphae, was supported by experiments which placed the constitutively
expressed gene in the SGC. A reduction in transcription of at least five-fold was
observed when this housekeeping gene was integrated at different positions in the SGC.

The data from the SGC experiments indicates that the clustering of genes can
serve to regulate gene expression, although the mechanisms of this regulation remain to
be defined. The physical linkage of aflatoxin biosynthesis genes may likewise have a
regulatory role and studies designed to establish such a phenomenon and in turn to
determine the nature of the regulation, may eventually lead to a broader understanding of
the expression of secondary metabolism and the ability to manipulate its expression in
microorganisms. With knowledge about aflatoxin gene regulation still being acquired, it
seems prudent to validate aflatoxin reporter constructs as reliable indicators of wild-type
gene expression, which of course this work attempts to do with the nor-I/uidA reporter

fusion.

19

MATERIALS AND METHODS

Strains, Plasmids and Culture Conditions

Plasmid DNA was propagated in E. coli DH5a F" [F ’/endA1 hstI 7 (r,‘m,,+)
supE44 thi-I recAI gyrA (Nal') DrelAI (lacZYA-argF),,69:(m8O DlacZ M15)] (Gibco
BRL, Life Technologies, Inc. Gaithersburg, MD). The A. parasiticus nitrate reductase
(niaD) mutant strain NR1 (Homg Dissertation, 1990; Liang et al., 1996), was derived
fi'om the wild-type aflatoxin producer NRRL 5862 (SU-l; ATCC 56775). NR1 was used
as the recipient strain in all fungal transformation experiments. The plasmids
pAPGUSNNA and pAPGUSNNB contain the same nor-1 /uidA translational fusion, and
were constructed for analysis of uidA expression in NR1. These vectors were created by
ligating the 9.6-kb, XhoI digested fusion construct pAPGUSN, with the 7.4-kb XhoI-Sall
fragment of pSL82 (Homg et al., 1990) (Figure 2). The pSL82 fragment contains the A.
parasiticus niaD gene as a selectable marker. The plasmid pGAPN2 (Liang et al., 1997)
was used in transformants as a control for constitutive expression of GUS. It contains an
A. flavus B-tubulin promoter fused to the uidA gene, and the A. parasiticus niaD gene as a
selectable marker. Bacterial strains harboring plasmids were stored in 20% glycerol at
-80°C. Plasmids used for fungal transformation were purified by CsCl density gradient
centrifugation (Ausubel et al., 1987).

For the analysis of RNA, protein, and aflatoxin in time-course studies, the
following culture conditions were used: 250 m1 Erlenmeyer flasks, coated with
Sigrnacote (Sigma Chemical, St. Louis, MO), and containing 100 m1 of YES broth (2%
yeast extract [Difco Laboratories, Detroit, MI], 6% sucrose, pH 5.8) and five 6 mm glass

beads (Fisher Scientific, Pittsburgh, PA), were inoculated with 2x106 viable conidia

20

 

 

 

 

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1.1-.13:

Figure 2. Construction of pAPGUSNNA and pAPGUSNNB reporter plasmids. (A)
pAPGUSN was derived from pGLGUS-l (Wu, 1995) and contains a nor-I/uidA
translational fusion. The solid red boxes represent the 5’ and 3’ regions of the nor-1
gene. The 5’ region is approximately 3-kb and includes the sequence encoding the first
21 amino acids of the NOR protein. The 3’ region is approximately 1.8-kb and contains
sequence encoding the final 6 amino acids of the carboxyl terminus of the NOR protein,
the translation termination codon and the predicted polyadenylation site of the nor-I
transcript (Trail et al., 1994). The red hashed region represents the uidA structural gene
(Jefferson et al., 1986) and begins with the sequence encoding the third amino acid
(arginine) of GUS. The black box indicates an ampicillin-resistance gene (cloned from
Promega’s pGL2-Basic) for selection in E. coli. (B) pSL82 contains an 8.2-kb SaII
fragment of A. parasiticus genomic DNA. The blue arrow represents the A. parasiticus
niaD gene, which is used as a selectable marker in fungal transformations. The black
arrow represents the arnpicillin-resistance gene from pUCl9. A sticky-end ligation was
performed between the 7.4-kb XhoI-SalI fragment of pSL82 and the XhoI linearized
pAPGUSN vector, to create the pAPGUSNN plasmids. (C) pAPGUSNNA is oriented
with both niaD and the uidA fusion being transcribed off the same DNA strand, while (D)

pAPGUSNNB has these two genes transcribed in opposite directions.

21

 

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(2x104/ml). Sigmacote was applied to prevent fungal growth on flasks and glass beads
were used to help maintain uniform culture conditions by interfering with mycelia]
aggregation (Skory et al., 1993). Cultures were incubated in the dark at 29°C, with
shaking at 150 rpm for 24, 48, or 72 hr. Mycelia were harvested at the appropriate time
points by vacuum filtration on Miracloth (Calbiochem-Novabiochem, San Diego, CA),
and 65% - 85% of mycelial mat was immediately frozen in liquid nitrogen. The
remaining 15% - 35% of the mycelia were thoroughly dried at 70-80°C and then used for
dry weight determination of the culture. The culture filtrate and frozen mycelia were
stored at -80°C.
Fungal Transformation

A homologous transformation system for A. parasiticus, using the niaD gene as a
selectable marker, has been developed (Oakley et al., 1987; Homg et al., 1990), and was
used in this study with modifications. Approximately 2.5x108 NR1 spores were
inoculated into a 250 ml Erlenmeyer flask containing 100 ml Czapek-Dox (CZ) broth
(Difco Laboratories) supplemented with 1% Bacto Peptone (Difco Laboratories).
Cultures were incubated in the dark at 29°C with shaking at 150 rpm for 18 hr. Two
flasks of mycelia were used for each transformation. Mycelia were harvested by vacuum
filtration using Miracloth, and then mixed with 10 ml CZ broth. Ten ml (5 mg/ml) of
Novozyme 234 (Interspex Products, Foster City, CA) in reaction buffer (1.1 M KCl, 100
mM NaCitrate, pH 5.8) was gently mixed with mycelia for 5 hr at 29°C in the dark.
Protoplasts were separated from cell wall debris by centrifugation at 160 x g, 25°C for 60
sec (IEC PR-6000 Centrifuge), and the supernatant was filtered through 29 mm mesh.
The filtrate was centrifuged at 3,000 x g, 4°C for 15 min (Sorvall SS-34 rotor).

Protoplasts were kept on ice and washed 3X with 1 ml protobuffer (600 mM KCl, 50 mM

23

CaClz, 10 mM Tris-HCl [pH 8.0]) and finally suspended in 110 pl protobuffer. 1-10 ug
of circular plasmid DNA in 10 ul of TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) was
added to 100 pl of protoplast suspension. 50 111 of a filter-sterilized PEG solution (25%
polyethylene glycol 3350, 600 mM KCl, 50 mM CaClz, 10 mM Tris-HCI [pH 7.5]) was
gently mixed with the protoplast/DNA suspension and then placed on ice for 20 min. An
additional 850 pl of PEG solution was added and the transformation mixture incubated at
25°C for 30 min. Transformants were selected on CZ agar supplemented with either 600
mM KCl or 20% sucrose as an osmotic stabilizer. Agar plates were incubated in the dark
at 29°C for 3-5 days.
Spore Preparation

Transformants selected for further analysis were subjected to three cycles of
single spore isolation. One cycle consisted of the following: A single transformant was
allowed to sporulate on CZ agar for 1-2 wk in the dark at 29°C. Asexual conidiospores
were dislodged from the colony with a sterile spatula and water. The crude spore
suspension was filtered through sterile 8 pm fiberglass (Corning, Corning, NY) in a 10 cc
syringe (Becton Dickinson, Sparks, MD). The spore concentration was determined with
an improved Neubauer hemacytometer. The spore suspension was then appropriately
diluted for the plating of 1-10 spores per CZ plate. Plates were incubated as described
above for approximately 3 days. Mycelia from a single colony were transferred to a fresh
CZ plate to complete the cycle. After the completion of three cycles, large scale
preparations of spores from certain transformants were performed. This entailed
inoculating a 150 cm2 tissue culture flask (Corning) containing 100 ml of Potato
Dextrose Agar (Difco Laboratories) with the appropriate spores. Flasks were incubated

as above for 2-3 wks. Spores were dislodged by spinning a sterile stir bar, in

24

approximately 50 m1 of water, over the colony. The spore suspension was filtered
through 8 pm fiber glass in a 60 cc syringe (Becton Dickinson). Spores were pelleted by
centrifiigation at 2,600 x g, 25°C for 10 min (Sorvall GSA rotor). Three to five ml of
20% glycerol was used to resuspend spores. The spore suspension was aliquoted, frozen
in an ethanol/dry ice bath, and stored at -80°C. Spore viability counts were performed on
frozen stocks by serial dilutions of a spore sample and plating in triplicate on CZ agar.
Plates were incubated as above and colony counts performed after approximately 3 days
of growth.
Fungal DNA Isolation and Southern Blotting

250 m1 Erlenmeyer flasks containing 100 ml YES broth were inoculated with
spore stocks and incubated in the dark at 29°C with shaking at 150 rpm for approximately
48 hr. Mycelia were collected by vacuum filtration on Miracloth. A fraction of the
mycelia was exposed to liquid nitrogen and macerated with a mortar and pestle. DNA
was extracted according to a modified procedure based on a phenol-chloroform protocol
for mammalian DNA isolation (Ausubel et al., 1987). Mycelia] powder was mixed with
an extraction buffer (10 mM Tris-HCI [pH 8.0], 150 mM NaCl, 25 mM EDTA, 0.5%
lauryl sulfate, 100 ug/ml Proteinase K [Boehringer Mannheim]) at 50°C with gentle
agitation for approximately 20 hr. The crude sample was extracted with phenol-
chloroform-isoamylalcohol (25:24:1) and then precipitated with ammonium acetate and
ethanol at -20°C. DNA was pelleted by centrifugation, dissolved in TE, and treated with
DNase-free RNase A (Boehringer Mannheim, Indianapolis, IN). Further purification was
achieved with a series of two phenol-cloroform-isoamylalcohol (25:24: 1) extractions,
followed by precipitation, redissolving in TE, and treatment with RN ase A. The

appropriate dilutions were then performed on the sample and the DNA concentration

25

determined with a Genequant RNA/DNA calculator (Pharrnacia Biotech, Bridgewater,
NJ).
For Southern blotting, approximately 8 pg of each DNA sample was digested

with the appropriate endonuclease (Boehringer Mannheim, Gibco/BRL) and loaded into a
0.8% agarose gel (leBE buffer [89 mM Tris, 89 mM Boric Acid, 2 mM EDTA], 0.5
pg/ml ethidium bromide). Electrophoresis was performed for approximately 15 hr at 50
volts and the gel was subsequently treated with 5 volumes of 250 mM HCl in order to
partially depurinate DNA fragments to facilitate DNA transfer. Further gel treatment and
blotting to Nytran membrane (Schleicher & Schuell, Keene, NH) was performed
according to standard methods (Maniatis et al., 1989). DNA transfer was allowed to
continue for 20-24 hr, and then ultraviolet irradiation was used to crosslink DNA to the
membrane (Stratagene UV Stratalinker 1800, La Jolla, CA).
Fungal RNA Isolation and Northern Blot Analysis

Mycelial samples stored at -80°C were macerated with a mortar and pestle in
liquid nitrogen. RNA was extracted from 100 mg of mycelia] powder with one ml of
TRIzol reagent (Gibco/BRL) using the recommended manufacturer procedures. RNA
concentrations were determined using a Genequant RNA/DNA calculator. Northern blot
analysis was performed according to standard procedures (Ausubel et al., 1987; Maniatis
et al., 1989). 30 pg of total RNA from each sample was loaded into a denaturing, 1.2%
agarose gel (lxMOPS [40 mM 3-(N-morpholino)-propanesulfonicacid, [pH 7.0], 10 mM
sodium acetate, 1 mM EDTA], 800 mM formaldehyde). RNA was separated by
electrophoresis at 70 volts for 3 hr and then transferred to Nytran membrane by capillary
action. RNA transfer was allowed to continue for 20—24 hr and the nucleic acid was

subsequently crosslinked to the membrane with the UV Stratalinker 1800 at standard

26

crosslink settings.
Nucleic Acid Hybridization

For prehybridization, membranes were incubated in hybridization solution (50%
forrnamide, SXSSC [750 mM NaCl, 75 mM sodium citrate, pH 7.0], leE [50 mM Tris-
HCl [pH 7.5], 0.1% sodium pyrophosphate, 1% lauryl sulfate, 0.2% polyvinylpyrolidone
40,000, 0.2% ficoll 400,000, 5 mM EDTA, 0.2% bovine scnun albumin], 150 pg/ml
salmon sperm DNA) at 42°C for 3-5 hr in rotating vessel. Probe DNA was gel purified
with GELase (Epicentre Technologies, Madison, WI) and labeled with a-dCTP 32P (NEN
Life Science, Boston, MA) using a random primed DNA labeling kit (Boehringer
Mannheim). Radioactive probes were separated from free nucleotides using a Nuc Trap
Push Column (Stratagene). Denatured probe was added to hybridization solution and
incubated with the membrane, in a rotating vessel, at 42°C for 10-15 hr. Membranes
were washed under high stringency conditions (0.1% lauryl sulfate, 0.1xSSC, 65°C), and
exposed to X-Ray film (Kodak XAR-S) at -80°C.
Qualitative and Quantitative GUS assays

To rapidly screen for the ability of a transformant to express GUS activity, a
qualitative GUS assay (Kolar etal., 1991; Verdoes et al., 1994) was used. Transformants
were transferred from selective medium to plates containing an aflatoxin-inducing, solid
growth medium (YES), and then overlaid with circular 82 mm Nytran Plus membranes
(Schleicher & Schuell). Plates were incubated in the dark at 29°C for 36-48 hr.
Membranes, with attached fungal colonies, were removed from plates and exposed to
liquid nitrogen for 30 sec. Membranes were allowed to thaw and then incubated in a
GUS substrate solution (60 mM Nazi-IP04, 40 mM NaH2P04, 10 mM KCl, 1 mM MgSO,,

0.27% D-mercaptoethanol, 0.04% X-GLUC [Gold Biotechnology St. Louis, MO]) for up

27

to 24 hr.

For quantitative GUS analysis, samples of mycelial stocks stored at -80°C, were
macerated with a mortar and pestle in liquid nitrogen. Approximately 100 mg of crushed
tissue was mixed with 500 pl of chilled GUS lysis buffer (50 mM NaHzPO4 [pH 7.0], 10
mM EDTA, 0.1% Triton-X400, 0.1% lauryl sulfate, IOmM B-mercaptoethanol, 25 pg/ml
phenylrnethanesulfonyl fluoride [PMSF]) and centrifuged at 12,000 x g at 4°C for 10
min. The protein concentration of the supernatant was determined by the Bradford
method (Bradford, 1976) using bovine serum albumin as a protein standard and Protein
Assay Dye Reagent (Bio-Rad laboratories, Hercules, CA) according to the
manufacturer’s instructions. One pg of total protein fi'om each extract was then incubated
in a 200 p1 volume of 2 mM 4-methylumbelliferyl-[B-D-glucuronide, at 37°C, for 0, 3, 5,
6, 9, 10, 12, 15, and/or 30 min as necessary. GUS reactions were terminated with the
addition of 800 pl of 200 mM Na2CO3. To determine GUS activity, the fluorescence of
each reaction mixture was measured at an excitation wavelength of 365 nm and an
emission wavelength of 455 nm, with an SLM-4000 fluorometer (SLM Instruments,
Champaign, IL).

Western Blot Analysis

Frozen mycelial samples were macerated with a mortar and pestle in liquid
nitrogen. Approximately 400 mg of powdered tissue was mixed with 300-400 pl of
chilled GUS lysis buffer (described above with the addition of 1 mM PMSF, 1 pg/ml
leupeptin, and 2 pg/ml peptain A). Cell material was incubated on ice for at least 30 min.
The extract was then centrifirged at 12,000 x g at 4°C for 30 min. The protein
concentration of the supernatant was determined as described above. Discontinuous

SDS-PAGE and Western blot analysis were performed with the Mini-Protean II

28

electrophoresis apparatus and the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad
Laboratories) according to manufacturer’s procedures. 60-75 pg of total protein from
each sample was loaded into a 4% acrylamide stacking gel (125 mM Tris-HCI [pH 6.8],
0.5% SDS) and electrophoresed through a 12% acrylamide separating gel (375mM Tris-
HCl [pH 8.8], 0.5% SDS). Proteins were transferred to a polyvinylidene difluoride
(PVDF) membrane (NEN Life Sciences), and then incubated for 1 hr at 25°C in blocking
solution (150 mM NaCl, 0.05% Tween 20, 1% blocking powder [Schleicher & Schuell]).
Primary antibody incubations were performed in blocking solution at 25°C for 45 min,
with either rabbit a-NOR (Zhou, 1997) at a concentration of 2 p g/ml or rabbit a-GUS
(Clontech Laboratories, Palo Alto, CA), at a dilution of l : 1600. A goat a-rabbit, alkaline
phophatase conjugate (Schleicher & Schuell) at a dilution of 1:10,000, was used for all
secondary antibody incubations (30 min, 25°C, in blocking solution). Washes were
performed in 150 mM NaCl, 0.05% Tween 20, after each antibody incubation.
Secondary antibody was detected by incubation in chromogenic substrate solution (30 ml
water, one BCIP/NBT Substrate Tablet [Schleicher & Schuell]).
Polymerase Chain Reaction Analysis

A PCR assay was developed for the screening of fungal genomic DNA in order to
identify transformants with specific vector integration events. The PCR reactions
included 200 ng of genomic DNA, primer (1 pmole/ pl), 3.5 mM MgC12, 800 mM dNTPs,
and 0.05 units/ pl Taq polymerase (Perkin Elmer). The reaction volume was 50 p1.
Samples were amplified in the GeneAmp PCR System 9600 (Perkin Elmer, Norwalk,
CT) as follows: 1 min annealing at 68°C, polymerization for 4 min at 72°C, and
denaturation for 1 min at 95°C. 40 cycles were used per reaction. The initial

denaturation was for 2 min at 95°C, and the final polymerization was extended for 10

29

min. Primers IL 99 (5’TTT CAC GGG TTG GGG TTT CTA CAG G3’) and IL 100
(5’GAC GGG GAA CCT CTT TAC AAA CAT C3’) were used for the identification of
5’ nor-1 pAPGUSNNB integration events. Primers IL 102 (5’CGC AAG GTG AGG
GTT CGA ACC GAG G 3’) and IL 103‘ (5’CCG CAG CAG GGA GGC AAA CAA
TGA A3’) were used for the identification of 3’ nor-1 pAPGUSNNB integration events.
Aflatoxin Analysis

The concentration of aflatoxin in culture filtrates was determined by direct
competitive ELISA (Ram et. al., 1986). The rabbit polyclonal AFB, antibody and AFB,-
horseradish peroxidase conjugate (AFBl-HRP) were kindly provided by J. Pestka,
Michigan State University. 125 pl of a 1:900 dilution of (Jr-AFBl in coating buffer (15
mM Na2CO3, 35 mM NaHCO,) was applied to Irnmulon 4 microtiter plates (Dynatech
Laboratories, Chantilly, VA), and the plates then dried for 18 hr at 42°C in a forced air
oven (Fisher Isotemp Oven 338F). Plates were washed eight times (four times in each
orientation) in a Skatron A/S Microwash II instrument (Skatron, Lier, Norway) using
0.02% Tween 20 in PBS. Plates were incubated with 300 pl of blocking solution (1%
bovine serum albumin in PBS [pH 7.2]) at 37°C for 30 min to eliminate nonspecific
binding. Plates were washed as described above. A 1:300 dilution of AFB,-HRP was
prepared in blocking solution, and then mixed with culture filtrates in a 1:1 ratio (a total
volume of 100 pl). The mixture was prepared in the microtiter plates and then incubated
in the dark at 37°C for 1 hr. After incubation, plates were washed with the PBS, Tween
20 solution eight times in each orientation using the NS Microwash II instrument. 100
pl of chromogenic substrate solution (42 mM citric acid [pH 4.0], 0.03% H202, 420 mM
2,2’-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid]) was used as an indicator of

peroxidase activity and was incubated in plates at 25°C for 4 min. Color reactions were

30

terminated with 100 p1 of 300 mM citric acid. Color intensity was analyzed using a

Vmax spectrophotometer at 405 nm (Molecular Devices, Sunnyvale, CA).

31

RESULTS

Transformation and screening of A. parasiticus

Three separate transformation experiments of NR-l were performed using the
pAPGUSNNA and pAPGUSNNB reporter vectors. Because both vectors contain the A.
parasiticus niaD gene as a selectable marker, transformation mixtures were plated on CZ
agar with nitrate as the sole nitrogen source. The transformation frequencies in each

experiment are presented in Table 1. All transformants from the second and third

Table 1. NR-l transformation frequencies from three separate experiments.

 

 

 

 

 

Transformation pAPGUSNNA pAPGUSNNB
First 287 cfus/6.1 pg DNA/1.0x107 322 cfus/6.5 pg DNA/8.0x10"
protoplasts protofists
Second 355 cfus/6.1 pg DNA/5.9x10° 492 cfus/6.5 pg DNA/9.6x10°
protoplasts . protoplasts
Third 891 ems/6.1 pg DNA/1.1x107 706 cfus/3.3 pg DNA/1.4x107
protoplasts protoplasts

 

 

 

 

experiments were selected on CZ agar supplemented with sucrose as an osmotic
stabilizer. These colonies had a morphology that was similar to the NR-l parent strain.
However, in the first transformation experiment, KC] was used as an alternative osmotic
stabilizer and this experiment resulted in transformants with two colony morphology
types. The first morphology type was characterized by green pigmentation of the
conidiospores and was similar to the phenotype of the parent strain. The second
morphology type of the first transformation experiment was distinguished by yellow
spores, highly concentrated in the center of the colony. A plate from the first
transformation experiment would either contain all green-spored or all yellow-spored

colonies, which suggested that the unique environment in an individual plate was a factor

32

in morphology determination. In most cases, when a yellow-spared colony was
transferred and reincubated, it assumed the typical green-spored phenotype.

In each of the three transformation experiments, a number of the m'aD+ colonies
were chosen in order to screen for their ability to express GUS activity in a qualitative
assay (Figure 3 and Table 2). This was a colorimetric assay where the substrate X-Gluc
was cleaved by the GUS protein in situ. Cleavage of X-Gluc by GUS resulted in a blue
color that was indicative of reporter gene expression. None of the pAPGUSNNA
Table 2. The ratios of GUS+ transformants from three separate transformation
experiments. The plasmids pAPGUSNNA and pAPGUSNNB were used to transform

NR-l. A qualitative GUS assay was used to screen m’aD+ colonies fiom these
transformations, for their ability to express GUS activity on an aflatoxin inducing media

(YES agar).

 

 

 

 

Transformation NR-I/pAPGUSNNA NR-l/pAPGUSNNB
GUS’“ GUS+
First 0/ 15 ' 4/ 15
Second 0/32 9/32
Third 0/81 22/102

 

 

 

 

 

transformants examined expressed GUS qualitatively. Although this result was
interesting, our major goal was to develop a feasible reporter enzyme system in A.
parasiticus, and therefore the pAPGUSNNA transformants were not fimher analyzed.
Since DNA sequence analysis of the reporter vectors was not performed, nor was the
expression of GUS activity in E. coli examined, the lack of activity from pAPGUSNNA
transformants may have been due to DNA mutation in the plasmid. Another possible
explanation for nonexpression in pAPGUSNNA transformants, is that the proximity of
the nor-1 promoter to unknown cis-acting factors in this particular orientation of the
reporter vector, repressed uidA transcription in the fungus.

Qualitative GUS analysis of transformants from three pAPGUSNNB

33

Figure 3. Qualitative GUS assay of fungal colonies transformed with pAPGUSNNB. A
sterile toothpick was used to transfer fungal transformants onto an aflatoxin inducing
medium (YES agar). The inoculated agar was overlaid with a nylon filter and then
incubated at 29°C for 36-48 h in the dark. The filter with the accompanying fungal
growth, was subjected to a freeze thaw cycle, using liquid nitrogen, in order to break
down cell walls and membranes. After a thawing period of five minutes, the nylon
membrane was then incubated for 30 min in a GUS substrate solution. The freeze-thaw
cycle enhances the opportunity for substrate-enzyme interaction by eliminating physical
barriers to ligand binding. The blue colonies resulted from cleavage of the colorimetric
substrate X-Gluc by B-glucuronidase, and were indicative of transformants with
pAPGUSNNB integrated into the nor-1 chromosomal region. The non-blue or GUS"
strains were characterized by fusion integration into the niaD region of the NR-l

chromosome.

34

 

 

 

 

Figure 3

35

 

transformations suggested that 27%, 28%, and 22% of the screened colonies expressed
GUS. In order to help explain the low ratio of GUS expressors in the pAPGUSNNB
transformant population, a number of GUS+ and GUS' transformants were chosen for
genetic analysis.

Southern Hybridization Analysis

The integration of transforming DNA can occur at either homologous or
heterologous locations in the genomes of filamentous fungi. Restriction enzyme analyses
of the nor-1 and niaD regions of the A. parasiticus chromosome were performed in order
to develop Southern hybridization schemes used to identify transformants with
pAPGUSNNB single crossover integrations into either the chromosomal nor-1 or niaD
genes (Figure 4). GUS+ and GUS‘ transformants were selected from previous
experiments and evaluated according to these schemes.

Initial experiments identified three types of transformants (Figure 5), each
characterized by a different integration event and each event predicted by the schemes
displayed in Figure 4. Transformants D8D1 and D8D6 were GUS+ and represent single
crossover integration of pAPGUSNNB into the 5’ region of nor-I . The intensity of the
hybridization signals from the D8D1 samples in Figures 5A, 5B, 6A, and 63 suggested
that multiple integration of the vector occurred in this transformant, while the D8D6
transformant appeared to carry a single copy of the vector. Transforrnant D8D3 was
GUS+ and represented single crossover integration of pAPGUSNNB into the 3’ region of
nor-I . Transforrnant D8D4 was GUS‘ and represented single crossover integration of
pAPGUSNNB into the niaD chromosomal region. It is likely that the GUS" transformant
D8E5 was also a niaD integrant, but the data presented in Figure 5 did not completely

confirm this characterization.

36

 

 

IF.
\

\_V‘"' I‘I'cn.‘

 

'€
‘ u
k
:- rqn: 1"":

figure 4
mforr
region 0:
W fra
memos
gradient

iiappee

Figure 4A. Schematics for Southern hybridization and PCR analyses of niaD”
transformants. To determine whether the pAPGUSNNB vector integrated into the 5’
region of nor-I , a Southern blot of a Xhol chromosomal digest was probed with a 750-bp
ScaI fragment from the 5’ region of nor-I . This probe is common to both vector and
chromosome. A single crossover of pAPGUSNNB into the 5’ region of nor-l was
predicted to produce two Southern hybridization signals of 19-kb and 3.4-kb, with the
disappearance of the 5.8-kb wild-type hybridization signal.

The analysis of the 5’ nor-1 pAPGUSNNB transformants by PCR was performed
using primers represented in this figure by golden arrows. One primer was designed to be
unique to the vector and to anneal to the 5’ region of the uidA gene. The second primer is
unique to chromosomal sequence and anneals to DNA in the 5’ region of nor-1 . A 3.1-kb
arnplicon should result only when DNA from a 5’ nor-1 pAPGUSNNB integrant is used

as template.

37

 

 

 

 

 

 

 

 

38

Figure 4B. Schematics for Southern hybridization and PCR analyses of niaD+
transformants. To determine whether the pAPGUSNNB vector integrated into the 3’
region of nor-I, a Southern blot of Sea] chromosomal digests was probed with a 900-bp
ClaI fragment from the 3’ region of nor—1 . This probe is common to both vector and
chromosome. A single crossover of pAPGUSNNB into the 3’ region of nor-I was
predicted to result in two Southern hybridization signals of 4.0-kb and 4.4-kb, with the
disappearance of the 3.0-kb wild-type hybridization signal.

The analysis of the 3’ nor-I pAPGUSNNB transformants by PCR was performed
using primers represented in this figure by golden arrows. One primer was designed to be
unique to the vector and to anneal to the 3’ region of the uidA gene. The second primer is
unique to chromosomal sequence and anneals to DNA in the 3’ region of nor-I . A 2.1-kb

arnplicon should result only when DNA from a 3’ nor-1 pAPGUSNNB integrant is used

as template.

39

 

 

 

 

 

 

no: soils
.v. Vw§M\~-...o= Q3: . ~35: .
TIAItlirlilIY " t lTiAI-Ili
aoraoao 40$... 4% 40 40 a...
e..- m
. 78: x

 

:88 .23.
8a... .3
2.3 .38 .5... .84..
28. .
on._¢8w £88 .82..
. ”Wm.
_ . s .
38m 83.
. a... .88.
2.8 _. .228 .89.

40

Figure 4C. Schematics for Southern hybridization analysis of niaD+ transformants. To
determine whether the pAPGUSNNB vector integrated into the niaD region of the NR-l
chromosome, a Southern blot of Sal] chromosomal digests was probed with an 800-bp
SaII-flrol fragment from the niaD chromosomal region. This probe is unique to the
chromosome. A single crossover of pAPGUSNNB at niaD was predicted to yield an 11-

kb Southern hybridization signal, with the disappearance of the 8.2-kb wild-type

hybridization signal.

41

i 1‘!

3. 0.5»:—

 

 

 

 

_ 8:... _ 8:3 _ fl-.. _
_ Q3: {33:82 _ Q3: _
l . Iii
a... a... 83.... a... «A...
_ _
_ QB... _
“oEOmoEoEo
f/éno
mam 000.: “WWW WWW

mzzmaofiq

 

42

Figure 5A. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants. To determine whether the pAPGUSNNB vector integrated into the 5’
region of nor-1, a Southern blot of XhoI chromosomal digests was probed with a 750-bp
ScaI fragment fi‘om the 5’ region of nor-I . This probe is common to both vector and
chromosome. A single crossover of pAPGUSNNB into the 5’ region of nor-1 was
predicted to produce two Southern hybridization signals of 19-kb and 3 .4-kb, with the
disappearance of the 5.8-kb parental strain hybridization signal. The sizes of the DNA
fragments are shown on the left and the DNA marker sizes are indicated on the far right.

NR1 served as the parental strain control. These data indicate that transformants D8D1

and D8D6 are 5’ nor-1 integrants.

43

 

 

 

 

 

1 9-kb

 

Figure 5a

Figure 5B. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants. To determine whether the pAPGUSNNB vector integrated into the 3’
region of nor-1, a Southern blot of ScaI chromosomal digests was probed with a 900-bp
ClaI fragment fi'om the 3’ region of nor-1. This probe is common to both vector and
chromosome. A single crossover of pAPGUSNNB into the 3’ region of nor-1 was
predicted to result in two Southern hybridization signals of 4.0—kb and 4.4-kb, with the
disappearance of the 3.0-kb parental strain hybridization signal. The sizes of the DNA
fragments are shown on the left and the DNA marker sizes are indicated on the far right.
NR1 served as the parental strain control. These data indicate that transformant D8D3 is

a 3’ nor-1 integrant.

45

 

 

 

 

5.4-kb

4.4-kb
4.0-kb

3.0-kb

 

 

 

 

Figure 5b

46

Figure 5C. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants. To determine whether the pAPGUSNNB vector integrated into the niaD
region of NR-l chromosome, a Southern blot of SalI chromosomal digests was probed
with an 800-bp SalI-Xhol fragment from the niaD chromosomal region. This probe is
unique to the chromosome. A single crossover of pAPGUSNNB at niaD was predicted
to yield an 11-kb Southern hybridization signal, with the disappearance of the 8.2-kb
parental strain hybridization signal. The sizes of the DNA fragments are shown on the left
and the DNA marker sizes are indicated on the far right. NR1 served as the parental

strain control. These data indicate that transformant D8D4 is a niaD integrant.

47

 

 

1 1-kb
8.2-kb

 

Figure 5c

48

Figure 6. Southern hybridization analysis of NR-l and NR-llpAPGUSNNB
transformants. To confirm the presence of the of the uidA reporter gene in the niaD
selected transformants, Sea! (A) and XhoI (B) chromosomal digests were probed with a
670-bp MluI fragment from the uidA gene. The sizes of the DNA fragments are shown
on the left and the DNA marker sizes are indicated on the far right. NR1 served as the
parental strain control. These data illustrate that each of the transformants contains the

reporter gene, and that the NR-l parent strain is negative for uidA.

49

no.

 

5.8—kb

4.0-kb

 

 

 

19-kb
1 1-kb

 

 

Figure 6

50

 

Once the experimental schemes were tested, a larger group of GUS+ and GUS’
pAPGUSNNB transformants was assayed to identify the integration events (Figures 7
and 8). Ten of eleven (91%) GUS’ transformants examined were either 5’ (four) or 3’
(six) nor-1 integrants. The exception is transformant 15, and although its hybridization
patterns could not corifirm nor-1 integration of the vector, this possibility still remains.
Six GUS“ transformants were shown to be either single crossover integrations at niaD

(two) or double crossover integrations of the selectable marker (four). The hybridization

‘I'o ‘._ __

pattern of transformant 15 was not indicative of integration at niaD.
PCR Analysis

A PCR assay was developed to efficiently screen a population of transformants
for those isolates with reporter genes located in the nor-1 region of the chromosome. The
assay requires one primer unique to the vector and one primer unique to the chromosomal
DNA. When the primers anneal to the template in close proximity to each other, an
amplicon can be produced. If the sequence in the region of interest is known, then the
site of vector integration can be surmised by the size of the amplicon produced under
stringent conditions. In the assay, one set of primers was used to determine a 5’ nor-I
integration event (3.1-kb amplicon) and another set was used to determine a 3’ nor-1
integration event (2.1-kb amplicon). The assay scheme is illustrated in Figures 4A and
43. Several transformants were screened with this assay (Figure 9) and the interpretation
of the data from the assay is supported by Southern hybridization analyses (Figures 7 and
8).

As indicated in Figure 9, transformants 50, 59, 61, 68, 85, and 96 produced a 2.1-
kb amplicon with primers JL 102 and JL103. The production of this specific amplicon

suggests that these transformants have pAPGUSNNB integrated into the 3’ region of nor-

51

1 (refer to Figure 4B for PCR schematics). Transformants 8, 34, 67, and 73 produced a
3.1-kb amplicon with primers IL 99 and IL 100. The production of this specific
amplicon indicates that these transformants have pAPGUSNNB integrated into the 5’
region of nor-1 (refer to Figure 4A for PCR schematics).
Time Course Analysis of NR—l/D8D3

Transformant D8D3 was grown in an aflatoxin-inducing medium (YES broth) and
evaluated for nor-1 and uidA expression at 24, 48, and 72 h after inoculation. Mycelia
dry weight (Table 3A, Figure 10), nor-I and uidA RNA (Figure 11), NOR and GUS
Table 3A. Time course analysis of transformant D8D3. The dry weights of D8D3 and
control cultures were determined at 24, 48, and 72 h after inoculation in aflatoxin
inducing medium (YES broth). D8D3 was tested in triplicate while only single cultures

of the control strains were harvested for analysis. The data were reported in mg of
mycelia.

 

 

 

 

 

 

 

 

 

 

 

Strain 24 hours 48 hours 72 hours
NR-l 205 1,150 1,510
NR-l/pSL82 207 1,010 1,660
NR-l/pGAPNZ 279 1,230 1,470
NR-l/D8D3A 227 1,080 1,690
NR- 1 /D8D3B 238 1,230 1,700
NR-l/D8D3C 251 1,210 1,660

 

 

protein (Figure 12), and GUS activity and aflatoxin production (Tables 3B, 3C and Figure
13) were compared in this study. NR-l, NR-l/pSL82B, and NR-l/pGAPNZB served as
controls in the experiment. The pGAPN2B vector contains a constitutively expressed A.
flavus b-tubulin promoter fused to the uidA gene, and the A. parasiticus niaD gene as a
selectable marker. The NR-l/pGAPNZB transformant had vector integration at the niaD
locus (data not shown). The location of integration of the pSL82 plasmid in transformant
NR-l/pSL82B was not identified. Growth of the four strains, as determined by dry
weight analysis (Figure 10), was similar during the course of the study. Between 24 and

52

Haul”
I

48 hours of incubation, each of the cultures was in log phase growth. A transition from
log phase to stationary phase was observed for each culture between 48 and 72 hours.
Previous studies have shown that it is during this transition that nor-1 expression and
aflatoxin production are first detected in culture (Skory et al., 1993; Trail et al., 1995).
Time Course Analysis of NR-l/D8D3 Transcripts
The accumulation of nor-1 transcript was detected by Northern blot analysis at 48
and 72 h afier inoculation in all strains (Figure 11A). These data indicate that NR-l and in
D8D3 express a relatively high level of nor-1 transcript when compared to the pSL82 and i
pGAPN2 transformants. NR-l/pGAPNZ is clearly the weakest expressor of nor-I
transcript.
The timing of uidA transcript accumulation in D8D3 (Figure 11B) mimicked that
of nor-1, with detection of transcript only at 48 and 72 h after inoculation. NR-l and
NR-l/pSL82 were negative for uidA transcript at all time points. The NR-l/pGAPN2
transformant is positive for uidA transcript at 24, 48, and 72 h. Figures 11C and 11D
demonstrate the consistency of D8D3 transcription from replicate cultures. The Northern
blot data suggest that nor-1 transcription is more efficient than nor-1 /uidA transcription in
D8D3, despite the use of the same promoter to regulate the expression of these two genes.
The specific activities of the nor-I and uidA probes in these experiments were roughly
equivalent.
Time Course Analysis of NR-l/D8D3 Proteins
Nor-1 and GUS proteins were detected by Western blot analysis in mycelial
extracts of D8D3 at 48 and 72 h after inoculation (Figures 12A and 12B). This pattern of
protein accumulation was consistent with the accumulation of nor-I and uidA transcripts

from the same mycelia samples. Western blot analysis failed to detect GUS protein in

53

Figure 7. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB GUS+
transformants. Eleven GUS+ transformants were analyzed. A) Six (50, 59, 61, 68, 85,
and 96) demonstrating the characteristic hybridization pattern of 3 ’ nor-1 integrants. B)
Four transformants (8, 34, 67, and 73) exhibited a hybridization pattern consistent with
pAPGUSNNB integration into the 5’ region of nor-1 . The probes used in these
hybridization analyses were the same as those described in Figures 5A and 5B. The sizes
of the DNA fragments are shown on the left. NR1 served as the parental strain control.
These data do not allow for definite conclusions to be drawn about the location of

pAPGUSNNB in the chromosome of transformant 15.

54

4.4-kb
4.0-kb

 

 

 

3.0-kb

 

1 9—kb

5.8—kb

 

 

3.4-kb

 

Figure 7

55

Figure 8. Southern hybridization analysis of NR-l and NR-l/pAPGUSNNB
transformants. Six GUS‘ transformants were analyzed. A) Two (10 and 20) produced the
characteristic hybridization pattern of pAPGUSNNB integration into the niaD region of
the chromosome. Transformant 15 (GUS*) does not have a hybridization pattern
indicative of niaD integration. The probe used in this hybridization analysis was the

same as that described in Figure 5C. The sizes of the DNA fragments are shown on the
left. NR1 served as the parental strain control. B) A 670-bp MluI fragment of uidA was
used as a probe to confirm the presence of the reporter gene in the two niaD integrants.
NR1 served as the parental strain control. The absence of signal from the four remaining
GUS’ transformants (25, 35, 40, and 45), suggests that these transformants have double

crossover integration of the selectable marker.

56

MQY’H 3‘ s
A i.
‘.

 

 

 

 

 

 

 

 

Figure 8

57

Figure 9. PCR assay for identifying transformants with reporter constructs integrated
into the nor-I region of the NR-l chromosome. A) Primers IL 102 and JL103 were used
in this assay to produce a 2.1-kb amplicon, specific for pAPGUSNNB integration into the
3' region of nor-1 . B) Primers IL 99 and IL 100 were used in this assay to produce a 3.1-
kb amplicon, specific for pAPGUSNNB integration into the 5' region of nor-1 .

Arnplicon size is indicated on the left, and the DNA marker sizes are shown on the far
right. NR-l and transformants 10 (single pAPGUSNNB crossover integration at niaD)

and 15 (integration site not defined) were used as negative controls in this experiment.

58

 

 

o
A 69%
u 23.1-kb
.. 9.4-kb
6.6-kb
4.4-kb
2.3-kb
2-1-kb 2.1-kb
B
3.1 -kb

 

Figure 9

59

Figure 10. Dry weight analysis of transformant D8D3 and control strains. Mycelia were
harvested (see Methods) at 24, 48, and 72 h after inoculation in aflatoxin-inducing
medium (YES broth). A portion of each sample was dried for weight determination. For
the control strains, the points on the graph represent data from a single culture. D8D3

was grown in triplicate and the error bars indicate the standard error.

60

Dry Weight (mgs)

 

1750 J _A_ NR,
1500 ‘ + NR-1lpSL82
1250 4 + NR-1/pGAPN28

—o— NR-1IDBD3
1000 —

      

750 "

500 4

250 4

 

 

 

 

Hours

Figure 10.

61

 

 

Figure 11. Northern hybridization analysis of nor-1 and uidA transcripts in transformant
D8D3 and control strains. 30 pg of total RNA was loaded into each gel lane. Each
autoradiogram represents RNA isolated from mycelia after growth for 24, 48, and 72 h in
aflatoxin-inducing medium. A and C) Blotted RNA was hybridized with a PCR
amplified 780 bp nor-1 probe. The target DNA for this amplification was nor-1 cDNA.
The nor-1 transcript is approximately 1.3 kb. B and D) Blotted RNA was hybridized
with an approximately 1.7 kb amplification product of the uidA gene. The predicted nor-
I/uidA fusion transcript is approximately 2.2 kb. This estimate is based on the size of the
uidA gene in the fusion and the nor—1 transcription initiation site and polyadenylation site
which are also included in the fusion (Trail et al., 1994). Transcript sizes are indicated on
the left and RNA marker sizes are indicated on the far right. NR-l was used as the
parental control strain. pGAPN2 was used as a control for constitutive uidA expression.
pSL82 was used as a transformation control and was uidA'. D8D3 A, B, and C (in 11 C,

D) are replicate samples.

62

 

”If bfll iiD‘BIHim ad."

 

48 hr 72 hr
«6" <8

24 hr
<23"

 

 

1.4-kb
A _. *0

 

 

 

fl 2.4-kb

 

 

nor-1
1 .3-kb

UIdA ’
2.2-kb ’

b
b.“
A
1

 

 

 

 

2.4-kb
' O a: I I

 

 

nor-1
1 .3-kb

uidA
2.2-kb

D

Figure 11

63

 

 

 

Figure 12. Western blot analysis of Nor-1 and GUS proteins in transformant D8D3 and
control strains. Protein was electrophoresed through a 12% separating gel. 60 — 75 pg of
extracted protein was loaded into each gel lane. Each blot represents protein isolated
from mycelia after grth for 24, 48, and 72 h in aflatoxin inducing-medium. Only the
sections of the blot containing proteins of relevant sizes are included in this figure. A and
C) Blotted protein was probed with a-NOR antibody. The Nor-1 protein is
approximately 31 kDa (Zhou, 1997). B and D) Blotted protein was probed with a-GUS
antibody (Clontech Laboratories). The size of the Nor-I/GUS fusion protein, as
determined by these data, is approximately 73 kDa. The estimated size of this fusion
protein is similar to the wildtype E. coli GUS protein which is also approximately 73 kDa
(Jefferson et al., 1986). Protein sizes are indicated on the far left. D8D3 A, B, and C (in

12 C, D) are replicate samples.

64

Figure 12

 

 

 

 

 

 

f WW / WW I q! \
Q q, é q, Q q,
950%? $5636? §o§§b
Q Q Q Q Q Q Q Q Q
a—NOR
31 kDa \‘ "'-
or—GUS \.
73 kDa
72 hr 48 hr 24 hr
f A N F A N f A N
‘T Q7 0 v- Q) 0 v Q7 (2
S’oé’é’oé’ 53’ as: .43" §
0 Q Q Q Q Q Q Q
or—NOR -,
31 kDa
or—GUS TR:- ..........-.,....... H.-. -31. __ «non: *
73 kDa specrfic

*The signals at 24 hours are non-specific, however those at 48 and
72 hours are GUS specific.

65

 

 

Figure 13. Analysis of GUS activity and aflatoxin production in transformant D8D3 and
control strains. Mycelia were harvested (see methods) at 24, 48, and 72 h after
inoculation in aflatoxin-inducing medium (YES broth). A) One pg of protein from each
mycelia sample was used to determine GUS activity. The substrate for GUS analysis was
4-methylumbelliferyl-B—D-glucuronide. B) Culture filtrate was used for the

determination of aflatoxin levels. For the control strains, the points on the graph
represent data from a single culture. D8D3 was grown in triplicate and the error bars

indicate the standard error.

66

 

 

 

 

 

 

 

 

 

88:-mz IoI
~2u<0&.-mz III
848..-”? 1.1
Tmz Lei

#008

10000?

I Doom?

I oooom

 

 

000mm

(Iw/fiu) urxomuv

 

 

 

   

88.5.2 IOI
~2u<o&.-mz III

«33.4.2 Ibl
fizz lei

 

row

19

ION

row

 

 

(nw-v BwIUIw/seIowu) Asimov sno

67

NR-l/pGAPNZ extracts at any time point although the presence of uidA transcript and
GUS activity were established in these cultures. Northern hybridization analysis
indicated that the level of uidA transcript and GUS activity were relatively low in the
pGAPN2 transformant during the course of the study, and apparently the Western
technique lacks the sensitivity to detect this low yield of GUS protein.
The GUS protein was only identified in D8D3 at the 48 and 72 h time points. A
protein with similar size, but slightly larger than GUS, displayed cross-reactivity with a-
GUS and was seen with the 24 h samples in Figure 128 and 12D. The lack of uidA
transcript and GUS activity in D8D3 at these tirnepoints supports the interpretation of this
signal as nonspecific protein.
The Nor-1 protein was identified in all samples at 48 and 72 h after inoculation.
The expression of Nor-1 was weakest in the NR-l/pGAPN2 strain, and although the
representation of the data in Figure 12A failed to show this weak expression, the Nor-1
protein in NR-l/pGAPNZ could be identified in the original blot. The low level of Nor-1
in NR-l/pGAPNZ is probably due to the weak transcription of nor-I demonstrated in
Figure 11A. Figures 12C and 12D demonstrate the consistency of D8D3 translation from
replicate cultures.
Time Course Analysis of NR-l/D8D3 Protein Activity
Nor-1 protein activity was indirectly assayed by measuring aflatoxin
concentrations. Both aflatoxin (Table 3C and Figure 138) and GUS activity (Table 3B
and Figure 13A) were first detected in D8D3 after 48 h of fungal growth. This is
consistent with the accumulation of nor-1 and uidA transcripts (Figure 11) and Nor-1 and
GUS proteins (Figure 12) from D8D3 mycelia samples. Although GUS activity in D8D3

increased slightly fi'om 48 h to 72 h, a greater increase in aflatoxin levels during this

68

 

Table 33. Time course analysis of transformant D8D3. The GUS activities from the
mycelia of D8D3 and control strains were determined at 24, 48, and 72 h. D8D3 was
tested in triplicate while only single cultures of the control strains were analyzed. The
data were reported in nanomoles 4—methylumbelliferone/min/mg of protein.

 

 

 

 

 

 

 

 

 

 

Strain 24 hours 48 hours 72 hours
NR—l No detection No detection No detection
NR-l/pSL82 No detection No detection No detection
NR-l/pGAPNZ 1.39 1.89 1.47
NR-l/D8D3A No detection 21.4 21.1
NR-l/D8D3B No detection 20.4 20.1
NR-l/D8D3C No detection 20.7 28.8

 

 

 

Table 3C. Time course analysis of transformant D8D3. The aflatoxin concentrations in
the filtrates of cultures of D8D3 and control strains were determined at 24, 48, and 72 h.
D8D3 was tested in triplicate while only single cultures of the control strains were
analyzed. The data were reported in ng/ml as determined by direct competitive ELISA.

 

 

 

 

 

 

 

 

 

 

 

Strain 24 hours 48 hours 72 hours
NR-l < 50 3,440 13,512
NR-l/pSL82 < 50 680.3 2,186
NR-l/pGAPNZ < 50 233.8 369
NR-l/D8D3A < 50 5,367 25,015
NR-l/DSDBB < 50 5,141 20,256
NR-l/DSD3C < 50 3,974 18,474

 

 

period was observed. This apparent discrepancy in proportion is likely due to the
stability of aflatoxin and its continuous production from active enzymes, and would
probably be absent if Nor-1 activity was measured directly. The pSL82 and pGAPN2
transformants were relatively weak producers of aflatoxin, as might be expected when
comparing the nor-1 transcripts (Figurel 1) and Nor-1 proteins (Figure 12) of these strains
with those of D8D3 and NR-l.

GUS activity was detected in NR-l/pGAPN2 at all three time points. At 48 h
after inoculation, D8D3 expressed 11 times more GUS activity than GAPN2 and at 72 h,

D8D3 expressed 48 times more activity. Since uidA transcript levels were similar in the

69

D8D3 and GAPN2 transformants (Figure 11B), the lower level of GUS activity in
GAPN2 is likely the result of relatively inefficient translation of the [3-tubulin fusion
protein (Figure 12B). GUS activity was absent in NR-l and NR-l/pSL82 at all time
points.

Table 4. GUS phenotypes of pAPGUSNNB transformants on media supportive (YES)
and non-supportive (CZ) of aflatoxin production. Qualitative GUS assays (see Methods)

were performed on fungal colonies after 48 h of grth on both types of media. Site of
vector integration is indicated rn parenthesis.

 

 

 

 

 

 

 

 

 

 

Strain YES CZ

D8D1 (nor-I) GUS+ GUS’
D8D3 (nor-1) GUS+ GUS‘
13804911210) GUS‘ GUS'
D8D6 (nor-1) GUS+ GUS’
D8E5 (niaD*) GUS' GUS‘

 

Table 5. GUS activities in NR-l transformants. Mycelia from each of the strains were
harvested at 48 hrs after growth in aflatoxin inducing medium (YES broth). One mg of
protein from each sample was used to determine GUS activity. The data were reported in
nanomoles 4-methylumbelliferone/min/mg of protein. The GUS activities for the
transformants that are presumably genetically identical to D8D3 were reported in an
experiment separate from the analysis that produced the GUS activity data for the D8D4
and D8D6 strains. Transformant D8D6 has the GUS reporter fusion located in the 5’
region of nor-1 whereas D8D4 has the reporter integrated at niaD (Figure 5).

 

 

 

 

 

 

 

 

 

Strain Sample A Sample B Mean

D8D6 78.6 65.2 71.9

D8D4 No detection No detection NA
D8D3 (50) 19.6 19.0 19.3
D8D3 (59) 20.4 16.6 18.5
D8D3 (61) 16.0 19.2 17.6
D8D3 (68) 15.7 14.6 15.2
D8D3 (85) 11.0 12.5 11.8

 

 

 

 

 

7O

 

DISCUSSION

Biological strategies for eliminating pre-harvest aflatoxin contamination of crops
are directed toward the development of atoxigenic fungal strains or toxin-inhibiting
plants, which will be successful in the agricultural environment. The long-term success
of this type of strategy requires an understanding of the plant-fungal relationship. The
interaction between plant and fungus can be efficiently monitored with reporter gene
fusions as has been demonstrated in other plant-pathogen relationships (Couteaudier et
al., 1993; Maor et al., 1998; Olivain and Alabouvette, 1997; Spellig et al., 1996). With
regard to aflatoxin elimination, the ability to assay for aflatoxin reporter enzymes in plant
tissue inoculated with fungal spores should allow for rapid screening of compounds
and/or plant strains with the ability to impair toxin production. Experiments involving
mutagenesis of the promoter region of reporter constructs should also aid in identifying
cis-acting regions and trans-acting factors essential for the regulation of a gene of interest.
Knowledge of aflatoxin regulatory control mechanisms should prove valuable in the
development or identification of anti-toxin compounds. A prerequisite of such reporter
studies, is the analysis of fusion expression to confirm that the wild-type gene of interest
and the derived reporter construct have a similar pattern of regulation.

Our experiments have demonstrated that the nor-1 luidA reporter construct and the
wild-type nor-I gene are regulated in a similar manner in strain D8D3. Under aflatoxin-
inducing conditions, the timing and pattern of transcript and protein accumulation from
the expression of these two genes were similar (Figure 11 & 12). Data from the same
time course experiment also showed that the timing of aflatoxin production and GUS

activity was identical (Figure 13), while the pattern of aflatoxin accumulation was

71

consistent with the levels of GUS activity at 48 and 72 hrs (i.e. relatively high GUS
activity precedes high levels of aflatoxin production). Furthermore, when qualitative
GUS assays were performed on a medirun not supportive of aflatoxin production (CZ),
GUS activity in D8D3 was not detected (Table 4). To demonstrate the reproducibility of
GUS expression from transformants that are presumably genotypically identical to D8D3
(Figure 7), selected isolates were cultured under identical growth conditions and then
were assayed for GUS activity (Table 5). The difference between strains did not exceed
65% GUS activity (strains 50 & 85). The overall data support the use of the nor-I/uidA
fusion as a reliable reporter for nor-1 expression and for nor-1 promoter analysis.

Our data have also suggested the presence of a position-dependent expression
mechanism Operating in the nor-1 region of the A. parasiticus chromosome. The absence
of detectable GUS activity in 77% of pAPGUSNNB transformants (Table 2) had
indicated the possibility of such a mechanism. Subsequent genetic characterization of
GUS" transformants (thirteen) showed all had reporter fusion integration into the nor-1
locus (Figures 5 and 7), while GUS' transformants were found either to be double
crossover integrations of the selectable marker (four), or pAPGUSNNB integrations into
the niaD chromosomal region (three) (Figures 5 and 8). The lack of GUS activity in
transformants with the reporter construct located outside the nor-1 region supports the
existence of a position-dependent expression mechanism. Studies with a ver-I/ur’dA
fusion (Liang et al., 1997) have also demonstrated position-dependent expression of an
aflatoxin promoter, and together the data indicate that the phenomenon may apply to a
substantial area of the aflatoxin gene cluster. However, in both of these studies the GUS'
phenotype in single crossover recombinations, was the result of vector integration in the

m'aD chromosomal region. This locus has been shown to be both positively and

72

negatively regulated in A. parasiticus (Chang et al., 1996), thus lack of GUS expression
in this region could be the result of local regulatory factors. These results indicate, that it
is critical for future nor-1 /uidA expression experiments to have integration of the
pAPGUSNNB vector be targeted to the nor-I region of the NR-l chromosome.

The targeting of reporter constructs to specific chromosomal locations for
promoter analysis has been advised by other investigators wary of position-dependent
expression of fungal genes (van Gorcom er al., 1986; Hamer and Timberlake, 1987;
Tirnberlake and Marshall, 1989), but the mechanisms responsible for the phenomenon
have not been elucidated. It has been hypothesized that enhancer elements may be
responsible for the position effect (Kinsey and Rarnbosek, 1984). The presence of this
type of positive cis-acting factor having regional control over transcription of the
aflatoxin gene cluster is plausible, since removal of aflatoxin reporter fusions from the
cluster results in the GUS’ phenotype. However, experiments involving the SpoCl gene
cluster (SGC) in A. nidulans (Miller et al., 1987) suggest that position dependent
expression of ftmgal genes may not be so simply explained.

In this work, removal of a cluster gene to novel chromosomal positions resulted in
elevated cluster gene transcription in certain cell types. The authors hypothesized that a
repressive cis-acting element with activity over large distances was a possible explanation
for their data. This argument was supported when a constitutively expressed gene was
positioned at different locations within the SGC and a reduction in transcript levels was
detected. The possibility of a negative regulatory mechanism, similar to the SGC model,
operating in the aflatoxin gene cluster is unlikely, since our experiments have shown an
opposite effect (i.e. reporter expression occurs when the fusions are localized to the

cluster, and the selectable marker (niaD), when placed in the cluster, is expressed without

73

 

 

 

any detectable difference in morphology between transformants with different fusion
integration sites).

The study by Miller et a1. (1 98 7) also postulated that chromatin conformation was
a factor in the repressive effect on cluster genes. This was based on the observation that
genes at the border of the SGC were transcribed more efficiently than centrally located
genes under repressive conditions. If such a conformational control mechanism exists in
the aflatoxin gene cluster, it has to be reconciled with experiments by Flaherty and Payne
(1997) that showed that by constitutively expressing aflR (positive aflatoxin regulatory
gene) under aflatoxin inducing and non-inducing conditions, aflatoxin biosynthetic genes
were transcriptionally activated. In the F laherty and Payne (1997) experiments, an aflR
structural gene was fused to a constitutively expressed promoter and then introduced into
A. flavus strains. The chromosomal position of the aflR construct was not reported in this
study.

The placement of an extra copy of aflR (Chang et al., 1995) and the constitutive
expression of aflR (F laherty and Payne, 1997) in the A. flavus chromosome both appear
to overcome the repressive effect of nitrate on aflatoxin gene transcription. However
F laherty and Payne demonstrated that under these derepressing conditions, aflatoxin
production in nitrate containing media was still dramatically decreased. Again, the
position of the integrating aflR vectors was not reported in these studies. When
considering a chromatin conformation control mechanism for the aflatoxin gene cluster, it
is interesting to note the central position of the aflR regulatory gene in the cluster. A non-
coordinately expressed cluster gene (L8B) (Miller etal., 1987) can also be found in a
central location in the SGC, and its pattern of expression indicates it may too serve a

regulatory role in cluster transcription. Additional studies placing aflatoxin reporter

74

fusions at noncluster sites other than niaD are being performed in our laboratory to
confirm the position-dependent expression of aflatoxin genes. Also, the construction of
an aflR/ur’dA fusion and analysis of its expression at different chromosomal locations
under aflatoxin inducing and repressing conditions is being planned, and should aid in
elucidating aflatoxin regulatory mechanisms.

The targeting of the nor-1 /uidA reporter fusion to the 3’ nor-1 region, as opposed
to the 5’ nor-1 region, will also be important to future reporter expression analysis.
Transformants with 3’ nor-1 integrations of pAPGUSNNB will more likely maintain the
integrity of any in vitro modifications of the nor-I/uidA promoter during recombination.
For example, if a mutation in the nor-Ilur'dA promoter is to be evaluated in the fungus,
the position of a 5’ nor-1 integration of the vector could result in the mutated promoter
regulating nor-I expression instead of uidA expression. A 3’ nor-1 integration would
avoid this potential outcome. Two screening assays were developed to aid in the
identification of transformants with 3’ nor-1 pAPGUSNNB integrations. The first is a
qualitative GUS assay (Figure 3) adapted from the literature (Kolar et al., 1991; Verdoes
et al., 1994) and can be used as an initial screening assay to identify transformants with
integrations in the nor-I locus. This assay takes advantage of the position-dependent
expression of the nor-1 /uidA fusion in the NR-l chromosome and should eliminate 75%
(Table 2) of potential transformants from further analysis.

The remaining transformants will likely have pAPGUSNNB integrated in either
the 5’ nor-1 or 3’ nor-1 chromosomal regions. A novel PCR assay (Figures 4A & 4B)
was developed to identify transformants of both types and is designed with one primer
specific to plasmid DNA and one primer specific to chromosomal DNA. In this study, all

transformant types identified by PCR (Figure 9) were confirmed by Southern

75

hybridization analyses (Figures 5 and 7).

We conclude that the expression of the nor-1 /uidA reporter fusion is similar to the
expression of the wild-type nor-1 gene in A. parasiticus, and can therefore be used as a
reliable indicator of nor-1 expression and aflatoxin biosynthesis. In addition, the
qualitative GUS assay and PCR assay developed in the course of this research can be

adapted for further use in the study of position-dependent expression of fungal genes.

76

 

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