abundance DNA Aspetgillus flavipes...
Transcript of abundance DNA Aspetgillus flavipes...
Distribution, abundance and analysis of polymorphic microsatellite DNA in Aspetgillus flavipes and Pjdhium ulomum
Marcel Alexander Femandez
A thesis submitted to the Faculty of Graduate Studies
in partial fulfilment of the requirement for the
degree of Doctor of Philosophy.
Department of Microbiology
University of Manitoba
Winnipeg, Manitoba
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ABSTRACT
The objective of this research project was to develop tools for strain
identification in the fungi Pythium ultmum and A s ~ l l u s flavipes- The former
is a well-known plant pathogen, and the latter is important in the pharmaceutid
industry. The principal drive was to identify genetic loci wntaining simple
sequenœ arrays (microsatellites) which could be amplified from nurnerous
isolates to reveal polymorphisms. but the RAPD approach was also used for
Aspergillus tla vipes.
A genomic library of P. ultimum (BR471 ) was screened with d(GT)* and
d(CT)g probes and from the data it was estimated that a d(GT/CA) motif occurs
at least once every 86 kb and that a d(CT/GA) motif ocairs at least every 137 kb
on average. A clone hybridizing to the d(GT)g probe was chosen, and
sequenced to reveal a 200 bp region with five d(GT/CA). motifs interspersed
with unique and repeütive sequenœs. Primers complernentary to flanking
sequences were employed to amplify the regions from afl P. ultimum var.
ultimum isolates tested (25) and from one isolate P. ultimum var. sparangiiferum.
Four other species of Pythium tested did not produœ amplification products.
Many length polymorphisms were detected in the amplification products of al1
isolates tested. Three of these isolates w r e charaderized by sequencing the
polymorphic region. Variance in the number of d(GT/CA) dinucleotides as well
as a deletion extending into the sequence flanking the d(GT/CA) array was
O bserved.
For strain differentiation in Aspergillus flavipes, amplification of genomic
DNA *th single random prirnen (RAPD) produced unique sets of
eledrophoretic profiles for each of the nine isolates of the species. Thirteen
primers were used in the study. The assay was show rot to be very sensitive
to the purity, age or the concentration of template DNA, but it was sensitive to
the temperature profile protocol, Mich cannot be adequately reproduced from
one PCR instrument to another. Reproducible profiles for each isolate showed
extreme polymorphism with very few coincident bands between pairs of isolates
and none shared by al1 nine isolates.
The genome of A. flavipes was investigated using hybridization of a
genomic library w-th four synthetic oligonucleotide probes d(GT)g, d(CT)a,
d(AT)s, and d(GC)s. Results from genomic dot blots showed the existence of
abundant d(GT1CA) and d(CTIGA) simple sequence motifs, but no apparent
d(ATKA) or d(GC/CG). Self hybridization of the later probes may have interfered
with the screening.
Hybridization of the probes to Southem blots of restriction profiles of
genomic DNA revealed that the simple sequence motifs are widely dispersed in
the genorne, that their distribution is highly polymorphic, and that some of them
may be members of repetitive DNA families.
In order to assess the feasibility of using simple sequenœ motifs
as genetic markers in A. flavipes, D M Aagments from library clones Hihich
hybridized to the simple sequence probes d(GTb and d(CT)* were cloned and
iii
sequenced. Primers Ranking the simple sequenœ repeats were synthesized
and used in the polymerase chain reaction for amplification of two d(GT/CA) and
two d(CT1GA) motifs. Sequencing of the polyrnerase chain reaction products
showed that d(GT1CA) and d(CTIGA) loci are polymorphic as a cansequence of
site specific length variation lacated within the dinucleotide repeat. Alignment
of the sequenœs obtained for each locus showad that the flanking regions of
these motifs are highly conserved. Howver, minor difFerences in sequence
homology were identified in the regions flanking the simple sequence motifs. Of
the nine isolates studied, only three produœd a major PCR product. The
ineffediveness of the primer pairs to amplify similar locus among other isolates
may have arisen from differences in the flanking regions-
One micmsatellite locus contained a ~(GTICA)~~J repeat. This repeat
showed the largest degree of length variation in comparison to the other loci
investigated. These results support the hypothesis that long simple sequence
repeats show a greater amount of length variation in contrast to short motifs
M ich show less or none. In another simple sequenœ loci, two d(CT1GA) motifs
w r e found flanking a d(GT1CA) motif. Sequence comparison of this locus to
that in other isolates of A. flavipes showed that polymorphism of a simple
sequenœ motif can include its flanking regions. Usually dinucleotide
polymorphisms are confined to their repeated units, but in this locus the flanking
regions can conhibute to simple sequence polymorphism.
This is the first report of the amplification of simple sequence motifs in a
fungus. The high degree of variation exhibited by these isolated loci
demonstrate the value of simple sequenœs in distinguishing isolates of A.
flavi@es and P. ultimum. The abundance and the amount of information derived
from these types of markers together with the ease by which they can be
identified make them ideal markers for genetic linkage studies, physical
mapping, population studies and varietal identification-
ACKNOWLEDGMENTS
Este trabajo esta dedicado a mi padre Dr. Raul O. Femandez, sin ti est0 no
seria posible, te quiero mucho. Great appreciation to Dr. Gien Klassen M o
provided a creative working environment, tremendous amount of helpful
comments and making this work possible. I also wish to thank the members of
my cornmittee, you have provided important commentary to help accomplish my
research. I thank al1 faculty of the department, you have made me feel very
welwme. Thank you, fellow students and staff, you al1 provided a kind and
wann wrking place. My experience here was most positive, fruitful, and exciting.
TABLE OF CONTENTS
Page
..................................................................................................... ABSTRACT.. ,. i
.............................................................................. ACKNOWLEDGMENTS .... .. .v
........................................................ ......-............-...... TABLE OF CONTENTS ... vi
............................................................................................. LIST OF TABLES. -x
LIST OF FIGURES ............................................................................................ xi
............................................................................. LIST OF ABBREVIATIONS.. xiv
INTRODUCTION ........................-...................................................................... 1
.................................................................................. LITERATURE REVIEW-.. 4
.......................................................................................... Introduction ..-5
.............................................................. Survey of molecular markers. --.7
..................................................... Minisatellites and simple sequenœs 12
Properties of mini and microsatellite DNA sequences .......................... 14
Localization of mini and microsatellites .................................................. 16
Mechanisms accountable for minisatellite and microsatellite
polymorphisms ........................................................................ 1 7
................................................ Arnplified microsatellites.. - 1 8
Functional importance of minisatel lites and simple sequemes.. ........... -21
................................................................................... Telomers.. .21
.............................................................................. Centromeres.. -22
. . Transcn ption.. ..... ., ....................................................................... 22
. . Transcnptional regulation ............................................................ 23
............................................................................ Recombination -23
. . .................................................................................. Replication -24
................................................................... Molewlar markers in Fungi -25
.......................... Classification of the genus Pythium ....A
................................................. Classification of the genus Aspergillim 36
................................................... PCR-amplified microsatellites in fungi 39
............................ MATERIALS AND METHODS ......... .. ... ... .. ................ -40
Pythium ultimum strains ......................................................................... 41
Aspergillus flavipes strains .................................................................... -41
.......................................................... .............. Culture methods ..... -46
.............................................. Genomic DNA extraction and purification -46
RNAse treatment of nucleic acid preparations for RAPDs .................... -48
................................. Oligonucleotides probes and sequencing primers 48
........................................................................... RAPD PCR conditions -51
DNA dot blotting and fixation of DNA to nylon membranes .................... 52
............................................. DNA digestion and elechophoresis ...... .. 52
5' end labeling of oligonucleotides ... ... ............................................ -53
3' end labeling of oligonucleotides probes using
.................................................................................... digoxigenin UT? -53
Southem blotting and hybridizations ..................................................... -54
Construction of the Pythium uîümum and Aspefgillls flavipes
. . ...................................................................................... genomic Iibrary 55
Amplification of the EMBL3 genomic Iibrary .......................................... 59
...................................................... Plating the EMBL3 genomic library -60
EMBL3 genomic library plaque blotting ................................................. 60
. * ................................................................... Selection of positive clones -61
Large scale isolation of phage DNk ...................................................... 62
Subcloning DNA fragments from phage clones into
pBluescript plasmid (Ml3 Ks +) .............................................................. 63
Cloning of PCR products ........................................................................ 65
Purification of plasmid DNA ................................................................... 65
Screening clones with PCR ................................................................... -66
Construction of deletion clones ............................................................. -67
Fragment amplification of simple sequence motifs ................................. 68
......................................... Sequencing cloned PCR products ........... .. -68
Sequencing of PCR products ............ ... ............................................ -69
DNA sequenœ analysis ........................................................................ -71
RESULTS AND DISCUSSION ........................................................................ 73
CHAPTER 1 . Simple Sequence Motifs in P . ultimum ........................... 73
Introduction.. ......................................................... -74
.................................................................. Results -76
............................................................ Discussion -92
CHAPTER 2 . Use of RAPDs to DifFerentiate lsolates of
....................................................................... A . flavipes 97
......................................................... Introduction -98
................................................................ Results -100
......................................... .......... Discussion ....... -114
CHAPTER 3 . Simple Sequence Motifs in A . flavipes ........... ~SSSSSSSSSSSSSSS1 18
Introduction ............... ..... ................................... 119
................................................................. Results 121
Discussion .......................................................... 1 6 7
CONCLUSIONS .............................................................................................. 173
...................................................................................... LITERATURE CITED 180
UST OF TABLES
INTRODUCTION
........ Table 1 : Hybridization based DNA fingerprinting studies in fungi 27
................... Table 2: PCR based DNA fingerprinting studies in fungi 3 0
MATERIALS AND METHODS
Table 3: Strains of Pythium .................................................................. 42
.............................................. Table 4: Strains of Aspergiilus flavipes -45
........................... Table 5: Oligonucleotides and sequencing primers -49
................................... Table 6 . Primers used in RAPDs experirnents -50
........ Table 7: DNA motifs used in sequence analysis of cloned DNA 72
CHAPTER 1
Table 8: Primers for amplification of simple sequenœ motifs .............. -80
Table 9: Sequence analysis of cloned DNA from P . ultimum .............. 81
CHAPTER 2
Table 1 O: Results of working prïmen used in RAPDs
........................................................................... A . flavipes 103
CHAPTER 3
Table 1 1 : Simple sequenœ motifs in S . cemvisiae. ................ .. .. 131
Table 12: Primers for amplification of simple sequence motifs ........... 132
Table 13: Plasmid constnicts of cloned PCR products ............ .. ........ 133
Table 14: Sequence analysis of pdGT1, pdGT2, pdCT1, and
........................................ .................... ...... pdCT2 ... ...... 134
LIST OF FIGURES
CHAPTER 1.
Fig. 1. Restriction digest of lambda DNA clone and
................................................ southern blot analysis.. -84
Fig. 2. Nucleotide sequence of DNA from P. ultimum
containing several d(CA/GT) simple sequence
repeats.. ........................................................................ .û6
Fig- 3. Polyacrylamide gel eledrophoresis of PCR products
.................. ............ from various isolates of P. ultr'rnum ... 88
Fig . 4. Pol yacrylamide gel electrophoresis of PCR products
frorn various isolates of P. ultimurn ............................... 90
Fig. 5. DNA sequence alignment of PCR products from
.............................................. 3 isolates of P. u/timurn,.. -92
CHAPTER 2.
Fig. 6. The effect of old and newly prepared DNA
template from A. flavipes in RAPDs .............................. 104
Fig. 7. The effect of RNAse treated and non-RNAse treated
.................. DNA template from A. flavipes in RAPDs. -1 06
Fig. 8. The effect of concentration of template DNA
from A. flavipes in RAPDs.. ....................................... .1 08
F ig. 9. RAPDs DNA analysis in difFerent isolates of
........... A. flavipes. ,.., .................................................. 1 1 0
Fig. 10. RAPDs DNA analysis in different isolates of
A. flavi,pes. ......-.. S.SS.S..S...SS.S...SSSSS.S....S -....--..-.....-....-..--... 112
CHAPTER 3.
Fig. 11. Autoradiogram genomic dot blot hybridizations with
d(GT)9 and d(CTl9 probes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -1 35
Fig. 12. RFLP analysis of isolates of A. flsvipes wïth
d(GTI9 and d(CT)9 probes.. . . . . . . . . . . . - - -. -. .. . -. . . . ..---.. -... - -. . . -1 37
Fig. 13. Restriction digests of lambda DNA clones and
hybridizations to d(GVs probe ..............---..--.---.-......... 139
Fig. 14. Restriction digests of lambda DNA clones and
hybridizations to d(CT)s probe,.. .-.-. .... . ... .. . . --..-.. . .--. .-. ... 141
Fig. 15. Plasmid constnicts containing lambda DNA
inserts with simple sequenœ DNA ....... ..... .. ...... ...... 143
Fig. 16. Sequence of plasmid pdGT1 containing d(GTICA)
motif ...... . . ....... ..... .... .... ....... .... . ...... ... .. . ... . . .... . ... . .... ....... .. 145
Fig. 17. Sequenœ of plasmid pdGT2 containing d(GT/CA)
mot if... ... . . . .. ... .-,... . . . . . ..,,. .-.... .. . . . . . . . . .. ... . .. . . . . . . . . . . . . . . . . . . . . . .. . -147
Fig. 18. Sequenœ of plasmid pdCTl containing d(CTIGA)
motif. ... ..... ..... ....... .,...,.. ..,......,....-... ..... ... ...... . ..... .... .... ... 149
Fig. 19. Sequenœ of plasmid pdCT2 containing d(CT1GA)
motif ........................................,..................-.. ................. 151
Fig. 20. PCR amplification of GT1 and GT2 simple sequenœ
motifs in different isolates of A. flavipes and
hybridization to d(GT)9,. ................................................. 1 53
Fig. 21. PCR amplification of CTI and CT2 simple sequence
motifs in different isolates of A. flavipes and
hybridization O d(Cvg .................................................. 1 55
Fig. 22. Plasmid constnicts of cloned PCR products containing
simple sequence motifs ................................................. 157
Fig. 23. DNA sequence alignment of GT1 simple sequence
............................................................................. motifs f 59
Fig. 24. DNA sequenœ alignment of GT2 simple sequence
........................................................................... motifs.,. 161
Fig. 25. DNA sequence alignment of CTI simple sequenœ
........................................................................... motifs.. -1 63
Fig. 26. DNA sequence alignment of CT2 simple sequenœ
.............................................................................. motifs 165
xiv
A
bp
C
CTAB
cm
DNA
dATP
DMSO
dNTP
EDTA
G
9
h
l PTG
kb
L
mg
min
mL
mm
mM
adenine
base pairs
cytosine
hexadecyltrimethyl ammonium bromide
œntimetre
deoxyri bonucleic acid
Z-deoxyadenosine 5'-triphosphate
dimethyl sulfoxide
Zdeoxyribonucleoside 5'4riphosphate
eth y lenediamine-tetra-acetic acid
guanine
gram(s)
hour(s)
isopropylthiogaladoside
kilobase pairs
litre(s)
milligram(s)
minute(s)
millilitre($)
millimetre(s)
mil timolar
nm
PCR
pfu
pmole
RAPD
RFLP
rDNA
RNA
'Pm
SDS
SSC
T
ug
UL
um
UV
v/v
w/v
X-gai
Y
nanomette
polymerase chain reaction
plaque foming unit(s)
pico mole@)
random amplified polymorphic DNA
restriction fragment length polymorphism
ribosomal DNA
ribonucleic acid
revolutions per minute
sodium dodecyl sulfate
sodium saline citrate
thymine
m icrograrn(s)
micro1 itre(s)
m icrometre(s)
ultraviolet
volume/volume
weiçht/volume
5-bromo-4-chloro-Sindol yl-b-D-gaIaÇtoside
Cytosine or Thymine
Introduction
The research presented here describes the isolation and characterization
of DNA molewlar marken in fungi. In partiwlar, the examination of
microsatellite DNA type sequences will be investigated. The organisms used in
these experïments (Pythium ultimum and Aspetgîllus fla vipes) have important
economic and environmental impacts in our environment The research
conducted in this thesis is important for several reasons: (1) To date no research
has been conducted to determine if microsatellite DNA sequences in fungi are
polymorphic; (2) Little evidenœ conœming the behaviour, composition and
abundance of microsatellite DNA sequences have been addressed; (3) and the
analysis of a large groups of fungal isolates using microsatellite DNA markers is
rare.
Only recently has the use of ONA molewlar markers gained much
attention. With the advent of these new molewlar markers, strategies for
investigating the genome of organisms have been developed. New techniques
have helped detennine genome characteristics (Le. quantitation of repetitive
DNA) and assisted in the development of human and mouse genome maps (Dib
et al. 1996). The research presented here is a step towards the understanding of
general characteristics of microsatellite DNA sequences in the genomes of
Pythium ulümum and Aspetgillus flevipes.
The objectives in this thesis are: (1) to isolate microsatelfite DNA
sequences, (2) to examine the polyrnorphic nature of microsatellite ONA, and (3)
to quantify microsatellite DNA sequenœs in P. ulamum and A. flavipes. An
additional related objective will be to examine the use of npid amplified
polymorphic DNA (RAPD) as another approach for differentiating isolates of A.
t7a vipes-
introduction
Examining the relatedness and genetic diversity between or within
different species, populations, and individuals is a main chore for numerous
disciplines of biological science. In the past, classical strategies of assessing
genetic variability such as comparative anatorny, morphology, ernbryology and
physiology have been supplemented with molecular techniques. These
encompass the characterization of chernical constituents (e-g. plant metabolites)
and, most importantly, the analysis of rnacromolecules. Development of
"molecular markers," that are based on polymorphisrns found in proteins or DNA,
has exceedingly simplified the investigation in a variety of disciplines such as
taxonomy, ecology, phylogeny, genetics, and plant and animal breeding
programs. Morphological characters have long been applied to identify
species, famil ies, and genera (Fal wner 1 981, Epplen et al. 1 991 ). Unlike
molecular markers, morphological characters are often firmly influenced by the
environment, and consequently, special breeding programs and experimental
designs are required to distinguish genotypic from phenotypic variation.
For the most part, allozymes have been the molecular markers of choice,
but attention has increasingly œntred on the DNA molecule as a source of
informative polyrnorphisrns. Due to the high information content of the DNA
sequence, sequenœ information can be exploited for the study of genetic
divenity and relatedness arnong organisrns. A wide diversity of techniques to
visualise DNA sequence polyrnorphisms have been developed in the past few
years, and molecular markers have been derived from these techniques.
The temi "DNA fingerprinting" was introduœd by Jeffreys et al. (1985), to
denote a tedinique for the simultaneous detedion of many highly variable DNA
loci by hybridization of particular muitilocus "probesaa to electrophoretically
separated restricted DNA fragments. Several alterations of the basic technique
have appeared and comparable strategies have been developed. Most
importantly, DNA polymorphisms became detectable by use of the polymerase
chain reaction (PCR). Some methods are still tened DNA fingerprinting, but
the tens "DNA profiling," and "DNA typing" are also being used.
There are particular properties which would be generally desirable for a
molecular marker. The following list shows some of the important features for
molecular markers:
1. Highly polymorphic behaviour
2. Codominant inheritance (penits discrimination betwen homo and heterozygotic states in diploid organisms)
3. Repeated occurrence in the genome
4. Even distribution throughout the genome
5. Selectively neutral behaviour (Le. no pleiotropic effects)
6. Easy access
7. Fast and easy assay
8. High reproducibility
9. Cost effective
No molewlar marken are obtainable yet that fulfill al1 of these criteria, but *th
the availability of nurnerous different types of marker systems, most of these
mentioned aiteria can be accomplished (Eppien et al. 1993)
Survey of molecular markers
Protein markers are a frequently used in molecular techniques. The
procedure involves the electrophoretic separation of proteins followed by
specific staining of a distinct protein subclass. While some studies utilize
protein patterns, more than half of protein markers are represented by
allozymes.
Allozyme electrophoresis has been successfully used in many organisms
from bacteria to numerous animal and plant species since the 19608s, and has
been reviewed in detail by May (1992). However, there are a number of
limitations to allozyme studies. With allozymes, a new allele will only be
detected as a polymorphism if a nucleotide substitution is a consequence of an
amino acid substitution, which in its tum affects the eledrophoretic mobility of
the studied molecule.
Polymorphisms at the DNA level may be studied by several means. The
most direct approach is the detemination of the nucleotide sequence of a
defined region and the alignment of this sequence to an orthologous region in
the genome of a similar related organism (Hillis et al. 1990). The informative
analysis of this data Gan be adapted to distinct levels of discriminatory potential
by choosing appropriate regions of the genome. This type of analysis is largely
applied for evaluating medium and long distance relatedness in phylogeny, but
occasionally it is also utilized for population studies (Hoelzel and Green 1992).
The sequencing approach has been extremely facilitated by the advent of
PCR (Saiki et al. 1988), which makes it feasible to isolate homologous DNA
sequences from many organisms. Primen are developed on the basis of
sequence information for conserved parts of the DNA, and the desired target
sequences are arnplified. The PCR produd is sequenœd directly or after
cloning (Hoelzel and Green 1992).
An alternative means for assessing DNA sequence variation is the
analysis of restriction fragment length polymorphisms (RFLPs). Digestion of a
particular DNA molewle with a restriction enzyme results in a reproducible
group of fragments of defined lengths. Point mutations wthin the recognition
sequence of the restriction enzyme utilized as well as insertions or deletions wi-Il
result in a changed pattern of restriction fragments and may bring about a
screenable polymorphism among different genotypes. Hybridization based
fingerprinting, which actually depicts a particular case of RFLP analysis,
involves the digestion of genomic DNA ~ Ï t h restriction enzymes and separation
of the fragments using electrophoresis on a gel. This gel is Southem blotted on
to a membrane and particular fragments are made visible by hybridization with a
labelled probe (Southem 1975). Two primary differenœs exist between the
RFLP techniques and hybridization based fingerprinting: (1 ) DNA fingerprinting
utilizes multilows probes, creating complex banding patterns, whereas RFLP
probes are generally locus specific. (2) DNA fingerprinting is customarily
perfomed with non species-specific probes that identify ubiquitous ocairri-ng
sequences such as minisatellites, whereas RFLP probes are usually species-
specific
RFLP anal ysis of nuclear 0 NA common l y uses s pecies-s pecific probes
which are obtained from a cDNA or genomic Iibrary of the investigated species,
or a close relative. RFLPs have been identified and employed in molecular
marker assisted selection in breeding programs and map based cloning of genes
(Nienhuis et al. 1987, and Tanksley et al. 1989). Additional application areas for
RFLPs are phylogenetic studies and cultivar identification (Gebhardt et al. 1989,
Rajapakse et al. 1992, Debener et al. 1990, Dowling et al. 1990, Song et a'
1 988). In addition to probes generated from cDNA or genomic library clones,
ribosornal DNA (rDNA) (wding regions 18s. 5.8s and 25s) is frequently used as
a source for RFLPs. Sinœ polymorphisms in rDNA are easy to detect due to the
high abundance of these sequences the same probes can often be utilized in
different species because of their conserved coding sequences. Many reports
have shown the use of these sequences as a good source of DNA
polymorphisms (Hamby and Zimmer 1992, Kim and Marby 1991, Leam and
Schaal 1 987, Nybom et al. 1 992, Saghai-Maroof et al. 1 984, Buchko 1 996).
Other studies involving RFLPs make use of mitochondrial DNA (mtDNA)
in animals and chloroplast DNA (cpDNA) in plants. 60th types of DNA are
present in several to hundreds of copies per cell. There are hm principal
approaches for studying RFLPs in cytoplasmic DNA The first is to extract
mtDNA and cpDNA separately from the nuclear DNA (Milligan 1992 and
Tegelstrom 1992). Cytoplasmic DNA is then digested with partiwlar restriction
enzymes and efedrophoresed on agarose or polyacrylamide gels, RFLPs are
then directly detected by ethidium bromide or silver staining. The second
approach is to isolate and digest the total DNA of the organism, followed by
electrophoresis and Southem blotting of the restriction fragments. The
cytoplasmic DNA is then visualized by hybridization with a particular labeled
probe.
The development of molewlar markers based on hybridization usually
involves RFLP analysis. RFLP analysis is largely distinguished from
hybridization based fingerprinting by the type of probe used to reveal
pol ymorphisms (Jeffreys et al. 1 985). These multilows probes are
characterized by more or less regular arrays of tandemly repeated DNA motifs,
as a result, a complex banding pattern is usually produced (Jeffreys et al.
1985).
Two classes of multilows probes are mainly used. The first comprises
cloned DNA fragments or oligonucleotides which are complementary to
"minisatellites" (Le. tandem repeats of a basic motif of about 1 0 to 60 bp)
(Jeffreys et al. i 985). The second is exemplified by oligonucleotide probes
which are mplementary to "simple sequences" (Ta- and Renz 1984) or
"microsatellites" (Litt and Luty 1989)( i.e. tandem repeats of very short motifs,
mostly 1 to 5 bp). With both kinds of probes, a high degree of polyrnorphism
among related genotypes is regularly observed, and has been exploited for
many studies of genome analysis.
With the advent of PCR, numerous variations of the basic PCR strategy
were advanœd (Innis et al. 1990). It became apparent that PCR would also be
useful for the detection of DNA polymorphisms. Initial efforts to reveal DNA
polymorphisms made use of specific primers wmplementary to rewgnized
sequences. These experiments demonstrated that primers wtiich are
complementary to flanking regions of minisatellite and simple sequences loci
produce highly polymorphic amplification products. This kind of polymorphism
has turned out to be partiwlarly useful for studies of population genetics and
human and mouse genome mapping (Dib et aL 1996).
Additional strategies made use of semispecific primers, which are
complernentary to repetitive DNA elements. For human genome analysis, a
plentiful dass of randomly interspersed DNA elements called "Alu repeats" was
used for this purpose, and "AIu-PCR" unveiled considerable levels of
polymorphism (Ledbetter et al. 1990). As an alternative to interspersed repeats,
primers complementary to other repetitive sequence elements were Iikewise
successfully used for the generation of polyrnorphisms. Such sequenœs
encompass intronlexon spliœ junctions (Weining and Langridge 1991 ), tRNA
genes (Welsh and McClelland 1991 ), 5s RNA genes (Kolchinsky et al. 1991 ),
zinc finger protein genes (Unkles et al. 1992), as well as mini and mimsatellites
(Heath et a' 7 993. LiecMeldt et al. 1992, Meyer et a' 1 993a, Meyer et al.
1 993b).
Another approach makes use of one or two short, GC-rich primers of
arbitrary sequence to generate ?CR amplification products from genornic O N k
This technique, which does not need any sequenœ information, was called
random arnplified polymorphic DNA (RAPD) analysis (Williams et al. 1990).
Variations of this technique include the arbitrarily primed polymerase chain
reaction (AP-PCR) (Welsh and McClelland, 1990), and DNA amplification
fingerprinting (DAF) (Caetano-Anolles et al. 1991). As is the case with
minisatell ites and microsatell ites the pol ymorphic nature of the amplified DNA
fragments is paralleled by a polymorphic nomenclature. A common term,
"multiple arbitrary amplicon profiling" (MAAP) has been used to describe the
collective characteristics of al1 these techniques (Caetano-Anolles 1992).
Minisatellites and simple sequences
Repetitive DNA is an intemal elernent of eukaryotic genomes and may be
classified as either tandemly repeated or interspersed. For interspened
repeats. the repeated DNA motifs take place at multiple sites throughout the
genome. Tandem repeats, on the other hand, contain arrays of two to several
thousand basic motifs which are arranged in a head-to-tail fashion. Though this
kind of organization is also exhibited by sorne genes (e-g. the transcription units
for histone and ribosomal RNA), the majority of tandem repeats probably consist
of non-coding DNA (Hentschel and Bimstiel 1981 ).
Tandem repeats rnay be classified according to the length and copy
number of the primary repeated element, as wll as their genomic localization.
Satellite DNA is so called because of its ability to separate from bulk DNA by
buoyant density gradient centrifugation. Satellites wntain many repetitions
(usually between 1000 and more than 100,000 copies) of a basic motif and they
fom very long, frequently heterochromatic (tightty coiled) stretches of DNA
(Yunis and Yasmineh 1971 ). The length of a repeat unit may Vary between 2
and several thousand bp. but repeat units of 100 to 300 bp are most ordinarily
observed. Satellites regularl y occur at few genomic loci.
The term "minisatellites" was coined in 1985 to illustrate another family of
tandemiy organized repeats (Jeffreys et al. 1985). This class of DNA is
composed of shorter motifs (usually I O to 60 bp) and exhibits a lower degree of
repetition at a given locus. Minisatellites may fom "families" with related
sequences and occur at numerous loci in the genome.
Tandem repeats are made up from very short (between 1 and about 5 bp)
motifs have been dubbed "simple sequenœs" by Tautz and Renz (1 984). These
sequenœs have also been referred to as microsatellites (Litt and Luty, 1989).
Other names include, "simple repetitive sequences" or "simple tandem repeats"
(Edwards et al. 1990). Microsatellites ordinarily consist of short motifs, with a
comparatively low degree of repetition, and have a dispersed distribution over
the genome in eukaryotes (Tautz and Renz 1984).
Another tandem repeat vas t e m d "midisatellite" by Nakamura et al.
(1 987). DNA in this arrangement combines typical properties of satellites (Le.
long array of repeats at a single genomic locus) and minisatellites (Le. variable
number of tandemly repeated 40 bp sequenœ).
Properties of mini and microsatellite DNA sequences
A collective property of mini and microsatellite tandem arrays is that
identical or related motifs ocair at multiple genomic sites (Le. these sequences
share the properties of both tandemly repeated as well as interspened DNA).
Moreover, different minisatellites and microsatellites occur frequently
intermingled with each other (Weber 1990, Amour et al. 1990). Together Gth
the accumulation of point mutation wi-thin repeat units, the intemingling of
different types of repeats may wnclude in DNA sequenœs which are cryptically
simple (Tautz et al. 1 986) ( Le. their repeat structure is more or less hidden).
S ince tandem repeats in general, and minisatellite and microsatellite-like
sequences in particular, are characterised by highly fluduating copy numbers of
identical or closely related basic motifs, this class of DNA polymorphism was
called 'VNTR" (variable number of tandem repeats) by Nakamura et al. (1 987).
The existence of tandem repeats containing very short (1 to 6 bp)
sequence motifs was recognised in the early 197Qs (e.g. (TAGG)n repeats in
satellite DNA of a hennit crab) (Skinner et al. 1974). Sinœ then, many studies
have been undertaken on the occurrence and distribution of this kind of DNA in
human, animal, fungal, plant and bacterial genomes (Beckmann and Weber
1992, Greaves and Patient 1985, Hamada et al. 1982, Lagercrantz et al. 1993,
Miklos et al. 1 989, Stall ings et al. 1 991, Tautz and Renz 1984, Tautz et al. 1 986,
Weising et a' 1991 ). The general consequenœ of these studies was that simple
sequences or microsatellites are ubiquitous constituents of most eukaryotic
genomes, Mi le they are scarce or absent in prokaryotes (Gross and Garrard
1986).
The work of Epplen and colleagues (1 986), showad that hybridization
probes complementary to simple sequence motifs can be successfully used in
generating DNA polymorphic markers. While probes used in this oligonucleotide
fingerprinting technique probably recognized microsatellite-like target
sequences, the nature of the detected polymorphisms was not clear. Since
sequenced microsatellites are usually not much longer than about 100 bp, and
the size of the fingerprint fragments detected by simple sequence
oligonucleotide probes ordinarily ranges from 1 kb to more than 10 kb, the
detected polymorphisms are probably not only based only on variable numbers
of microsatellite repeat units. Cloning expenments demonstrated that different
types of simple sequences are customarily intemingled with each other and with
other types of (tandem as ml! as interspersed) repeats (Amour et al. 1989,
Broun and Tanksley 1993, Kaukinen and Varvio 1 992, Zischler et al. 1992). It
remains to be resolved whether the concerted action of a mixture of distinct
classes of repeats or an as yet unkncnm mechanism is responsible for the
development of these polymorphisms.
Levels of polymorphism revealed by DNA fingerpfinting wïth both
minisatellite and simple repetitive oligonucleotide probes rely on several factors:
(1 ) the investigated species, (2) the repeated sequence motif employed, (3) and
the restriction enzyme employed. However, it is not clear which features are
important for a probe to reveal polymorphisms (Sharrna et al. 1994, Weising et
al. 1992, and Zeh et al. 1993).
Locakation of mini and microsatellites
The genomic dispersion of GC-rich minisatellites in humans as wll as in
several birds and mammalian genomes has been studied (Jeffreys et al. 1986,
Wetton et al. 1987, Amour et al. 1989, Wells et al. 1989). Sequence analysis
has show that minisatellite arrays are often intemingled with different types of
repetitive DNA, distinctively with interspersed repeats. In humans, in situ
hybridization unveiled a prevalent localization of minisatellites close to telomeres
(RoyIe et al. 1988, Vergnaud et al. 1991 ). However, such dispersions were not
found in other species (e-g. mouse, Julier et al. 1992, cattle, Georges et al.
1 991 , and tomato, Broun et al. 1 993). The data implied a clustered rather than
dispersed distribution of minisatellites throughout the genome, wïth a propensity
for reg ions that are generally rich in repetitive DNA. Conversel y, microsatellites
appear to be more evenly dispersed throughout eukaryotic genomes. Studies
using m icrosatel lite probes showed that d(GT/CA). repeats are evenly
dispersed throughout the human (Weissenbach et al. 1992), mouse (Dietrich et
ai. 1992), and rice genorne (Wu et al. 1993).
Mechanism that account for minisatellite and microsatellite polymorphism
Minisatellite and microsatellites are customarily characterized by high
meiotic mutation rates, which chiefly conœms the number of repeats. (Jeffteys
et al. 1 988, Jeffreys et ai. 1 990, Kelly et al. 1 989). Interestingl y, mutation rates
are positively correlated with total size of the array, not only in minisatellites but
also in microsatellite sequences (Amour et al. 1992. Gray and Jeffreys 1991,
Caskey et al. 1992, Weber 7990). 60th types of repeats remain invariant for
long periods of time if they cany a few repeated motifs. However, as soon as
the tandem copy number surpasses a certain threshold, the likelihood for further
change is greatly enhanced (Richards and Sutherland, 1992).
The molecular foundation of both minisatellite and simple sequence
variability is still a mater of controversy. Conceivable mechanisms include
replication slippage, transposition, recombinational events, and unequal
exchange between sister chromatids at rnitosis/rneiosis or between homologous
chromosomes at meiosis (Jarrnan and Wells, 1989, Jeffreys et al. 1993, Wolff
et al. 1991 ). The slippage hypothesis implicates slipped-strand mispairhg of the
newly replicated strand during the replication process (Levinson and Gutman
1987). In vifm experiments demonstrate that replication slippage can actually
result in considerable amplification of a given simple sequence repeat
(Sdilotterer and Tautz 1992).
Several lines of evidenœ have lent support to the recombination
hypothesis: (1 ) a collection of minisatellite wre sequenœs share homology
the bacterial rewmbination signal chi (Jeffreys et al. 1985a, Jeffreys et al.
1985b) (2) minisatellite-like sequenœs have been discovered at sites of meiotic
crossing over (Chandley and Mitchell 1988) (3) both minisatellites and
microsatellites a d as recombinational hot spots in transfected mammalian cells
(Wahls et al. 1 990). Taken together, recombinational processes as well as
repl ication slippage may positive1 y contribute to minisatellite and simple
sequence variability. Howver, other unidentified mechanisms rnay also be
involved, particularly in the case of amplification of trinucleotide based
microsatellites associated with some human genetic diseases (Caskey et al.
1992, Richards and Sutherland 1992, Kunkel 1993, Orr et al. 1993. Wang et al.
1 994).
Amplified microsatellites
After the sequences of minisatellite or simple sequence flanking regions
are known. locus-specific primers for DNA amplification using PCR can be
attempted. This approach combines the high infomativeness of minisatellite
and simple sequenœ loci the ease and speed of the PCR technique. The
polymorphic behaviour of an individual, defined minisatellite, or simple sequence
stretch is largely a consequence of its variable number of tandemly repeated
sequence elements. Consequently, amplification of this stretch with flanking
primers should result in a polymorphic, highly informative band derived from an
individual locus. The successful application of this technique was established
for human minisatellites (Boerni-nkle et al. 1989, Hom et al. 1989, Jeffreys et al.
1988), as wll as for simple sequenœ loci (Litt and Luty 1989. Smeets et al.
1989, Tautz 1989, Weber and May 1989). These initial experiments showad
that (1 ) single loci are amplified, resulting in one or hiuo bands depending on the
homo- or heterozygous structure, (2) many different-sized alleles exist in a
population, and the level of heterozygosity is notably high, and (3) these
markers are transmissible in a Mendelian fashion and can be used for linkage
and segregation analysis.
Simple sequenœs have two main advantages over minisatellites in this
kind of analysis. Foremost, they are short (typically 20 to 40 bp) and easy to
amplify. Minisatellite arrays often are too long (i.e. 0.5 to 30 kb) for efficient
amplification (Jeffreys et al. 1988). Second, stretches of simple sequences are
more evenly dispersed over the genome than minisatellites. Weber (1 990)
investigated the infomativeness of microsatell ites of the (GTICA). type. Weber
showed that the level of polymorphism exhibited by PCRamplified
microsatellites rely on the num ber of the "pure" (Le. unintempted), tandeml y
repeated motifs. Below a certain threshold (Le. 12 CAiepeats in this
investigation), the microsatellites were not considerably polymorphic. Above this
threshold, however. the likelihood of pol ymorphism increased with length. In
contrast to RAPDs, in PCR of microsatellites restrictive annealing conditions can
be applied. This ensures high levels of reproducibility, and eliminates problems
regarding cornpetition between primer$ and errors during amplifications in
RAPDs.
These marker techniques which have refined high resolution genetic
maps for human (Weissenbach et al. 1992), and mouse genomes(Love et al.
1990), are mainly based on the use of microsatellite DNA sequences. PCR
amplification of microsatellites has also been successfully utilized for the
anaiysis of plant genomes (Aùkaya et al. 1992, Lagercrantz et al. 1993,
Morgante and Olivieri, 1993, Senior and Heun, 1993, Wu et al. 1 993, Zhao and
Kochert 1993). In the course of these experiments, extensive database research
unveiled that the relative abundance of different microsatellite motifs in plants
and animals dïfFer considerably (Lagercrantz et al. 1993, Morgante and Oliviefi
1993). For example, the d(GT/CA), repeat is one of the most frequently
occurring microsatellites in humans and many mammals (several tens of
thousands of copies) (Beckmann and Weber 1992, Hamada et al. 1982,
Stallings et al. 1991). In contrast, (ATKA). is the rnost common microsatellite in
plants, while d(GT/CA)" is relatively rare (Lagercrantz et al. 1993).
Though the convenience of PCRamplified microsatellites over other
types of markers is promising, there are partiwlar limitations. Most importantly
the identification of an informative microsatellite locus, and identification of
suitable primer sequences is even more wmbenome and expensive than in the
generation of locus-specific pol ymorphic hybridization probes. Another
drawback is that enzymatic amplification of dinucleotide repeats commonly
results in a cluster of bands which are separated from each other by two or more
bp intervals and may cause disaepancy on the actuel sire of the PCR products
produced. The extra bands are thought to be the result of slippage events. This
is thought to occur during DNA replication by the Taq polymerase (Smeets et al.
1 989).
Functional importance of minisatellites and simple sequences
The functionality of minisatellite and simple sequences for eukaryotic
genomes is not well understood. Whereas both types of sequences confonn
with the concept of "selfish DNA (Dwlittle and Sapiema 1980, Orgel and
Crick 1980), their ability to multiply in the absence of counterselective pressure,
probable structural and functional roles, nevertheless have been implicated.
Telomeres
Telomeric repeats, are found at the ends of eukaryotic chromosomes,
and depict a special class of simple sequences. A basic motif of 4 to 10 bp,
which exhibits a marked base asymmetry (one strand is GA-rich and the other
strand is CT-rich), is repeated several hundred to thousand times and foms a
single-stranded 5' overhang at the ends of each chromosome. Telomeres are
created by the action of a specific DNA polymerase named telornerase. Using an
intemal RNA molewle as the template, this enzyme adds additional telomeric
repeat units to the end of existing telorneres. Telomeric sequenœs are a rare
example of simple sequences retaining clearly defined functions: they protect
the chromosomal ends from degradation and fusion process and compensate for
DNA loss due to unfinished replication of chromosomal ends. Functional impact
of telomeric repeats on nuclear architecture is reviewad by Blackburn (1 991 and
1 992).
Centromeres
The majority of diromosomal centromeric regions most Iikely consist of
repeated sequences. The simple repeated element (GGAAT), was recently
described in yeast and human œntromeric regions (Grady et al. 1992). This
repeat displays an unusual DNA conformation and has a elevated affÎnity for
specific nuclear proteins. The discovery of highly reiterated repeats reminiscent
of degenerate telomere sequenœs in plant centromeric regions also propose
that simple repeats may be a general structural component of centromeres
(Richards et al. 1 991 ).
Transcription
With some exceptions (e-g- multigene families, ribosomal and t RNA
genes, transposable elements), repetitive DNA is commonly thought to be
transcriptionally silent. While this is most likely also tnie for the majority of
minisatellites (Swallow et al. 1 987). short stretches of simple sequenœs
(trinucleotide repeats) such as GGT in glycineiich and CCA in proline-rich plant
proteins have been show to be transwïbed (Condit et al. 1986). Simple
sequence motifs have been shom to be transcribed and translated in several
human genetic disorders (Caskey et al. 1992, Richards and Sutherland 1 992).
Except for contributing large nurnbers of identical amino acids within specific
proteins, the functiona! impact of simple sequence transm.ption is as yet
uncertain.
Transcriptional regulation
Minisatellites or simple sequenœs located in DNA wntrol regions are
capable to enhance or diminish the transcription rate of neighbouring genes
(Glaser et al. 1990, Hamada et al. 1994, Lu et a' 1993, Naylor and Clark 1990,
Spandidos and Holmes 1987). For example, poly(CA) was found to enhance the
expression of genes in transfected mammalian cells (Hamada et aL ?984), and
two (CT/GA)n motifs were identified as important stimulating elements of a
Drosophila heat shock gene promoter (Glaser et al. 1990, Lu et al. 1993). A
negative effect on transcription was obsewed by Naylor and Clark (1 990), in
Wich a (GTICA). motif upstream of the promoter region of the rat prolactin gene
abolished transcription of a reporter gene in transfected mammalian cells. In
these experiments, the formation of Z-DNA in the upstream region containing the
(GTICA). motif was attributed ta the inhibition of gene transcription.
Recombination
Minisatellites as well as simple sequenœs (especially (GTICA). repeats)
have been assigned a functional role as recombinational hot spots in humans,
however this view is controversial (Wahls et al. 1990). Research conducted by
Wahls et al. (1990) demonstrated that the insertion of minisatellite sequenœs in
plasmids stimulated recombinational events that allowed the integration of the
plasmid into the genome of wltured human cells. The presence of minisatellite
DNA in a plasmid caused a 13.5 fold increase in the frequency of integration into
the hostos genome wmpared to a plasmid without minisatellites.
Replication
Simple sequence repeats have been discovered at a putative replication
origin in the slime mold Physanrm polyœphalum (Opstelten et al. 1989). This
group put fontvard a general hypothesis in which slippage of simple sequences
may yield locally unpaired areas of DNA that are recognized by replication
initiation factors.
In summary, tvvo main mechanisms have to be examined when showhg
how minisatellite and simple sequences can control cellular processes: (1 ) some
simple sequences (e-g . (GT1CA)n or (CTIGA). type) may exercise their biological
effects by adopting peculiar DNA conformations and thus altemating chromatin
structure (Vogt 1990) and (2) some repeats maybe recognized by regulatory
proteins. Sequence-specific binding of nuclear proteins to minisatellites (Collick
and Jeffreys 1 990, Collins et al. 1 991, Wahls et al. 1 991, Yamazaki et al. 1 992),
and simple repeats (Epplen et al. 1993, Gilmour et al. 1989, Yee et al. 1991 ),
was commonly deteded, either with single or double stranded DNA Taken
together, most minisatellites and simple sequenœs do indeed function selfishly
by having evolveâ strategies which guarantee their genomic survival.
Nevertheless, there is substantial evidence that certain repeats located at
specific genornic positions have acquired one or more of the specific functions
illustrated above.
Molecular markers in Fungi
Fungi are increasingly important for a diversity of industrial purposes, and
numerous species are serious pathogens of plants, domestic animals and
humans. In different areas of research, the precise and unequivocal
identification, discrimination, and characterization of fungal species, races,
isolates, populations and pathotypes is of prime significance. However, this is a
laborious or even unattainable task if the characterization relies only on growth
characteristics, rnorphological, sexual compatibility ,and biochemical criteria.
Molewlar marken have consequently been looked for and a extensive variety of
molewlar techniques are available to study genetic variation within fungi.
These techniques include analysis of allozymes, RFLPs, eledrophoretic
karyotyping, and hybridization-based DNA fingerprinting. Three kinds of DNA
probes have mainly been employed for fingerprinting studies in fungi: (1 ) random
repetitive DNA probes derived from the fungal genome under investigation, (2)
minisatellite probes, often derived form the human or wild type M l 3 phage
genome, and (3) synthetic oligonucleotide probes complementary to simple
repetitive sequences. The majority of these studies in fungi have depended on
cloned genomic probes (Brown et a/. 1 990, Hamer et al. 1989, Kistler et al.
1991 , Scherer and Stevens 19ûû). Microsatellites have extensively been
employed in fingerprinting of fungi (Meyer et al. 1994, Meyer et aL 1992, Meyer
et al- 1993a). The use of RAPDs have also been used in fingerprinting of fungi
(Meyer et al. 1 993a. Meyer et al. 1993b, Schonian et al. 1993).
Examples of fungal species for Hihidi DNA fingerprinting analysis has
been accomplished by hybridization to repetitive DNA probes, in addition to
minisatellites, simple repetitive sequenœs, and probes cloned from genomic
DNA, are listed in Table 1, and examples of fungal species in which DNA
fingerprinting has been accomplished vvith PCR-based rnethods employing
arbitrary primers, together with minisatellite and simple sequence primers, are
listed in Table 2,
Tabfe 1. Fungal species in Hihidi DNA fingerprinting analysis has been perforrned by h ybridization to repetitive DNA probes.
Fungal species Referenœ
Absidia glauca
Alternaria altemata
Arxula adeninivorans
Ascochyta pisi, A. rabiei
Aspergillus amstelodami, A. a wamo~, A. ficuum, A. flavus, A. fumigatus, A. giganteus, A. nidu/ans, A- niger, A. ochraceus, A. repens, A. restnctus, A. teneus, A. versicolor
Beauveria bassiana
Candida albicans, C. glabrata, C. krusei, C. lipolytica, C. parapsilosis, C. stellatoidea. C. tropicalis, C. utilis
Cocholiobolus carbonum, C. hetemstmphus, C. victonae
Colletotnchum coccsides, C. desttucüvum, C. gloeosponoides, CC. graminimla, C. lagenanum, C. lindemuthianum, C. magna, C. orbiculare, C. pisi, C.trifdii
Coptinus comatus
Meyer et al. (1 992)
Adachi et al. (1 993)
Lieckfeldt et al. (1 992) Meyer et al. (1 993)
Bienverth et al. (1 992) Kaemmer et al- (1 992)
Girardin et al. (1 993) Meyer et al- (1 991 ) Meyer et al. (1 992)
Hegedus and Khachatourians (1 993)
Fox et a' (1 989) Hellstein et al. (1 993) Lieckfeldt et al. (1 992) Lieckfeldt et al. (1 993) Wilkinson et al. (1 993)
Rodriguez and Yoder (1 991 )
Braittiwaite et al. (1 989) Correll et al. (1 993) Radriguez and Yoder (1991 )
Weising and Kahl (1 990)
Milgroom et al. (1 992)
Erysri,he gratninis Brown et al. (1 991 )
Fusamm avenareum, F. culmo~m, F. avenaœum, F. graminearum, F. laterifunn, F. spomtrichiella F. oxyspowm, F. poae, F. scirpi,
Hansenula anomala
Leptosphaena maculans
Mucor hiemalis, M. plumbeus, M. racernosus
Mymsphaerella fijienssis, M. graminimla, M. pinodes
Nectna haematococca
Ophiostoma ulmi
Parasitella simplex
Penicillium canescens, P- chrysogenum, P. citreoviride, P. c#rinum, P. aurantiogriseum, P. dupontii, t? expansum, P. glabrum, P. glandicola, P. janthinellum, P. lavendulum, P. minioluteum, P. variabile, P- vïndicatum
Phoma lingam
Phyoomyces blakesleeanus
Phytophthora aïrophthora, P. colocadae, P. hibernalis, P. ilik5s, P. infestans, P. megaspem?a, P. mirabilis, P. phaseoi
Kistler et al. (1 991 ) Manicom et al- (1 987) Meyer et al. (1 992) Monastyrskii et a' (1 990)
Walmsley et al. (1 989)
LieMeldt et al. (1 992) Walmsley et al- (1989)
Meyer et al. (1 992)
Boewinkle et al. (1 989)
Meyer et al. (1 992)
McDonald and Martinez (1 990) and (1 991 )
Rodriguez and Yoder (1991)
Hintr et al. (1 991 )
Meyer et al. (1 992)
Kuhls et al. (1 992) Meyer et al. (1 993) Wei he et al. (1 990)
Rodriguez and Yoder (1991)
Weising and Khal (1 990)
Drenth et al. (1 993) Goodwin et al. (1 989) Rodriguez and Yoder (1 991 Whisson et al. (1 992) SMnger et al. (1 991 )
Puccinia graminis
qrthium mamillatum
Saccharomyces cerevisiae, S- dairensis, S. delbnreckiL S. exiguus, S. fermenta6 S. Muyveri. S. unisponrs
Sclefvünia scler0tiomm
Septosphaen'a turcica
Stachybotrys chartarum
Tnchoâerma hamanum, T. longibranchiatum. T. polyspomm, T. pseudokoningii, T. reesei, K satumisporum, T- vinde
Usfilago maydis
Verticillium lecanii
Zygosacchammyces bailii
Anderson and Pryor (1 992)
Rodriguez and Yoder (1991)
Kunze et al- (1 993) Lieckfeldt et al. (1 992) Walmsley et al, (1 989)
Kohli et a/. (1 992)
Rodriguez and Yoder (1991)
Cooper et al. (1 992)
Meyer et al. (1 992)
LiecMeldt et al. (1 992) Meyer et al. (1 991 ), (1 992) (1 993)
Meyer et al. (1 991 )
Meyer et al. (1 991 )
Varma and Kwbn-Chung (1 992)
Meyer et al. (1 991 ),
Walmsley et al. (1 989)
Table 2. Fungal species in which DNA fïngerprinting has been perfomed with PCR-based methods using arbitrary pnmers, including minisatellite and simple sequenœ primers
Fungal species Reference
Absidia glauca
Acaulospora laevis
Agancus bispotus
Armillan'a bulbosa
Arxula adeninivorans
Ascochjda ra biei
AspergilI'us aculeatus, A. a wamori, A. candidus, A. carbonariusa, A. ellipticus,A. flavus, A. ibetidus, A. fumigatus A. giganteus, A. helicathrk, A. hennebergii, A. heteromorphus, A. intemedius, A. japonicus, A. nanus, A. nidulans, A. niger. A. ochraceus, A. phoenias, A. pulverulentus A- resinctus, A. temus, A. usami, A. ve~~icolor, A. wentii
A ureobasidium pullulans
Candida albicans, C. glabrata, C. guillietmondii C. haemulonü , C. knrsei, CC. lipolytica, C. lusitaniae C. parapsilosis, C. pseudotmpicalis, C. stellatoidea C. tmpicalis, C. utilis
LiecMeldt et al. (1 992) Wostemeyer et a' (1 992)
Wyss and Bonfante (1993)
Khush et al. (1 991 )
Smith et al. (1 992)
Lieckfeldt et al. (1 992) Meyer et al. (1 993)
Kaemmer et al. (1 992)
Girardin et al. (1 993) Meyer et a/. (1 991 ) Meyer et al. (1 992) AufauvreBrown et al. (1 992) Loudon et al. (1 993) Megnegneau et al. (1 993)
Bulat and Mironenko (1 992)
Van der Vlugt-Bennans (1 993)
Bostock et al. (1 993) Caetano-Anolles et al. (1991 Lehmann et al. (1 992) Meyer et al. (1 993) Niesters et al. (1 993) Sullivan et al. (1 993) Jones and Dunkle (1 993)
Colletotnchum acufatum, C. mgaifa, C. gloeosponoides, CC. graminicola, C. kahawae, C. magna, C. onbicuIam
Cronattium quetwum
Cryptococcus albidus, C. laurentii, C. neohmans
Erysiphe graminis
Fusarfum graminearum, F. oxyspomm, F. solani
Gigaspora margatita
Glomus caledonium, G. mosseae, G. versiforme
Gremmeniella abietina
Heterobasidion annosum, H. araucanae
Histoplasma capsulaturn
Hypoxylon truncatum
KIluyverumyœs ftagilis, K, lactis
Lentinula edodes
Correll et a' (1993) Freeman et al- (1 993) Guthrie et al. (1 992) Mills et aL (1992) Vaillancourt and Hanau (1 992)
Meyer et al- (1 993a) Meyer et al- (1 993b) Mitchell et al- (1 993)
Caetano-Anol les et al. (1 993)
McDonald and McDennott (1 993)
Kistler et al. (1 991 )
Wyss and Bonfante (1 993)
Wyss and Bonfante (1 993)
Hamelin et al. (1 993)
Garbelotto et al- (1 993) Fabritius and Karajalainen (1 993)
Strongman and McKay (1 993)
Kersulyte et al. (1 992)
Yoon and Glawe (1 993)
Lieckfeldt et al. (1 993)
Kwan et al. (1 992)
Leptosphaena maculans
Mymsphaerella @ensis, MM. graminimk, M. musimla
Neurospora crassa
Goodwin and Amis (1 993) Wostemeyer et. al. (1 992)
JohansonandJeger (1 993) McDonald and McDemott (1 993)
Williams et al. (1 990) Williams et al. (1 991 )
Parasitella simplex Wostemeyer et al. (1 992)
Penici/Iium aurantiognseum, P. canesœns, Durand et al. (1 993) P. chrysogenurn, P. citreoviride, P- citrinum, Kuhls et al. (1 992) P. dupontii, P. expansum, P. glabnrm, P. glandida, Meyer et al. (1 993a) P. islandicum, P. janthinellum, P. la vendulum, P. minioluteum, P. roqueforfii, P. variabile
Pyrenophora graminea, P. teres Reeves and Ball(1991)
Pythium ultimurn Francis and Clair (1 993)
Rhabdoclrine parken McCutcheon ef al. (1 993)
Rhizodonia solani Duncan et al. (1 993)
Rhodotorula rubra Meyer et al. (1 993b)
Saccharomyces bayanus, S. cerevisiae, Lieckfeldt et al. (1 993) S. delbrueckii, S. diastaticus, S. fermentati, Meyer et al. (1 993a) S. pastorianus, S. willianus
Wyss and Bonfante (1 993)
Suillus granulatus Jacobson et al. (1 993)
Tnchoderrna harnatum, T. hanianum, Lieckfeldt et al. (1 993) T. longibrachiatum T.pseudokoningii, T. mesei, Meyer et al. (1 992b) T. satumispoium, T. vin& Meyer et al. (1 992)
Schlick et al. (1 992a) Schlick et al. (1 992b)
Venturia inaequalis Sierotzki et al. (1 994)
Classification of the genui Pvu,ium
mhium belongs to the class of organisms refened to as the Oomycetes.
Members of this class are mostly aquatic and are thought to be related to the
heterokont algae (Barr 1 992). Cavalier-Smith (1 989) classif ied the Oomyœtes
along with the Hyphochytriomyœtes under the subphylum Pseudomycatina,
phylum Heterokonta, and the kingdom Chromista. Thus, the Oomycetes are not
included in the tnie fungi, Hihich are plaœd in a separate kingdom, Eumycota
(Cavalier-Smith 1 989). The Oomyœtes produce biflagellate zoospores with one
tinsel flagellum directed forward and one whiplash flagellum directed backward.
Their cell walls consist mainly of glucans and they contain cellulose as opposed
to chitin. They are diploid and their sexual reproduction is oogamous and
meiosis is gametangial (Alexopolous and Mims 1979). Aquatic Oomyœtes are
know for their parasitic behaviour on many small animals, algae, water molds,
and other aquatic organisms. Terrestrial forrns of the Oomyœtes are parasites
of plants, passing their entire life cycle in the host However, the production of
zoospores continues to be common, an indication of their aquatic ancestral
traits (Alexopolous and Mims, 1979).
The Oornyœtes are subdivided into six orders; Eurychasmales,
Saprolegniales, Lagenidiales, ihraustochytriales, Labyrinthulales, and
Peronospwales ( S p a w 1976). Of these, the Peronosporales are of great
ewnomic importance due to their destructive nature in plants. Further
classification of the Peronosporales is mainly based on the shape of the
sporangia and sporang iophores. Alexopolous and Mims (1 979) divided this
order into four families; Albuginaceae, Peronosporaœae. Peronophythoraceae,
and the Pythiaceae. Waterhouse (1973) published a key to the Pythiaceae that
included eigM genera; the most cornmon of #ese being Phytophthora and
Pythium. Members of the genus Phytophthora include many important plant
pathogens, such as Phytophthora intèstans, the cause of late blight of
potatoes. The genus Pythium includes more than 120 descrïbed species with
w-de distribution and host ranges (Dick 1 990, Plaats-Niterink 1 981 ). Members
of the genus Pyfhium live in the soi1 saprobically on dead organic matter or
parasitically on the young seedlings of great number of susceptible species of
economic seed plants. Taxonomy of the genus Pythium is mainly based on the
morphological charaders of the reproductive structures, such as zoosporangium
presence, shape, and size, zoospore production, oospore size and wall
thickness, and the number, shape, and orïgin of the antheridia (Plaats-Niterink
1981). Identification of some Pythium species is difficult due to the absence of
certain reproductive structures. Heterothallic species are among these and thus
the opposite mating types must be considered for proper identification. Pythjum
isolates that reproduce only asexually are considered the most diffiwlt to identify
(Plaats-Niterink 1 981 ).
Pythium ultimum is regarded as a major plant pathogen with wrldwide
distribution. It has been isolated throughout the United States (Miller et al. 1957,
McLauglin 1 946, Sprague 1 942) and Canada (Vaartaja and Agnihotti 1 969).
Tahiti (Scott, 1 960), South Ametica (Alvarez-Garcia and Cortes-Monllor 1 971 ),
lceland (Johnson 1971), M c a wager 1931, Ravise and Boccas 1969, Fifani
1 975), Europe (Kouyeas 1 977, Domsh et al. 1 968, Plaats-Niterink 1975, and
Cejp 1 961 ), Japan (Alicbusan et al. 1965) and Australia (Vaartaja and Bumbieris
1 964)-
Pythium ultimum affects a wide variety of plant species, especially in the
seedling stage, causing preemergence and postemergence damping off, root rot,
and l o w r stem rot (reviewed by Hendrix and Campbell 1973). P. uftimum is one
of the most prevalent of Pythium species found in the soi1 (Plaats-Niterink 1981).
In soils where it is predominant, plants not immediatefy killed by the pathogen
can go on to mature, but are likely to experienœ poor root development, stunting
and reduced yields. Reduœd plant vigour has been described as the only
above-ground symptom which may be manifested in crops infected with P.
ultimum (Yuen et al. 1991). Some of the hosts from WiCh P. ultimum has been
isolated are: tulips (Moore and Buddin 1937), sweet potato (Poole 1934), sugar
beet (Gindrat 1976), coffee (Filani 1975). and apple (Bielenin 1976).
Classification of the genus AspergiIIus
Members of the genus Aspetgilfus belong to the group of fungi called the
deuteromycetes, which also includes the genus Penicillum. The deuteromyces
are among the most widely distributed fungi in the world. They are of immense
importance in human affairs and have been studied intensively (Raper and
Fenneli 1965). In their monograph on the genus Aspe~giflus, Raper and Fennell
(1 965) accepted 132 species subdivided into 18 groups. The generic and
species concepts were cirwmsaibed in this monograph and it is still a valuable
source for species identification and is wrrently used today for the classification
of members in this genus.
The Aspergilli have been used for many years by the fermentation
industry for the production of citric acid and other organic acids, and have been
used for centuries in the preparation of soy sauce (Yong and Wood 1974).
Furthemore, Aspeq$lli are capable of utilizing an enormous variety of
substances for food because of the large number of enzymes they produce.
More recently the Aspergilli have been exploited for the production of enzymes
widely used in industry for the manufacture of a variety of materials. A wide
range of secondary metabolites are produced by these fungi, including the
potent carcinogenic aflatoxins. Aflatoxin was deteded in the 1960's when
100,000 or more turkey pullets died in England from a new disease tenned
'Turkey X-disease". The mold producing the powerful toxin was Aspe~gillus
navus and thus the toxin was named aflatoxin (reviewed by Raper and Fennell
1965). As human pathogens, members of the Aspegilli produœ an assembly of
diseases col lect ive1 y k n ~ as aspergilliosis (Rippon 1 974). Asperg illiosis of
the lungs is apparently the most serious of these diseases and is quite common
in birds and various mammals including humans (Rippon 1974).
Aspergillus flavipes was first describecl in 1 91 1 as a species of
Stengmatocysfis (Bainier and Sartory 191 1). Colonies are desaibed as having
colorless to yellowish aen'al mycelium. Conidial heads are desmibed as radiate
to loosely columnar with globose vesicles. One distinguishing characteristic in
classification of the genus Asper@lli is the color of conidia. Because conidia are
produced in such abundance, their color is a predominant feature. Aspergillus
colonies can appear to be black, brown, blue, yellow, or green, with the color
depending on the species and on the medium on which the fungus is growing.
This characteristic is an important feature in the classification of many of the
species associated with this genus. Conidiophores in A. &@es are pale yellow,
smooth, and phialides are biseriate. The teleomorph of A. tlavipes (Fennellia
flavipes) is characterized by numerous small cleistotheicia within such a large
mass of elongate to helical hulle cells (Wiley and Fennell 1973).
The use of non-morphological methods vvhich could be important for
Aspergilllus taxonomy were discussed by Fennell (1 977). In her review, Fennell
discussed important features like cell wall composition, proteins, pyrolysis
products, nucleic acids, amino acid biosynthesis, hydrocarbon metabolism and
inorganic elements, which could aid in the classification of the genus. Of al1
these techniques only the use of enzymes and nucleic acids are still considered
to be relevant for taxonomie purposes today.
With the advent of biochemical and molecular genetic techniques, new
approaches to the detedion and identification of different species of the
Aspergilli have b e n developed. Although A. flavipes is an economically
important fungus which could benefit from DNA fingerprinting to differentiate its
isolates ftom each other, no studies have been cwiducted to help identify and
characterire its DNA for these purposes.
Some of the isolates of A. flavipes in this study are patented due to their
economic value which involves the synthesis of the antidepressant imiprimine.
Imipfimine is still widely used today to treat several obsessive compulsive
disorders such as anorexia nervosa, and hyperactivity in children. Furthemore,
isolates of Aspergillus flavipes have been used to perfom various chemically
diffÏcult steps in organic synthesis, e-g. in the production of anticancer agents,
and insecticides, or in stereospecifïc oxidation of R-(-)glaucine, Davis and
Talaat (1 981 ).
PCR amplifiad microsatellites in fungi
To date no research has been conducted to verify that PCR amplification
of simple sequence DNA (microsatellites) is useful for typing of different strains
of fungi although the abundanœ and types of simple sequenœs has been
reœntly described to some degree in members of the genus A s ~ l l u s .
Although simple sequence DNA has shown important promise as a molecular
marker, the use of microsatellite sequences for DNA fingerprinting has been
addressed only reœntly in fungi.
Materials and Methods
Strains of Pythium ultimum usad in this study
All Pythium ulfimum isolates used in this study were acquired from the
Biosystematics Research Centre, Ottawa, ON, Canada or from the
Centraalbureau voor Schimmelwltures, Baam, Netherlands, and are Iisted in
Table 3.
Strains of Aspergillus tïavipes used in this study
The cultures of A s ~ l f u s flavipes were obtained from Apotex
Fermentation Inc. (Winnipeg, MB, Canada) and were provided on potato
dextrose agar slants. The swrce of these cultures was the American Type
Culture Collection (ATCC, Rockville, MD, USA). The information obtained for
these isolates is summarized in Table 4.
Table 3. Description of Pyfhium isolates used in this study.
P. ultimum. var. ultimum Papaya BR319
P. ultimum, BR406 Alfalfa
P. ultimum. var. ultimum Alfalfa BR41 8
P. ultimum. var. ultimum Soil BR471
P. ultimum BR583 Safflower
P. ultimum. var. ultimum Bean BR600
P. ultimum 8 ~ 6 1 2b Geranium
P. ulfimum. var. ulfimurn Pea BR638
P. ultimum. var. ultimum Pea BR639
P. ultimum. var. ulfimum Cucumber BR640
P. ultimum. var. sporangiifèrum Soil BR650
P. ultimum. var. ultimum unknown BR925
P- ultimum. var. uitimum Lepidiurn sativum CBS398.5 1 (neoty pe)
P. ultirnum. var. ultimum Apple root CBS488.86
California
Quebec
Quebec
Alberta
British Columbia
Ontario
Alberta
Alberta
Alberta
Spain
unknown
Netherlands
Poland
P. uitimum. var- uttimum CBS114.19
P. ultitnum. var- ultimum CBS305.35
P. ultimum. var- ultimum CBS378.34
P. ultimum. var. ulfr'mum CBS730.94
P. ultimum. var. ultimum CBS726.94
P. ultimum. var. ultimum 688728.94
P. ultimum. var. ulfimum CBS729.94
P. ultr'mum, var. ultimum CBS249.28
P, ultimum. var. ultimum CBS264.38
P- ultimum. var. ultimum CBS291.31
P. ultimum. var. ultimum CBS296.37
P. ultimum. var. ultimum CBS656.68
P. anhenomanes BR607
Gymnospenn seedling
Grass root
TiïfblÏum pratense
8eans
VVheat soil muck
VVheat soi1 loam
VVheat soil muck
Sinningia speaisa
Seedling of pinus
Yams
Pea
Lycopetsicon esculenturn
Maize
Pheseolus vulgaris
Netherlands
Nethsrlands
Netherlands
British Columbia
British Columbia
British Columbia
British Columbia
Netherlands
Netherlands
Netherlands
England
Netherlands
Manitoba
Netherlands
Cuwmber British Columbia
P. disimile BR1 60 Wheat Ontario
'Isolates with the "BR" prefix were obtained from the Biosystematics
Research Centre, Ottawa, ON, Canada. lsolates with "CBS'prefix were obtained
from the Centraalbureau voor Schimmelcultures, Baarn, Netherlands.
b Originally identified as P. sp. Type G, but shown to be P. ulomum var.
ultimum (Buchko 1 996).
Table 4. Designation of strain number and ATCC characteristics of isolates of A. flavipes used in this study
Number assigned in ATCC Isolate, Characteristics, Origin this study
ATCC 1030, Produces imiprimine, transformations of sesquiterpene lactone costunolid, produœs didehyroglaucine. Possibly from Thailand.
ATCC 1 1013, ER. Squibb 8 Sons MD 2472, US patent 2768928, produœs imiprimine, transformations of sesquiterpene lactone costunolid.
ATCC 13830, Takeda Pham. Ind. Ltd. Patented
ATCC 16795, Transformations of sesquiterpene lactone costunolid, produces imiprimine, Texas
ATCC 16805, lsolated from soil, Haiti.
ATCC 1681 4, lsolated form dairy products, Minnesota
ATCC 24487, Type culture, Haiti
ATCC 26499, lsolated from bird feces, produces glutamicine (flavipucine), Italy.
ATCC 481 36, lsolated fom grassland soil, North Dakota, USA.
Culture rnethods
From the margin of a vigorous wlture growing on malt extract agar, an
agar plug (approx 5 mm in diameter) was transferred to a 2 L shaker fiask
wntaining 200 mL - peptone yeast extract - glucose medium (PYG) containing 3
g glucose, 1 g peptone, and 1 g Difco yeast extract per litre, and allowed to grow
in shake culture for 2-3 weeks at room temperature for Pyfhium sp. and 1 month
for Aspergilius isolates. Myœlia was then harvested by vacuum filtration ont0
Whatman No. 1 filter paper (Whatman Laboratory Produds, Clinton, NJ),
thoroughly washed with distilled water, and then fieeze dried. Mycelia harvested
from two shaker fiasks (300 mg dry weight) was generally sufficient for DNA
extraction. lsolates used in these investigations and their sources are listed in
Tables 3 and 4.
Genomic DNA extraction and purification
Initially, large scale DNA extraction was employed whereby 4-5 1 L
shaker flasks were harvested and, immediatel y after washing , the mycelium was
extracted by grinding in a prewoled mortar with pestle for 20 min in the
presenœ of liquid nitrogen. A rapid proœdure was developed which also
required less mycelium for extraction of suitable amounts of DNA.
A DNA "mini" preparation proœdure based on the methods of Murray and
Thompson (1 980) and Kim et al. (1 990) was used to extract "polymerase chain
reaction grade" ENA (Saiki et a' 1988). Frozen myceliurn (1 W-200 mg) was
added to sterile Falcon polystyrene conical tubes (Bedon Dickinson Labmre,
Lincoln Park, NJ), each of vuhich contained 4 m l of iœ cold lysis buffer (1 50 mM
NaCI. 50 mM EDTA, 10 mM Tris, pH 7.4), 20 mglmL proteinase K (Sigma, St-
Louis, MO), and 9 g of acid-washed and bakeddry 0.5 mm glass beads (Braun
Melsungen). The mixtures were then vortexed for 2 to 3 min, and an additional 3
mL of lysis buffer was added to each tube. Sodium lauryl sulfate (SOS, Fisher
Scientific, Nepean, ON) was added to a final concentration of 1%, and the tubes
then incubated at 5S°C for at least 1 h. NaCl and hexadecyltrimethyl ammonium
bromide (CTAB. (Sigma Chemical Co., St. Louis, MO) were added to the tubes
to a final concentration of 1 M and 1 % respectively, and the tubes were then
incubated for an additional 30 min at 55OC. Next the glass beads were pelleted
by centrifugation at 2000 rpm for 2 min, and the supernatant transferred
aseptically to sterile 15 mL glass Corex tubes (Canlab, Winnipeg, MB), and the
CTAB-protein complex and SDS w r e removed by tm chlorofomi/isoamyl
alcohol (24:1, V:V) extractions. Approximately 1 00 mg of DNA was recovered
from each culture by precipitation wïth 2.25 volumes of 95% ethanol. This
miniprep method was self contained within separate sterile tubes for each
culture, thus cross contamination by DNA fmm different samples was avoided.
The DNA was redissolved in 150 to 500 uL of TE buffer (10 mM-TrisMCI; 1 mM-
EDTA; pH 7.6) and stored fmzen et -20°C. Although the quality (size range: 20-
40 kb) and yield of the DNA ~ w s somewhat variable, one miniprep procedure
yielded suffident DNA from each sample to carry out genomic RFLP and PCR
analysis.
RNAse treatrnent of nucleic acid preparaüons for RAPDs
Fifty rnicroliter aliquots of the DNA preparation for RAPD analysis was
incubated without or with 0.5 ug of RNAse A (Sigma) in 0.5 uL of RNAse buffer
(1 0 mM Tris-HCI, , 15 mM NaCI, pH 7.5) for 1 h at 20°C. The stock RNAse 0
solution (1 O mg/mL) had been boiled for 15 min to destroy DNAse.
Oligonucleotide probes and PCR primer$
Oligonucleotide primen used for the polymerase chain reaction (PCR)
amplifications and DNA sequencing are show in Table 5. Oligonucleotides
were obtained from the Department of Microbiology, University of Manitoba,
which w r e synthesised with the ?CR-MATE 391 DNA synthesizer (Applied
Biosystems, Foster City, CA). RAPD primers UBC series were purchased from
University of British Columbia, Nucleic Acid-Protein Service Unit n an couver,
BC, Canada) and OP series from Operon Technologies, Inc. (Alameda, CA)
(Table 6).
Table 5. Oligonucleotide probes and sequencing primers.
Name of oligonucleotide Sequence 3-3'
d(GVg probe GTGTGTGTGTGTGTGTGT
d(cT)~ probe
d(AT)o probe
~ ( G C ) S probe
Ml 3 pBluescript T7 primer A A ~ G A C T C A C I C A ~
Ml 3 pBluescript T3 primer ATZ!UCCCCrCACT!AAAG
Table 6. Primers used in PCR reactions for RAPDs
Name of oligonucleotide Sequenœ 5-3'
UBC4 primer
UBC-6 primer
OPC-2 primer
OPC4 primer
OPC-6 primer
OPC-8 primer
OPA-2 primer
OPA-3 primer
OPA-4 primer
OPA-5 primer
OPA-9 primer
OPA-1 O primer
OPA-13 primer
cc-cm
GTrCAGCCGTIC
RAPD PCR conditions
All PCR readions were conducted in a PTC-100 Programmable Thermal
Controller equipped with the hot bonnet fmm MJ Research, Inc., using Taq DNA
polymerase obtained from Promega. Reactions were conducted in 0.5 mL
regular (not thin walled) Eppendorf tubes with no ail added due to the use of the
hot bonnet Most readions were begun in late af€emoon, finished dufing the
night , and then were held at 4OC for up to 10 h before electrophoresis. The
PCR reactions were fun in 50 uL of volume consisting of 34.8 uL of sterïle HPLC
grade water, 5 uL Promega Taq Buffer (IOx), 4.0 uL, 25 mM Mg& (Promega),
final concentration 0.2 mM, wanned to 37OC before adding, 1 .O uL template DNA
(0, 0.1, 0.01, or 0.001 dilution), 1 .O uL primer (60 ng), and 0.2 uL Promega Taq
DNA Polymerase (1 unit). DNA and primer were added separately to the side of
the tube and spun d o m immediately prior to reaction. The reaction protowl was
as follows: 1 :94OC, 45 sec; 2:40°C, 1 min; 3:72OC, 2 min; 4:Repeat 1 -3 for 35
cycles; 5:72OC, 5 min; 6:4*C, hold. Electrophoresis and visualisation was
accomplished using 10 uL of each readion mixture electrophoresed on a 1.5%
agarose gel together w*th the BRL 1 kb ladder of size standards.
Electrophoresis was typically run for 2 h at 75 V. Ethidium bromide was added
to the gel prior to gelling. Photographs of completed gels were taken using a red
filter Ath the gel on a UV transilluminator (Fotodyne Incorporated, Mississauga,
ON). Polaroid 667 film was used.
DNA Dat blotüng and fixation of DNA to nylon membranes
Approximately 1 ug of total genomic DNA (sample listed in Table 4) was
denatured at 1 O°C for 5 min and then quickly chilled on ice. Samples w r e
spotted ~ Ï t h aid of a Pipetrnan (Gilson Pipetrnan P2) ont0 Hybond-N+
(Amersham International, Oakville, ON) membrane allowed to dry at ambient
temperature. DNA was fixed to the membrane using a 310 nm W source,
according to the manufacturer's instructions.
DNA digestion and electrophoresis
Endonuclease digestions were perfoned using enzymes obtained from
Pharrnacia (Canada) Ltd. Dorval, Que. and BRL (Bethesda Research
Laboratories Inc., Gaithersburg, MD), according to the manufacturer's
recommendations. Electrophoresis was camied out in TBE buffer (89 mM Tris,
89 mM boric acid, 2.5 mM EDTA, pH 7.6) on 15 X 20 X 0.4 cm horizontal 0.8 or
1 -2% agarose (Boehringer Mannheim Corporation, Indianapolis, IN) submarine
gels at 75 V/cm for 1 to 4 h. The BRL 1-kb (Bethesda Research Laboratories)
ladder las the molecular weight standard used to estimate fragment size. Gels
were stained for 15 min with ethidium bromide (0.5 mghL in TBE bufFer)
(Sigma) and illuminated with UV (31 0 nm) transilluminator (Fotodyne
Incorporated, Mississauga, ON), and photographed using Polaroid 667 film.
S'-end fabeleû oligonucleotide probes
Oligonucleotide primers were S-end labeled using T4 polynucleotide
kinase (Maniatis et al. 1 989). Synthetic oligonucleotides were synthesized
without a phosphate group at their 5' temini and then labeled by transfer of the
=P ftom [ a l p h a - = ~ ] d ~ ~ ~ . The readions were canied out at 37OC for 30 min and
stopped with 2 uL of 0.5 M EDTA, pH 8.0 (Sigma Chernical Co., St. Louis, MO).
The oligoprobes were then precipitated wïth 2.25 volumes of 95% ethanol and
each redissolved in 50 uL of TE buffer.
3' end labeling of oligonucleotide probes using digoxigenin UTP
lnitially oligonucl eotide probes for studies with P. ultimum were labeled
with [ a ~ p h a - ~ p l d ~ ~ ~ (Dupont, New Research Produds, Boston, MA) (Maniatis
et al. 1982). However, for analysis of A. flavipes, oligonucleotide probes were
developed using the digoxigenin UTP 3'end labeling system kit (Boehringer
Mannheim Corporation, Indianapolis, IN) according to the manufacturef s
instructions. To 100 prnoles of oligonucleotide, 4 uL of tailing buffer (1 M
potassium cacodylate, 125 mM Tris-HCI, 1.25 mglmL bovine serum albumin, pH
6.6), 4 uL of 25 mM CoClz solution, 1 uL of 25 mM DlG-ddUTP and 50 units of
terminal transferase in (0.2 M potassium cacodylate, 1 mM EDTA, 200 mM KCI,
and 0.2 m g h L bovine serum albumin, pH 6.5) were added in a 0.5 mL
microfuge tube. The final volume of the solution was adjusted to 20 uL with
distilled water. The readion mixture was incubated at 37°C for 30 min and then 5
uL of a 0.5 M EDTA solution was added to stop the labeling reaction. The
labeled oligonucleotide solution was then stored at -20°C and 1 uL was used for
hybrïdization readions.
Southem Blotting and Hybridizations
DNA which was digested restriction endonuclease(s) and fun on
agamse gels had been transfened ont0 Hybond-N nylon membranes
(Amersham) according to the manufacturer's instructions. Prehybridization of
the blots was at 42OC for 2 h in 1 M NaCl (Fisher Scientific) and 7 % SDS (Fisher
Scientific) with constant agitation. The probes were then added separately
(boiling was not needed for d(GT)9 and d ( C n probes, but the self annealing
d(Ang and d(GCk probes were boiled for 10 min prior to their addition) to the
hybridization fluid and incubated at specific ternperatures. d(GT)g and d(CT)g
probes were hybridized at 42OC, d(CG)s at 7S°C and d(AT)s at 35OC with slow
and continuous agitation for 12-14 h. d(AQ and d(GC) 9 probes were tested at
several different hybridization and washing temperatures (dom to 1 O°C below
those indicated). Following hybridization, the membrane las washed twice in W
sodium saline citrate (SSC; 0.1 5 M NaCI, 0.01 5 M sodium citrate, pH 7.0) at
room temperature for 5 min each, then three times in 2X SSC and 1 % SDS at
42°C for 30 min with constant shaking. Hybridization buffers containing DIG-
labeled oligonucleotides were stored at 4*C and reused up to 15 times. Blots
were prepared using Hybond-N nylon membrane (Amersham International,
Oakville, ON) according to the manufaduren instructions. Autoradiography
employed Kodak X-Omat RP film with a Dupont Hi-Plus intensifying screen at
-70°C for 4&96 h for radiolabeled probes. To deted DIG labeled
oligonucleotide probes, membranes were washed with 100 mL of washing buffer
(0.1 M maleic acid, 0.1 5 M NaCI, pH 7.0, containing 0.3% vhr Twen 20) for 5
min. The blot was then incubated for 30 min in 100 mL of blocking solution (1 %
blocking reagent, Boehringer Mannheim, 0.1 M maleic acid, 0.1 5 M NaCI, pH
7.0). Subsequently, 20 mL of fresh blocking reagent was added wntaining 2 uL
of a 75 mU/mL of anti-DIG-antibody. The blot was then incubated at room
temperature for 30 min with gentle agitation. The membrane was then washed
tuvice for 15 min with washing buffer and then equilibrated with detedion buffer
( 0.1 M Tris-HCI, 0.1 M NaCI, 50 mM MgCI2, pH 9.5) for 5 min. The membrane
was sealed between tw transparent plastic sheets wntaining 500 uL of
chemiluminescent substrate (1 uL to 500 uL vlv of CDPStar in detedion buffer).
The chemiluminescent buffer was spread evenly over the membrane by gently
applying pressure to the plastic bag containing the blot The membrane was
incubated at 37OC for 10 min and then exposed to Kodak X-Omat RP film from 2
to 60 min at ambient temperatures.
Construction of the Pytnium ultimum and Aspegillus flavipes genornic
li brades
A Lambda EMBL3 genomic libnry was constnided using a cloning kit
(Stratagene, La Jolla, CA) according to the manufacturers recommendations and
Sambrook et al. (1 989) as follows. High molewlar weight genomic DNA was
prepared using a large scale method as described above (Garber and Yoder
1 983). Conditions for partial digestion w*th Mbol (Bethesda Research
Laboratories Inc., Gaithersburg, MD) were established using 5 mg of total
genomic DNA, 0.05 units of endonuclease enzyme, and a series of incubation
times of 1 to 30 min at 37%. A large scale preparation of DNA, partially
digested with Mbol to a size range of 20 kb, was prepared by digesting 300 mg
of DNA 8.5 units of enzyme for 10 min. The reaction was then stopped by
placing on ice and adding 20 mL of 0.5 M EDTA, and the DNA was precipitated
with 2.5 M ammonium acetate (Fisher Scientific) and 2.25 volumes of 95%
ethanol. The DNA sample was redissolved in 250 mL of TE buffer, pH 8.0 and
stored at 4OC.
The isolation of 20 kb fragments was achieved by sucrose gradient
centrifugation as follows: 12 mL of a 1040% sucrose (Fisher Scientific) density
gradient was prepared in a 12.5 mL polyallomer tube (Beduiian Instruments,
Inc., Palo Alto, CA) using STE buffer (1 M NaCI, 20 mM Tris-HCI, pH 8.0, 5mM
EDTA). Then 225 uL of Mbol partially digested DNA were heated at 65OC for 1 O
min, cooled to room temperature and loaded on the sucrose gradient.
Centrifugation was perfomed at 26,000 rpm for 24 h at 20°C, using the SW41 Ti
swinging bucket rotor (Beckman).
Using the Fraction Recovety System (Bechan), approximately 200 uL
fractions were colleded in a sample tray. A sample of 1 O uL from eech third
fraction was mixed with 3 uL of stop solution (6.6% 0.04% bromophenol blue, 20
mM EDTA) and run on 0.4% agarose gel to detemine the inclusive fractions that
contained the 20 kb genomic DNA fragment The chosen fractions were diluted
with TE bufter, pH 8.0, so that the concentration of sucrose ~ w s reduœd to 10%.
The DNA was then precipitated 0.1 5 M sodium aœtate and 2.25 volumes of
95% ethanol, and washed with 70% iœ cold ethanol, Next, the DNA was
dissolved in 10 mL of TE buffer, pH 8.0, from which 1 uL was analysed by
agarose gel electrophoresis for a quality check.
The ligation of 20 kb genomic fragments to EMBW amis was perfomed
along with a control test insert, obtained from Stratagene, La Jolla, CA, in a total
volume of 5 uL. The test reaction included: 1.0 uL of Lambda EMBC3 vector pre-
digested with BamHIIEcoRI (Stratagene), 2.5 uL of 20 kb genomic fragments
(insert), 0.5 uL of 1 OX ligation buffer (0.5 M Tris-HCI, pH 7.6, 100 mM MgCI2,
100 mM dithiothreitol), 0.5 uL of 10 mM of MTP, pH 7.5, 0.5 mL (4 units) of T4
DNA ligase (Phanacia, LKB Biotechnology AB, Uppsala, Sweden). The ligation
reaction was inwbated at 4OC for 20 hl and stopped by heating at 65OC for 15
min, and stored a -20°C before packaging. To check the DNA ligation, g 1 uL
sam~le of Iigated Lambda a n s (EMBL3 + 20 kb insert) was mn on 0.4%
agarose gel, along wïth 1 uL of pre-digested Lambda ans, and luL of 20 kb
genomic fragments (insert).
In vitro packaging of Lambda DNA (genomic DNA-EMBL3 ans ) was
perfonned using Gigapack Il Gold packaging Extract (Stratagene, La Jolla, CA),
according to the manufadurers recommendations as follows. One set of
packaging extract from a -70°C freezer was removed and placed on ice. At the
same time a sonic extract was being t h m . The packaging extract was then
thawed quickly and 2 uL of ligated Lambda DNA were added and the tube was
placed on ice. To the tube, 15 uL of sonic extract w r e added and the contents
were mixed well and spun down quickly. The tube was then incubated at room
temperature for 2 h. 500 uL of phage dilution buffer (0.1 M NaCI, 0.02 M Tris-
HCI, pH 7.4, 0.01 M MgS04) and 20 uL of chloroforrn were added and mixed
gently. The contents wiere then spun briefiy to sediment debris, and the
supematant was stored et 4°C before titration.
A culture of E. cofi host baderium P2392 (Stratagene, La Jolla, CA) w s
grown in LB medium; 0.1 7 M NaCI, 0.5% yeast extract (DIFCO Laboratories,
Detroit, MI), 1 O h tryptone (DIFCO Laboratories, Detroit, Ml), supplemented with
10 mM MgS04 (Fisher Scientific) and 0.2% maltose (BDH; The British Dnig
Houses Ltd, Poole, England). The followïng day, 0.2 mL was subcultured into 10
mL fresh medium and allowed to grow by shaking for 2.5 h at 37OC. The cells
were spun down in a sterile screw capped centrifuge tube at 2000 rpm for I O
min. The supematant was then decanted and the cells were resuspended in 5
mL of sterile 10 mM MgS04. A series of EMBL3 genomic library serial dilutions
(1 O-', loJ, 1 04, 106) were done in 0.5 mL of phage dilution buffer (SM buffer).
Four sterile polypropylene tubes (Fisher Scientific) containing 0.2 mL of bacteria
(P2392) were set up and O. 1 mL of each EMBL3 genomic dilution was added
separately. After mixing by shaking, the tubes were incubated at 37% for 20 min
to allow the bacteriophage particles to adsorb. To the first tube, 3 mL of melted
0.7% NZY top agar (85.5 mM NaCl. 8.1 mM MgS04.7H20, 1 % casein
hydrolysate, 0.5% yeast extract, pH 7.5, 1.5 % agar; DlFCO Laboratories) was
quickly added and the content was immediately poured ont0 a labeled LB plate,
prewanned a 37%. The plate was aMrled gently to ensure an even distribution
of the bacteria and the top agar. The plates were left to stand at room
temperature for 5 min to allow the top agar to harden. They ware then inverted
and inwbated at 37% for 12-16 h to allow plaques to appear and be counted.
Based on the number of plaques in the four plates, the titre of the library, in
ternis of plaque forrning units per mL (pfuImL), was determined.
Amplification of the EMB W genomic library
A sterile tube containing 10 mL of LB broth, supplemented with 0.2Oh
maltose and 10 mM MgS04, was inoculated with a single colony of E. cdi
(P2392) and let grow ovemight shaking at 30°C. A tube containing 1 0 mL
of sterïle LB broth, supplemented with maltose and MgSO., was inoculated with
1 mL of bacteria grown ovemight. and incubated for 3 h by shaking at 37OC. The
bacterial cells w r e then spun dom at 2000 rpm for 10 min, and resuspended in
5 mL of sterile 10 mM MgSO.. Four sterile tubes containing 0.2 mL of plating
bacteria were set up and 100 uL of the genomic library preparation wore added
to each tube. The tubes were inaibated at 37OC for 20 min, and 3 mL of melted
NZY top agar were added to Uie first tube. The content was then poured ont0 a
LB plate and let stand to solidify for 5 min. The same procedure was cam-ed out
with the other three tubes. The plates were inwbated at 37% and plaques
began to appear after 8 h and matured after 12 h. To each plate, 3 mL of SM
bufFer were added and incubated ovemight at 4OC. The supernatant from the
four plates was mllected in a sterile tube and chloroforrn was added to a final
volume of 5%- The phage suspension was shaken for 15 min and the debris
spun down. The supernatant was kept, and hm drops of chlorofom were added.
This amplified library was titred as described previously, and 1 mL aliquots were
stored at 4OC. For long terni storage at -70°C, dimethyl suifoxide (DMSO) (Fisher
Scientific, Fair Lawn, NJ) was added to a final volume of 7%.
Plating the EMBL3 genomic library
To screen the P. ultimum and A. flaviws genomic library for the presence
of simple sequenœ motifs, approximately lo4 plaques ware plated on a 150 mm
diameter petri plate as follows. E, colr P2392 bacterial cells suitable for plating
were prepared as described previously for titration. To a sterile tube containing
0.4 mL P2392 cells, 250 mL of 1/10 genomic library dilution were added. The
tube was then incubated at 37OC for 20 min, and 8 mL of melted NZY top agar
were added, and the tube contents were immediately poured ont0 a 150 mm
diameter agar plate (LB). The plate was incubated at 37% for 12 h and then
stored at 4OC before plaque blotting.
EMBL3 genomic library plaque blotüng
Two 150 mm diameter HybondN membranes were carefully labeled *th
identification marking and date. The first membrane was plaœd on the agar
surface and, using a sterile needle, the edges of the membrane were marked by
piercing through the membrane into the agar. This ensured correct orientation of
plaques. The membrane was removed after 1 min and plaœd, colony side up, on
3hIM paper Matman International Ltd., Maidstone, England) soaked in
denaturing solution (0.4 M NaOH, 0.6 M NaCI). The membrane was left for 7
min, then placed on 3MM paper soaked in neutralizing solution (1 -5 M NaCI, 0.5
M Tris-HCI, 0.001 M Na2EDTA, pH 7.2). The membrane was left for 3 min, then
transferred to fresh 3MM paper soaked in neutralizing solution and left for
another 3 min. The same procedure was perfomed on the second duplicate
membrane, and the two membranes were washed by submerging for 1 min in W
SSC buffer. They were then air dried. colony side up, on 3MM paper. The
membranes were covered wïth Saran Wrap and exposed to UV light (320 nm),
for 2 min, colony side next to a UV transilluminator. They were then stored at
room temperature until ready to use.
Selection of positive clones
After hybridization to specific oligonucleotide probes as described
previously, the blots w r e exposed to Kodak X-OMAT film for 16 h. Positive
clones w r e identified as plaques that hybridized to the probe. Marks on the
autoradiograrns were aligned with the plates. Plaques which hybridized to the
probe were identified. The plaque was picked by using a Pasteur pipette
equipped a nibber bulb. Mild sudion mas applied so that the plaque,
together with the underlying agar, weis drawn into the pipette. The agar plug
containing the plaque was plaœd in a sterile Eppendorf tube containing 0.5 mL
SM b d e r and 3 drops of chlorofom. The agar fragment was let to stand at room
temperature for an hour to allow the phage particles to dif ise out of the agar.
An average plaque yielded 10' to 1 o6 phage particles that could be stored
indefinitely at 4OC without loss of viability. If the plaques wwe not well separated
it was necessary to repeat the screening process to ensure that virions were
derived ftom a single clone.
Large scale isolation of phage DNA
Lambda DNA from phage lysates was purified based on the rapid
biochemical method of Kaslow (1 986) as follows. A tube containing 1 O mL of LB
broth supplemented with 0-2% maltose and 10 mM MgS04 was inoculated with a
single colony of P2392 and let grow ovemight by shaking at 30°C. The next day,
1 mL of bacterial cells was mixed with 1 uL of eluted phage (1 0' to 108 pfulmL)
and 1 mL of 10 mM Mg& and inwbated at 37OC for 20 min. The mixture was
then transferred to 500 mL LB broth, supplemented O.ZOh maltose and 10
mM MgS04, and inwbated at 37OC by shaking for 8 h. Chlorofom was added to
2%. DNAse I and RNAse A (Sigma) were added to 1 mglmL, and solid NaCl was
added to final concentration of 1 M. After incubation at 37OC for 30 min, the
aqueous phase was clarified by centrifugation at 5000 rpm at 4°C for 10 min.
Solid polyethylene glycol (PEG 8000) (Sigma) m s added to 10% wlv and the
cloudy mixture stored at 4'C for at least one hour. The intact phage were
recovered by centrifugation at 5000 rpm at 4OC for 20 min and resuspended in 3
mL of SM buffer. DNAse I and RNAse A were added to 5 mglmL and 100 mglmL,
respedively. After a 30 minute incubation at 3?C, the phage w r e lysed by
adding 300 uL of 10% SDS containing 0.5 M EDTA (pH 8.0), and 20 uL of a
100 mg/mL proteinase K (GIBCO BRL) solution in sterile water and heating the
mixture to 68OC for 30 min. The phage DNA was extracted with equal volumes of
buffer-saturated phenol (Gl BCO BRL), phenollchloroforrn, and then chloroform
(Fisher Scientific), and precipitated by adding 0.5 volume of 5 M ammonium
acetate and 2.25 volumes of 95% ethanol. After storing on iœ for 15 min, the
precipitate was rewvered by centrifugation at 10,000 rpm at 4°C for 15 min. To
the dry pellet. 1.6 mL of HPLC grade &O, 0.4 mL of 4 M NaCl and 2 m l of 13%
PEG were added. The resulting precipitate was colleded &ter incubation on iœ
for 1 h. It was centrifuged at 10.000 rpm for 15 min, rinsed with 70% ethanol,
dried and resuspended in TE bufier to a final concentration of 1 ug/mL.
Subcloning DNA fragments from phage clone into the pBluescript plasmid
(Ml3 Ks +)
BamHl digestion of the phage clone LCA from the P. ultimum BR471
genomic library and four clones (LGT1, LGT2. LCTl , LCT2) from the A. flavipes
Iibrary, revealed that fragments produced were suitable in size to be cloned
into pBluescn'pt Ml3 Ks +. The plasmid wnstnicts wntaining simple sequenœ
inserts from P. ulamum BR471 was designated pLCA and those from A. flavipes,
pGT1, pGT2, pCT1, and pCT2. Various fragment sizes were subcloned into the
pBluesaipt Ks + vector (Stratagene, San Diego, CA) according to the
manufacturer's recommendations and Maniatis et al. (1 989) as follows.
Approximately 1 ug of pM13 plasmid DNA was digested with 5 units of BamHl
endonuclease in a total volume of 1 O uL, by incubating at 37% for 3 h. The
reaction was stopped by heating at 68OC for 15 min. Approximately 1 ug of
phage DNA was completely digested with 5 units of BamHl endonuclease, in a
total volume of 10 uL, by incubating at 37OC for 20 min. The reaction was
stopped by heating et 68OC for 15 min, and the DNA fragments w r e then
precipitated with 0.5 volume of 5 M ammonium acetate and 2.25 volumes of 95Oh
ethanol. The DNA was spun down and the pellet dried by vacuum. To ligate
BamHl fragments and pM13 plasmid vedor, the dried DNA pellet was
resuspended in 5 uL of pM13 pre-digested with BamHI. To the DNA mixture, 1
uL of 1 OX ligase buffer, 1 uL of 10 mM ATP (Sigma), 2 mL of HPLC grade H20,
and T4 ligase (Phamacia), were added to ligate the DNA fragments and the
plasmid. After incubating ovemight at lS°C, the reaction was stopped by heating
at 65°C for 1 5 min. To prepare competent JM109 cells (Stratagene), 0.2 mL of
an ovemight culture of E. coli was added to 10 rnL LB medium supplemented
with 10 mM MgC12. ARer shaking at 37OC for 2.5 h, the cells m e put on iœ for
20 min. They were then centrifuged at 3,000 rpm for 5 min at 4OC, and the pellet
was gently resuspended in 3 mL of 50 rnM CaCI2 and put on ice for another 20
min. The cells w r e again œntrihiged at 3,000 rpm for 5 min and resuspended in
0.5 mL of 50 mM CaCh and put on iœ ready for transformation- In a sterile
Eppendorf tube, 10 uL of ligation mixture were mixed with 200 uL of competent
cells (JMIOS), and put on ice for 15 min. The tube was then transferred to a
42OC water bath and inaibated for 1 min. After 10 min at room temperature, 1 rnL
of prewanned LBMg medium was added, and the mixture inwbated at 37OC for
1 h. The cells were collected by centrifugation and resuspended in 200 uL of LB
medium. LB-Ampicillin (Sigma) plates containing 40 mglmL Xgal (Sigma) and
24 nglmL IPTG (Sigma) were used to select for transformants and recombinants
as described in Sambrook et al. 1989.
Cloning of PCR products
Pfimers used for the amplification of simple sequences in A. flavipes
were designed to have BamHl sites at their ends. The resulting PCR products
were treated with BarnHi and cloned into the BamHl site of the pBluescript Ks+
vector by standard methods. Transfonnants were obtained as descfibed
previously.
Purification of plasmid DNA
Small scale purification of plasmid DNA was carried out using a Magic
Minipreps DNA Purification System (Promega Co., Madison, WI) according to
the manufacturer's recommendations as follows. A 10 mL ovemight culture of E.
ooli was pelleted by centrifuging for 5 min a 3,000 rpm and the cells
resuspended in 200 uL of Cell Resuspension Solution (50 mM Tris-HCI, pH 7.5,
10 mM EDTA, 100 mglmL RNAse A). After transferring the resuspended cells to
a microcentrifuge tube, 200 uL of Cell Lysis Solution (0.2 M NaOH, 1 % SDS)
were added. and the contents wre mixed by inverting the tube several times. To
the clear, lysed cell suspension, 200 uL of neutralizing solution (2-55 M
potassium acetate, pH 4.8) were added and mixed by inverting the tube. After
centrifugation at top speed in a microcentrifuge for 5 min, the cleared
supernatant was decanted to a new microcentrifuge tube. To the tube, 1 mL of
the Magic Minipreps DNA Purification Resin was added and mixed thoroughly.
Using a 3 mL disposable syringe, the DNA solution was fun through a Magic
Minicolumn and purified with 2 mL of Column Wash Solution (200 mM NaCI, 20
mM Tris-HCI, pH 7.5, 5 mM EDTA, Diluted 1 :1 with 95% ethanol). The
Minicolumn m s transferred to a 1.5 mL microcentrifuge tube and spun dom at
top speed for 20 sec to dry the resin. The Minicolumn was again transferred to a
new microcentrifuge tube and 50 uL of TE bMer was added. After 1 min, the
DNA was eluted by spinning the Minicolumn at top speed in a microcentrifuge
tube for 20 sec. The plasmid DNA was then stored at 4OC or -20°C.
Screening clones with PCR
To an Eppendorf tube, 20 uL of phage suspension and 20 uL of a stock
solution containing 2% CTAB and 2 M NaCl were added. The tube was then
inwbated at 55OC for 10 min, and the contents were extracted Mce with equal
volumes of chlorofom. The resulting supernatant contained DNA template that
could be used diredly in PCR amplification as describeci previously.
Recombinant clones were screened using T3 and T7 primers to amplify the
inserts. Southem blotting and hybridization was perfomed with either d(GQ or
d(CTk probes.
Constnicüon of deletion clones
The Erase-a-Base system (Promega) was used to construct deletion
clones of pGT1, pCT1, pCT2, following instructions given by the manufacturer,
minor modifications. In order to ensure that the plasmid DNA used was not
nicked, it was treated with T4 DNA ligase for 2 h at room temperature.
Approximately 5 ug of plasmid DNA was used to generate each set of deletion
clones. To produce a 3' overhang each subclone was digested a specific
restriction endonuclease. pGTl and pGT2 were digested wïth BstM, pCTl was
digested with Kpnl and pCT2 was digested wïth Sstl, this treatment produced a
3' overhang which is resistant to exonuclease III digestion. A 5' protniding end,
which is sensitive to exonuclease III digestion, was created by digestion of pGT1
and pGT2 wïth Xbal, pCT1 w-th EcoRI, and pCT2 with Xbal. Exonuclease III
digestion was perfomed at 3S°C and samples (up to 12) were wllected at 30
sec intervals. AI1 restriction sites used flank the insert and are found in the
multiple doning site region of pBluescript M l 3 Ks +. Deletion clones from each
interval were ligated and transfomed in to E. d i JM109 and screened for the
presenœ of their type of simple sequence using d ( G n or d(CQ probes.
Clones from each deletion time point w r e tested for hybridization to either
d ( G n or d(Cfk. Clones which were the last to hybridize to the simple
sequence probe were seleded and sequenœd with primers developed from the
vedor (T3 or T7).
Fragment amplification of simple sequence motifs
D NA reg ions contain ing specific simple sequence motifs were am pl Aed
using the polyrnerase chain reaction (PCR) (Saiki et a/. 1988) in a reaction
mixture of 100 uL total volume containing the followïng components: 10 uL 1 OX
Taq DNA polyrnerase reaction buffer (Promega Corp., Madison, WI.); 8 uL of
deoxyrïbonucleotide triphosphates (dNTP, Phannacia) mixture (stock
concentration 2.5 mM with final concentration of each dNTP, 200 mM); 1 uL (32
pmole) of each primer; 7 uL template DNA (50-100 ng of DNA); 78.5 uL ultrapure
m e r (HPLC grade, Fisher Scientific); and 0.5 uL (2.5 units) Taq polymerase
(Promega). The reaction mixtures were overlaid with mineraf oil (Paraffin ail,
Fisher Scientific) and subjected to 25-30 cycles in a Perkin Elmer-Cetus DNA
thermal cycler (Norwalk, CT) under the following general temperature conditions:
1 min at 93OC, 1 min at 55OC, and 2 min at 72OC.
Sequencing cloned PCR products
Approxirnately 10 ug of cloned PCR product in p8luescript Ml 3 Ks + were
dried down in an Eppendorf tube and sent to: DNA Technology Unit, Plant
Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan, Canada. Cycle sequencing was performed using an automated
ABI prism version 2.1 .O sequencing apparatw. Resolution up to 700 bases was
routinely achieved using either the T3 or T7 pnmers of pBluescrïpt M l 3 Ks +.
Early sequenœ (first 250 bases) was confimed by using standard sequencing
protocols as desu3bed previously.
Sequencing of PCR products
PCR products were purified by eledrophoresis in 0.6-1 % agarose
followed by freeze-squeeze extraction of bands by a method similar to that of
Tautz and Renz (1 983). modified by Hausner ef al. (1 992) as follows. After
staining *th ethidiurn bromide, bands were wt out of the gel and frozen at
-20°C. The gel plug was placed between two layers of parafilm (Amencan
National Can, Greenwich, CT) and gently thawd by steady finger pressure. The
expressed liquid was collected and made up to 1 M NaCl and 1 % CTAB. After
incubation at 55OC for 1 O min, two chlorofomi/isoamyl alcohol (25: 1 vfv)
extractions were done, followed by precipitation of DNA by the addition of 2.25
vol umes of 95Oh ethanol. Double-stranded templates were sequenced by a
method similar to a rapid denaturation-annealing-sequencing (RDAS) technique
suggested by L.E. Pelcher (personal communication), modfied by Hausner et al.
(1992). Approximately 1 ug of lyophilized template DNA w s dissolved in 3 uL of
water and rnixed with 12 uL of tricine buffer (0.6 M tricine (Sigma), 2% NP40
(Sigma), 100 mM MgCh (Fisher Scientific), 4 uL 0.6 N NaOH (Fisher Scientific),
and 5 uL of primer solution). The standard amount of primer was 5 pmol, but this
amount was adjusted to optimize sequencing for each of the primer$ used. The
mixture vms boiled for 3 min and then transfened immediately to an ethanol bath
at -70°C. The mixture was thawed on iœ, and 4 units of Sequenase (0.5 uL)
(United States Biochemical Co., Cleveland, OH) or T i polymerase (P hamacia)
in 4 uL of Sequenase dilution buffer and 1 uL of 100 mM dithiothreitol (Sigma),
and 2 uL of [ a l p h a - = ~ ] d ~ ~ ~ (1 mCi in 100 mL; Dupont) were added. Of this
mixture, 7.1 uL were added to each of the four sequencing temination mixes
prepared as prescribed by the manufacturer for Sequenase (see also Sambrook
et al. 1 989). After incubation for 7 min at 37%. the contents of each tube was
diluted wïth 9 mL of water and precipitation was perfonned by the addition of 51
uL of ethanol(95% ethanol made to 0.12 M sodium acetate). The DNA was
pelleted by centrifugation for 30 min in a tabletop centrifuge, the supernatant
decanted, and the ethanol evaporated by heating the tube in a waterbath. The
pellet was resuspended in TE buffer containing the sequencing stop solution
(Pharmacia) and loaded on the sequencing gels.
Sequencing reaction products w r e separated by electrophoresis using
6% polyacrylamide (polyacrylamide stock solution: 97.5% acrylamide (Bio-Rad
Laboratories, Richmond, CA) and 2.5% N,N'-methylene-bis-acrylamide (Sigma),
and 48% urea (BRL) denaturing gels (Maniatis et al. 1982). The preparation of
gels, including the preparation of the acrylamide solution and the cleaning and
taping of the sequencing gel plates, were as described by Sambrook et al.
(1 989). Two loadings were spaced approximately 2 to 2.5 h apart; this allowed
for determination of 250 to 280 nucleotide stretches. Gels were vacuum dried at
80°C and exposed for 1 4 days to Kodak X-OMAT fi lm at room temperature.
DNA sequence analysis
DNA sequenœ analysis was perforrned on phage cloned DNA Common
knom motifs were screened in the cloned sequenœ using a cornputer program
(Gene Runner version 2.0). The motif and sequence patterns are described in
Table 7.
Table 7. Nucleotide sequence analysis. The follom'ng DNA motifs were used to aid in characterising the cloned DNA sequences from either P. ulamum and A. flavipes
Nucleic Acid Motif' Pattern sequenceb
AP1 BlNOlNG SlTE AP2 BlNDlNG SlTE AP3 BINDING SlTE AP4 BlNDlNG SlTE CPl BlNDlNG SlTE CP2 BlNDlNG SlTE HSTF MAT-ALPHA1 MAT-ALPHA2 N F-1 GLUCOCORTICOID RECEPTOR CCAAT BOX TATA BOX CIEBP CREB GCN4 TARGET SlTE HOMEOBOX PROTN BNDNG SlTE INF-STIMULATED RESPONSE LARIAT CONSENSUS SEQUENCE OCTAMER SEQUENCE POLY-A SIGNAL SRF SPI BlNDlNG SlTE SPLICE JUNCT - DONOR SPLICE JUNCT - ACCEPTOR T CELL ELEMENT PU-BOX TRANSLATIONAL INlT SEQ
CCCCAGGC TGASTCAG GGGTGTGGAAAG YCAGCTGYGG YN(6)RRCCAATCA YAGYN(3)RRCCAATC CNNGAANNTTCNNG TTTCCTAATTAGGAAA TTTCCTTAlTNGGTAA TTGGMN(5)GCCAAT GGTACAN(3)TGTTCT CCAAT TTATA TGTGGAAAG TGACGTCA ATGASTCAT TCAATTAAAT RGGAANNGAAACT YNYTRAY ATITGCAT AATAAA GATGTCCATArrAGGACATC GGGCGG MAGGTRAGT VVYYYVYYYYYNYAGG GGGRlTïMA AAGAGGAAAA RNNMTGG
' Motifs cited in: Benjamin Lewin, Genes V, 1994. Oxford University Press. R: A or G, Y:C or T, M:A or C, K G or T, S:G or C, W A or T, N:A or C or G or T
RESULTS AND DISCUSSION
Chapter 1
Simple Sequence Motifs in Pjtthium ulfimum
INTRODUCTiON
The objective of this study is to identify and characterize a simple
sequenœ motif in P. ulamum BR471. ONA sequence of the microsatellite motif
will be used to show that differentiation of other isolates of P. uIfimum is
possible (Materials and Methods, Table 3). The abundance of d(GT/CA) and
d(CT1GA) type simple sequence and isolation of a simple sequence motif will be
descrïbed. The simple sequence motifwW be characterized and its use as a
polymorphic DNA marker m'Il be addressed by studying its variability in other
isolates of P. ulomum from many different geographical locations.
Simple sequenœs have proved to be a valuable tool in DNA
fingerprinting in rnany organisms. Furthermore, it is a widely accepted technique
which has show great potential in population genetics, inheritance studies,
genome mapping and other uses. No studies have been conducted in the
Oomyœtes to explore if microsatellite sequences are present and can be used
as a source of polymorphic markers. Furtherrnore, there is a large collection of
isolates of P. ultimum from around the w r l d and a simple way to differentiate
these isolates from one another is needed (Materials and Methods, Table. 3).
Since isolates of P. ulomum are so closely related it is difficult to distinguish
them using techniques such as RFLP (Martin 1989). A simple molecular
technique, such as PCR, -Id be of great importance to easily differentiate
these isolates.
This study m'Il show how one simple sequenœ array will be used as a
genetic marker to distinguish isolates of Pythium ulfimum. Furthemore, this
study provided the impetus to study microsatellite DNA sequences in a more
econornically important organism. namely Aspergillus flavipes.
RESULTS
Screening the P. uIfimum library with d ( G n and d(CTb
The abundance of d(GT1CA) and d(CT1GA) simple sequence in P.
ultimum 8R471 was estimated by screening approximately 200 plaques of a
genomic library with d(GT)B and d(CT)p radio-labeled oligonucleotide probes.
Results from these data suggest that d(GT1CA) simple sequenœ is more
abundant than the d(CT1GA) type. Forty-three signals indicative of hybridization
to d(GT)s and 27 for d(CT)9 demonstrate the abundance for these types of
simple sequence in P. ultimum BR471. The genome size of P. ulomum has not
yet been deterrnined but that of P sylvafrcum, a species cansidered to be
relatively closely related to P. ulfimum, has been estimated to be approximately
37 Mbp, although meiotic instability makes this estirnate somewhat uncertain
(Martin, 1995). If it is assumed that P. ultimum has a similar genome size, and
further that each library clone has an insert averaging 20 kb, then the 200
plaques screened represent about 10% of one genome. Therefore, the
estimated number of plaques per genome wu ld be 430 and 270 for d(GT/CA)s
and d(CT1GAk respectively. This would suggest that if the simple sequences
are evenly dispened, a d(GT1CA) motif occurs at least once every 86 kb (430
copies) and a d(CT/GA) motif every 137 kb (270 copies).
Cloning and characterization of simple sequence DNA
One plaque from the genomic library which showed strong signal
hybridization to the d(GT/CA) probe was seleded. DNA from this clone
designated LCA (lambda DNA containing d(CAIGT) type repeats) was extracted
and digested with BamHl restriction endonuclease (Fig. 1 ). Southem blotting
was perforrned on this restriction digest and the DNA fragment which hybridized
most strongly to the d(GTk probe (1 -01 kb) was purified and subcloned into the
BamHI site of pBluescript Ml 3 Ks +; this subclone was designated as pLCA
DNA sequencing was perfomied on this subclone using the T7 primer. and a
cluster of d(CA1GT) simple sequence motifs was found within the first 400 bp
adjacent to the TI primer site of pBluescript Ml 3. This cluster included the
foiiowing five motifs: d(CA/GT)41 d(CA/GT)* (CK)2(CA/GT)5. d(CAIGT)Ii.
d(CAGT)sl and d(CAIGT)6 (Fig. 2). Furthemore, 3 additional repeated
sequences were identified in this region (Fig. 2, boxed areas). These larger
repeats of 6 bp fall into the category of minisatellite DNA, due to their longer
repeated structure. However, s ine these sequences are not adjacent to each
other it is unlikely that they behave as minisatellite DNA and display length
polymorphisms-
Amplification of a simple sequence motif and sequence comparisons of
simiiar regions from different isolates and species of Pythium
To test whether the region containing d(GT/CA) repeats displayed length
polyrnorphisms, primers were construded flanking the simple sequence repeats
(Fig - 2 and Table 8). These primers (Table. 8) were used in the PCR in an
attempt to amplify similar regions in different isolates of P. ulamum and in other
species of Pythium (Fig. 3 and 4). Results from polyaaylamide gel
electrophoresis indicate that the d(CNGT) motif can be amplified from isolates
of P. ultimum and not from severai other species of Pythium. Amplification from
CBS730.94 (Fig. 4 4 lane 9) is weak, but the expected bands are present.
Attempted amplification for P. inegulare BR486 (Fig. 48, lane 1) did yield a faint
band at less than 200 bp, but it m s not similar to the products obtained from P.
ultimum- In several instances more than one band appeared in each lane,
probably due to slippage during amplification (Murray et al. 1993). The most
prominent bands from P. ulfrmum isolates BR600 BR406, and BR471 w r e cut
out of the gel and the DNA sequenœd. DRerenœs were noted in the central
region involving the d(CA/GT)ll and the d(CA/GT)s motifs. In isolate BR600, the
differenœ was the expansion of the d(CA/GT)ll to C~(CAIGT)~~, but for BR406
the region between d(CA/GT)li and ~(CAIGT)S was entirely missing
reduction of d(CNGT)ll to d(CA/GT), and the d(CA/GT)5 to a single d(CA/GT)
dinucleotide (Fig. 5).
Sequence analysis of cloned ONA from P. ulfimum BR471
A search of nucleotide motifs was perfomed on the DNA sequence from
P. ultimum BR471. Four transiational initiation sequences were located in the
sequenœ (Table 9). The DNA sequenœ was also used to screen for similar
sequences found in GenBank, but no homologies were revealed other than
homology to other d(CA/GT) dinucleotide motifs.
Stability of simple sequence m o m
The identified d(CA/GT) mot l in P. ulomum BR471 was used to study its
stability in the routinely propagated isolate BR600. lsolate BR600 was
propagated in 10 cm petri plates wntaining nuttient media and incubated at two
temperatures (1 5OC and 28OC). Every other day, after the cwnplete surface of
the medium was covered by growth, a smafl peripheral portion was removed
and subcultured on another petri plate. After growth for 3,129 h at 28% and
2,567 h at 1 5OC the genomic DNA was isolated from these cultures.
The simple sequence reg ion described earlier was amplified from genomic DNA
isolated from both cultures. Polyacrylamide gel electrophoresis did not reveal
any changes in the length of the simple sequence region as compared with
BR600 that had not been propagated for a long time period (data not shown).
Apparently, the d(CA/GT) motifs in P. ultimum do not expand or contract
significantly under these conditions, and could be considered to be relatively
stable.
Table 8. Primers used for amplification of d(CAIGT) motifs in isolates of P. ultirnum.
Primer namem Sequence S to 3
FCA RCA
a F:foward primer, R:reverse primer.
Table 9. Sequenœ analysis of pLCA
DNA Sequence Motif #Sites Nucleotide Position 5-3' pattern
Translation Init Seq
'Nucleotide pattern in Fig. 2, corresponding to its complement (CCATNNC).
Fig. 1. Restriction digest with BamHl of lambda DNA clone (LCA1) (lane 1 ),
and southem blot analysis to detemine which bands hybridize to the d(GT)9
probe (lane 2).
Fig. 2. Nucleotide sequenœ of DNA from P. ultimum BR471 containing severaf
d(CA1GT) simple sequence repeats. Simple sequenœs are indicated in bold
font. Arrows above sequences indicate location of primen used in PCR to
amplify the region containing the simple sequence motifs. Boxed areas indicate
a separate repeated motif observed in thb region.
Fig. 3. Polyacrylamide gel electrophoresis of PCR products from various
isolates of Pythium: Amw indicate molecular weight standards (based on the
BRL 1 kb ladder). Lanes 1 to 1 1, P- ultimum var. ultimum and P. ultimum var.
sporangiiferum isolates: 1:BR319,2:BR418, 3:BR47lI4:8R583 5BR600,
6:BR612, 7:8R638, 8:BR639, 9:BR640, and 1 O:BR65O (P. u. var.
sporangiiferum); 1 1 :BR925. Lanes 1 2 to 14, 1 2: P. coloraturn BR483, 1 3: P.
arrhenomanes BR607 and 1 4: P.dissimile BR1 60.
Fig. 4. Polyacrylamide gel eledrophoresis of PCR products from various
isolates of P. ultimum var. ultimum and one isolate of P. inegulate. Arrows
indicate molewlar weight standards, (based on the BRL 1 kb ladder). A: Lanes
1 to 14, 1 :CBS656.58:, 2:CBS296.37, 3:CBS291.31, 4:CBS264.38,
5:CBS249.28, 6CBS729.94, 7:CBS728.94, 8:CBS726.94, 9:CBS730.94,
1 O:CBS378.34, 1 1 :CBS305.35, 12:CBS114,19, 1 3:CBS488.86, and
14:CBS398.51. B: Lanes 1 to 7, 1 :BR4û6, 2:BR612, 3:BR600, 4:BR406,
5:BR471, 6:pLCA clone containing simple sequence insert from BR471.
7:Lambda DNA (LCA) containing simple sequence from BR471.
Fig. 5. DNA sequence alignment of PCR produds frorn 3 isolates of P. ultimum
var. ultimum: BR471, BR406, and BR600. Dashed lines indicate missing
nucleotides. The d(CA/GT) simple sequenœ motifs are indicated in bold font.
DISCUSSION
The search for microsatellite DNA sequenœs in P. ultimum, done by
probing the genomic library of P. ulomum BR471 with d(GT)o and d(CT)9
revealed that d(GT1CA) and d(CT1GA) motifs are present Although the
abundance of either type of these simple sequences was quite similar, a
predominance of d(GT1CA) over d(CT1AG) was obsenred- These results show
similarity to data obtained from organisms such as humans, maize, rice, yeast,
and Aspergillus Ilavipes (Lagercrantz et al. 1993, and this study ). This is the first
report of simpfe sequence motifs in any species of Pyfhium, or any other
mernber of the Oomycetes, and results obtained from screening the genomic
library indicate that the abundance of microsatellite sequences is similar to that
found in other eukaryotes.
To investigate the structure of simple sequence motifs and to see whether
they could be the source of polymorphisms useful in strain typing, a cloning and
sequencing project was done to isolate a d(CAIG1) amy from P. ultimum
BR471 - A lambda library clone (LCA) was identified by rneans of a (GT)s probe,
and a 1 kb BamHl fragment was subcloned for further characterization. Several
other BamHI fragments from LCA were also recognized by the probe (Fig. 1 ).
This indicates that the approximately 20 kb insert contains more d(CA/GT)
arrays than those locatted in the 1 kb fragment, and suggests that the calculation
of d(CA/GT) array abundance presented earlier represents a minimum estimate,
and that the adual ftequency of d(CA/GT) arrays may be several times higher
than that estimated-
Sequencing of the subclone (pLCA) resulted in the identification of
sequences that wu ld hybridize to the simple sequence probe, but instead of a
single array of d(CNGT) repeats, five separated short arrays w r e observed,
one of them an imperfect repeat (d(CAIGT)2 (CK)~(CA/GT)S) (Fig. 2). Simple
sequence motifs larger than 10 bp have been shown to be more polymorphic
than those under a length threshold of 8 to 10 bp (Weber and May 1989). Under
this size, microsatellite motifs are usually less likely to display length
polymorphisms (Weber and May 1986). A 6 bp minisatellite-type sequence
(GCACAA) was located intenpersed in the region containing the five d(CA/GT)
repeats. Minisatellite sequenœs are usually larger than 5 bp in length, and are
arranged in a tandem array (Jeffreys et al. 1985). The 6 nucleotide repeat found
in this region, were not tandemly arranged and should probably not be termed
minisatellite type sequences. However, it is important to note that these short
motifs are comrnon in other eukaryotic genomes, and microsatellite motifs are
usually found fianking minisatellite sequences. Furalemore, it is thought that
minisatellite sequences are produœd from microsatellite motifs, as the result of
recombination events (Wright 1994). The DNA sequenœ from P. ultimum
BR471 was utilized to search for homologies in the GenBank and EMBL DNA
sequence databases. No homologies were identified with the sequence from P.
ultimum. A search of wll established DNA motifs identified 4 translational
initiation sites (Table 9). The presence of these motifs may suggest that this
region of DNA could be transcribed and translated into a protein component,
howaver, additional transcriptional motifs would need to be identified to further
support this observation.
The main reason for the investigation of a simple sequence in Pythium
was the possibility that it would be a hypervariable region yielding
polymorphisms suitable for strain typing. With this in mind, primers annealing to
regions fianking the simple sequence arrays in P. ultimum BR471 w r e designed
and tested on a collection of P. ultimum var. ultr'mum strains, on one isolate of P.
ultimum var. sporangiiferum, and on several isolates from different species of
Pythium. Abundant amplification product was obtained from al1 but one P.
ultimum isolates (exception: lane 9. Fig. 4A) but not from other species of
Pyfhium. It appears that regions flanking the simple sequence region are highly
consewed across the species, but not in other species of Pythium.
PCR products generated from the amplification of the d(CAIGT)
microsatellite produced more than one band in several instances (Fig. 3 and Fig.
4A, 48). These additional bands have been previously identified and attributed
to slippage of 2 bp increments of the Taq DNA polymerase during the
amplification of tandem repeats (Murray et al. 1 993, Weber and May 1 989,
Ginot et a1.1996). These artifacts appearing as socallad "shadow bands" are
often co-amplified very efkiently and if the artifad is produœd during the eady
cycles of the PCR it can dominate the total yield of amplification products
(Murray et al. 1993). Another reason for the generation of additional bands rnay
be that there are other sites nearby to which the primers bind.
Companson of sequences from mhium ulamum isolates BR600. BR406
and BR471 revealed several interesting features. The sequenœ of the BR406
PCR pmdud revealed that a 22 bp stretch of DNA (d(CAIGT)ll to d(CNGT)5)
was missing. The deletion of flanking DNA in a microsatellite motif is unusual
since simple sequenœ length polyrnorphisms are generally wnfined to the
microsatellite DNA motif and not their flanking regions (Pardue et a' 1987,
Weber and May 1989). The event notiœd here may indicate that sequences
flanking microsatellite amys are prone to deletions. An examination of the
sequence of the PCR product generated in BR600 reveals an additional
d(CAIGT) dinucleotide compared to BR471. The expansion noticed between
BR471 and BR600 is typical of dinucleotide repeats, however, shorter
dinucleotide motifs are less likely to show a high degree of length variation as
compared to larger dinucleotide motifs containing 30 or more repeats (Pardue et
al. 1987, Weber and May 1989).
In summary. we have identified a polyrnorphic simple sequence region in
the genorne of P. ultimum var. ultimum BR471 Hihich can be amplified by the
?CR reaction in d i f rent isolates of P. ultlmum. Martin (1 989), attempted to
distinguish isolates of P. ullimum using mitochondrial DNA RFLP analysis.
Attempts to dinerentiate isolates of P. ulamum using this technique were mostly
ineffedive, with few observable polyrnorphisms. Martin wncluded that the
elaboration of a more seledive market was needed to help differentiate isolates
of Mhiurn and that mitochondrial DNA RFLP analysis does not yield sufficient
amounts of polymorphism to distinguish the isolates used in his study. The
results presented here demonstrate the usefulness a d(CA/GT) microsatellite
motif identified in P. ulamum BR471 as a genomic lows for dÏfferentiating
isolates of both varieties P. ultr'mum.
Chapter 2
Use of RAPDs to Differenüate Isolates of Aspergillus flavipes
INTRODUCTION
The aim in this study was to develop an approach to DNA fingerprinting of
isolates of Aspergillus flavipes that wu ld produce a unique and reproducible
pattern for each isolate. A secondary objective was the compilation of primers
useful for RAPD (Random Amplification of Polymorphic DNA) amplification of
each isolate and the refinement of the RAPD protocal for this particular
organism.
Specifically, the objective was to apply the RAPD technique to the
fingerprinting of a diverse collection of 9 isolates of A. flavipes (Table 2). An
approach to molecular typing of Aspergillus strains was developed by adapting
the Random Amplification of Polymorphic DNA (RAPD) method for this purpose.
The RAPD method has gained much reœnt interest as a way of generating
genetic markers in many organisms such as plants and fungi. The main
advantage of RAPD compared to microsatellites is the ease ~ Ï t h which new
polymorphic markers can be generated.
Genomic DNA was prepared from 9 isolates of A. flavipes, and d e r
removal of RNA, polymerase chah reaction (PCR) amplifications were done
using commercially available primers with random primer sequenœs (10 mers).
The protocol was developed by investigation of the effect of the amount of
template DNA , RNAse treatment of the DNA preparation, number of PCR
cycles, and other variables. Primer sets were screened and those producing the
most visible and diverse profiles of bands were chosen for the study.
Reproducibility of the profiles was assessed by side-by-side amplification
of the independently prepared DNA samples from the same isolate. This was
done for each isolate in this study.
lsolates of A. flavipes were reœived from Apotex Fermentation Inc. Some
of these strains are currently patented and aid in the production of several
important biological wmpounds. These compounds include: imiprimine, an
antidepressant (Hufford et al. 1981 ), sesquiterpene lactone, an anticancer drug
(Clark et al. 1978), flavipucine, an antibiotic (Findlay and Kwan 1972), and
chrysanthernic acid, a biodegradable insecticide (Miski and Davis 1988).
FurViennore. certain strains have helped in the organic synthesis of biological
compounds (Davis and Talaat 1981 ) e.g. transformations of arternisnic acid for
the treatment of dnig resistant malaria (Elmarakby et al. 1988). They have also
been shown to produce penicillin (Foster and Karow 1944, (Table 4).
RESULTS
Reproducibility
The main problem with the use of RAPDs is the frequently reported la&
of reproducibility of profiles. The most probable rationale is the critical
dependence of the amplification events on the temperature profile of the
instrument used. If more than one sample is amplified in the same fun and Vien
the results wmpared, this variable should be eliminated. To test reproducibility,
two independent DNA preparations from the same inowlum were prepared and
the DNA of each sample amplified in the same fun, Le. the cells were grown on
difierant days, and the DNA prepared on different days, but the amplifications
were done at the same time, in the same machine. The reproducibility results for
A. flavipes in general are good, the profiles are reproduced satisfactorily and
the technique should be adequate for comparing a test sample against a
referenœ sample when both have been amplified in the same run. Nine isolates
of A. flavipes (Fig. 6) were amplified with 8 different primers. lsolates were from
very diverse sources (Materials and Methods Table 4). Each isolate had a
unique set of profiles, and very few coïncident bands wuld be seen.
Effect of RNAse treatment
Figure 7 shows the consequence when a DNA sample is amplified before
and after RNAse treatment. The profiles resemble each other, but the RNAse
treated samples produce more intense higher molecular weight bands M i l e the
non-RNAse treated samples have low molecular weight bands that can not be
seen in the RNAse treated sarnples. In Iight of this, samples were routinely
treated with RNAse prior to amplification.
Effect of template amount
To detennine the amount of template DNA to use for primer screening ,
each DNA sample was amplifiecl with UBC4, using four dilutions: 0, 0.1. 0.01,
and 0.001. Representative results for hnio different DNA samples may be seen in
Figure 8. At O dilution (1 uL of the DNA sample used in the 50 uL assay)
usually no amplification products were obtained. At higher dilutions useful
profiles were usually obtained and these profiles w r e comparable even though
there was a 100-fold difference in the amount of template. As can be seen in
figure 8, most discrepancies are in the intensities of the faint bands. The 0.01
dilution was routinely preferred as the template for screening of the primers.
Background Amplification
Sometimes amplification products are observed in controls to which no
template had been added (data not shown). Presumably the template for such
amplifications would be trace contaminating DNA in the reaction mix.
The bands produced were unrelated to those produced in the presence of
template DNA, furthemore, spurious bands were never observed alongside the
bands produced from added template. A significant failure rate in performing
amplifications was obsenred. Occasionally, entire sets w l d show no products,
but then they would petforni well Wen repeated. These failures were attributed
to human emr, but they indicate that a high level of consistency and care is
required Men perfoming RAPD amplifications.
RAPD amplifications
Nine isolates of A. flaMpes were amplifed with 13 different primen (Fig. 9
and 10). lsolates were from very diverse sources, and some had no information
available conceming there origin (Table 4). The unique set of profiles, few
coincident bands generated and the failure of several isolates to generate bands
fith primers (Table 1 O) indicate the isolates of A. flavipes may be relatively
distantly related. A possible explanation for the many differenœs between the
isolates could be their diverse geographic origins. Consequently the various
strains of A. tlavipes may have developed different genome organizations with
respect to the RAPD primer sites tested here. Table 10 summarizes the data
obtained from the RAPD method (Fig. 9 and Fig. IO). The data obtained in
Table 10 show that UBC-6 and OPA-5 do not produce RAPD products in isolates
1,2, and 3, furthemore, these isolates produœd fewer RAPD products than
isolates 4,5,6,7,8,9. RAPD primers, OPC-2 and 0PA4 and OPA-13, generated
RAPD products in al1 isolates of A. flavipes. Isolates 4, 5, 6 and 7 produced
RAPD produds al1 the primers tested, furthemnwe, these isolates shared
more coincidental bands than other isolates of A. flavipes.
Table 10. Results of working primers used in RAPD experiments. 0: successfbl bnght bands, O: successful faint bands, .: unsuccessful
Primer lsolate of A. flavipes
Fig. 6. The effect of old (two months) and freshly prepared DNA template from
A. flavipes #4 in RAPDs. Two independently prepared samples of DNA from A.
flavipes #4 (N=new, O=old) were amplified wVth 8 primers and electrophoresed
side by side.
Fig. 7. The effect of RNAse treated and non-RNAse treated DNA template from
A. flavipes in RAPDs. A. flavipes #8 DNA was amplified before and after
treatment of the preparation with RNAse, using 6 prirners and then
electrophoresed side by side. N=non-RNAsed, R=RNAsed.
OPC-2 OPC-5 OPC-8 OPA-2 OPA-3 OPA-5 N R N R N R N R N R N R
Fig. 8. The effect of concentration of template DNA from A. flavipes in RAPDs.
Amplification of A. flavipes #6 with primer UBC4: lanes: 1, 2, 3, 4. Amplification
of A. flavipes #7 with primer UBC4: lanes 4, 5, 6, 7. Dilution factors: lanes 1
and 5; 0, lanes 2 and 6; 111 O, lanes 3 and 7; 111 00, lanes 4 and 8; 111 000.
Fig. 9. Agarose gel electrophoresis of isolates of DNA from isolates of A.
flavipes that were amplified with different RAPD primen. The isolates of A.
flavipes are indicated above each iane in the gel (isolates 1,2,3,4,5.6,7,8, and
9). L: BRL 1 Kb ladder size standards. RAPD primers utilized are Iisted above
lanes in the gel: UBC4, OPC-2, OPA-13. OPC-5, OPA-2, and OPA-3.
Fig. I O . Agarose gel electrophoresis of isolates of DNA from isolates of A.
flavipes that were amplified with different RAPD primers. The isolates of A.
flavipes are indicated above each lane in the gel (isolates 1,2,3,4,5,6,7,8, and
9). L: BRL 1 Kb ladder sire standards. RAPD primers utilized are listed above
lanes in the gel: OPA4,OPA-9, OPA-IO, OPC4, OPC-û, and UBC4.
DISCUSSION
The principal objective of this research was to develop an approach to the
DNA fingerprinting of Aspergillus isolates which could be applied to the problem
of identifying industrially important strains. The rnethod that was judged to be
most Iikely to yield different genetic profiles between closely related isolates,
and that wbuld be practical to apply to large numbers of isolates, was the
Randorn Amplification of Polymorphic DNA (RAPD) method developed by
Williams et al. (1 990).
The RAPD rnethod has gained much attention due to its ease of
application in the generation of DNA polymorphisms. Another important
consideration in the use of RAPDs is that no sequence information is needed for
primer selection. Other methods, especially RFLP (Restriction Fragment Length
Polymorphisms) were also considered, but M e n RAPDs were observed to give
satisfactory results, these w r e set aside. Efforts were likewise made to use the
simple sequences d(GT)9 and d(CT)g as primers, in hope that PCR amplicons
would possibly be generated. These efforts were not fruitful, however, the use
of d(GTk and d(cT)~ as primers has proven to be a valuable source of
polymorphisms in typing of other species of Aspergi/lus (Meyer et al. 1993,
Belkum et al. 1993).
It is crucial to the success of the RAPD rnethod that the profiles can be
consistently produced and that they are reproducible. The two main variables
that might be expected to interfere with reproducibility are the amount of
template DNA in the assay, and the purity of the DNA preparation.
This study showad that the assay was not very sensitive to the amount of
template within certain limits, and that the presence or absence of large amounts
of RNA did not significantly affect the profiles produced. The number of cycles
of amplification beyond 35 was also not significant (data not shown). It was also
shown that DNA prepared from independent cultures of the same isolate gave
virtually identical results as long as amplification was done on the same
instrument at the same time. Attempts at using a different machine (Perkin
Elmer-Cetus) resulted in non-reproducibility of profiles. For application of the
method to specific identification problems, it was important that test
amplifications and reference amplifications be fun at the same time, in the same
instrument. Another important factor which proved crucial in performing the
RAPD technique is the care in handling and setting up of the PCR reactions. It
was noticed that controls which aintained no DNA generated RAPO produds
which were visible during agarose gel electrophoresis. Thus extreme care and
caution is paramount Hihile perfoming these reactions, since additional bands
could generate inconsistent results. To circumvent problems associated with
contamination, DNA samples wwe prepared under laminar fiow and filtered
pipette tips w r e routinely used to inhibit aerosol foming in the barre1 of the
pipette.
This study has show that it is relatively easy to find a set of random
prirners that will amplify genornic DNA from isolates of A. tlavipes- Furthemiore,
complex profiles of bands were produced. The sets of profiles provided unique
and reproducible fingerprints for each isolate Wied, and thus the developed
approach is suitable for identification of strains for the purpose of patenting or
strain sewrity measures.
Although each isolate produced a unique profile, sorne band coincidences
w r e found in this study. There are two main implications flowing from this
finding. The first is that as isolates are more and more closely related, band
coincidences should increase, and the resolution of the method should also
decrease. Thus, for very closely related isolates, such as those in strain
lineages produœd during stain improvement, the rnethod rnight not be able to
find differences. On the other hand, the presence of coincident bands rnight
make it possible to develop species-specific marken. If a band occun in al1
isolates of a species, it could be isolated and used as a diagnostic probe for
prelirninary species assignment. This wu ld be extremely useful in situations
where the species identity of an isolate is in doubt,
In sumrnary, the profiles obtained wre higbly feproducible; differences
were mainly in the intensity of minor bands. Furaiemore, the RAPD method
represents a quick and reliable tool for establishing the amount of genetic
variability in strains of A. flavipes. Fingerprints wnsisting of 13 profiles for each
isolate were generated and compared. Each isolate was found to be unique wïth
respect to the other isolates in the study. For A. flavipes very few coincidenœs
were observed. The uniqueness for each set of profiles that was achieved in
this study indicates that the approach developed here wïll be useful for
identification of strains for the purposes of patenting, culture sewrity, and also
possible in the process of keeping track of strains during strain improvement
wrk.
In the next chapter the use of a recently developed method for DNA
fingerprinting be described, namely, microsatellite DNA analysis.
Microsatellite DNA sequences have been shown to be very useful tool in
applications of DNA fingerprinting. To amplement data produœd by RAPDs,
microsatellite DNA analysis be employed as another useful tool to further
distinguish isolates of A. flavipes.
Chapter 3
Simple Sequence Motifs in AsperglIus flavipes
INTRODUCTION
The aim in this study is to estimate the abundance of different types of
simple sequences in A. flavrpes and to investigate the feasibility of using simple
sequences for DNA fingerprïnting of strains. The abundanœ of d(GT/CA) and
d(CT/GA) microsatellite DNA in A. flavipes will be wmpared to that contained in
the complete nucleotide sequence of Sacchammyces œrwisiae, which has
been currently made available on the lntemet (Virtual Genome Center,
University of Minnesota). Studies of simple sequenœs in P. ultimum showed
that they can be the source of polymorphic markers to differentiate many
isolates. Thus, the same approach developed for P. ultimum wilf be attempted
A. flavipes. Investigations will be conduded into the different types of
simple sequenœ motifs and their polymorphic behavior in different isolates of A.
flavipes.
The isolates of A. flavipes utilized in this study include ones that are
patented or are economically important for production of many biological
cornpounds (as described previously). One important compound produœd from
A. flavipes is imiprimine. lmiprimine is commonly used today for treatment of
symptoms of depression and obsessive-compulsive disorders.
The development and identification of molecular markers based on
microsatellite DNA sequenceo is becoming a important method for strain
verificationfidentification, and an aid for the identification of desirable traits.
Today, many plant and animal breeding programs incorporate microsatellite
DNA markers to help identify and select desirable genetic traits (Biosystems
Reporter, Perkin-Elmer, 1 996).
RESULTS
Presence of simple sequence in the genomes of isolates of A. flavipes
The presenœ of simple sequence in the genomes of 9 isolates of A.
flavipes was determined by genomic dot blots probed with DIG-lsbeled d(GTl9,
d(CTk, d(AT)g and d(GC)9 oligonucleotides (Fig. 1 1 A,B.C, D). Approximately 1
ug of genomic ONA from each isolate was applied to the membrane as a target
for the probe. The same amount of pBluesaipt Ml 3 Ks + plasmid DNA was
spotted on each membrane as a negative control (under lane 1, Fig. 11
A,B,C,D). The positive control spot for d(GTI9 hybridization (under lane 1, Fig.
11A) consisted of about 1 ug of cloned DNA fom P. u l ~ m known to contain
abundant d(GT1CA) motifs (pLCA, Fig. 2). For the d(CT)g hybridization (under
lane 1, Fig. 1 1 B) the plasmid pCT1 was used as a positive control due to the
high amount of d(CT1AG) simple sequence (Fig. 18). For the d(AT)s and d(GCk
hybridizations (under lane 1, Fig. 1 1A and 1 7 B respectively), 60 pg of d(AQ
and d(GC)s were applied to the hm membranes respectively. Oark spots
indicative of hybridization to the oligonucleotide probes were observed for al1 the
positive wntrols, but the degree of intensity for d(AT)@ and d(GCl9 was much
less than that for the other probes (under lane 1, Fig. 1 l A and 1 18). A possible
reason for the lower intensity for d(AT)@ and d(GCk will be addresseci in the
discussion section of this chapter. Examination of the dot bfots which w r e
probed with d(GT)@ and d ( C n probas reveals a significant difference in the
intensities of the spots among the isolates (Fig. 1 1 A and1 1 8). The differenœ
betwaen the intensities for each isolate may reflect that the number of d(GT1CA)
and d(CT1GA) motifs are different, a finding which supports the notion that these
sequences are polyrnorphic.
Estimation of the abundance of simple sequence motifs in A. flavipes
The genomic library constnided from A. ffavipes (ATCC No.16795) was
plated, and approximately 2000 plaques were observed. Plaque Iifts were
hybridized to d(GT)o, d(Cn, d(AT)9 and d(GC)g probes, producing
approximately 800 and 600 signals for d(GT)s and d(CT)@ respectively, and no
detectable signals for d(AT)s and d(GClg were observed (data not show).
Although attempts w r e made to resolve the problems associated with using
d ( A n and d(GCI9 probes by varying the hybridization temperature, and
stringency during blot washes were made, no observable hybndization was
noticed with these probes.
The abundance of d(GT1CA) and d(CT1GA) can only be estimated
because the genome size of A. flavipes is not knowri. If it is assumed that the
genome size is similar to that of A. neer, 38 Mbp (Keller et al. 1992), and that
the average size of the library inserts is 20 kb, the 2000 plaques screened
represent about 1 genome, and the numbers of d(GT1CA) and d(CT1GA) motifs
per genome are at least 800 and 600 respecüvely. The abundanœ of these
motifs wwld be greater if more than one simple sequenœ motif ocwrred in any
of the 20kb library inserts. If microsatellite motifs are evenly dispemed
throughout the genome of A. flavipes it can be inferred that a d(GT1CA) motif
ocwrs about once every 47 kb and a d(CT/GA) motif, once every 63 kb.
Abundance of GTs Cf, AT and OC type microsatellites sequences in
the genome of Sacchammyces cerevisiae
The Virtual Genome Center database penited easy access to search this
for simple sequenœ motifs in S. œrevisiae. A search was conducted using the
S. œrevisiae database for homology to 9,8,7,and 6 bp repeats for d(GT/CA),
d(CT/GA), and d(ATlTA) motifs, and 9,8,7, 6, 5, and 4 bp repeats for d(GC1CG)
motifs. The number of matches to each locus was determined and tabulated in
Table 11. The results from the search indicate that d(ATiTA) is the most
abundant type of simple sequence followed by d(GTICA), d(CT1GA) and
d(GC1CG).
Southem blot analysis of nine ATCC isolates of A. flavipes
In order to confin that d(GT/CA) and d(CT1GA) motifs are interspersed in
the genome and to show that their distribution is polymorphic within the species,
genomic DNA from eight different isolates of A. flavipes was digested with
BamHI, agarose gel electmphoresed, Southem blotted, and probed with either
d(GTI9 (Fig. 124 or d(CT)9 probe (Fig. 128). In each lane (lanes 1 to 9. Fig.
12A and 128) numerous bands appear. The positions of these bands indicate
that d(GT1CA) and d(CT/GA) motifs are dispersed in the genomes of each strain
of A. flavi's. Furthemore, the presenœ of several prominent bands in most
profiles rnay also suggest that some of the simple sequenœ motifs may be
present in the fom of a repetitive gene family.
Cornparison of the profiles for each strain (lanes 1 to 9, Fig 12A and 128)
fails to reveal any sirnilar patterns of bands or even any obviously coincident
bands that are widely shared. This observation suggests that BamHl sites are
highly polymorphic in A. tlavipes, and probing of genomic digests with abundant
interspersed elements such as d(GT)@ or d(CT)o is successful in exhibiting such
polymorphisms.
Isolation of simple sequences
Four lambda clones were isolated by hybridization of d(GT)g and d(CT)g
probes to the genomic library of A. flavipes ATCC 16795. Two lambda clones
which hybridized to the d(GT)o probe w r e named LGTI, LGT2 and two lambda
clones that hybridized to the d(CT)g probe were named LCTI, and LCT2. The
DNA from each lambda clone was isolated, treated with the endonuclease
BamHI, and obsented by agarose gel electrophoresis (fane 1, Fig. 13A, 138 and
fane 1, Fig. 14A, 14%). The agarose gels containing the DNA fragments were
Southem blotted and hybridized with d ( G n (lane 2, Fig. 1 3A, 138) and d(CT)g
probes (lane 2, Fig. 144 148). In figure 148, lane 2, an additional faint
hybridization signal is noticed above the 5.0 kb signal. Although no visible band
is seen in the agarose gel the faint signal may be due to partially restrided
lambda DNA Mich can cause a smeanng effed and the appearanœ of
additional bands. Lambda DNA fragments which hybridized to the oligo probes
(d(GT)B and d(CT)@) were purified and subcloned into the BamHl site of
pBluescript Ml 3 Ks + and designated pGT1, pGT2, pCT1, and pCT2 (Fig. 15).
Localization of simple sequence
To detemine the sequenœ and location of the simple sequenœ motifs in
each lambda subclone, deletion cloning was performed. Deletion cloning
involves the directional deletion of nucleotides in a particular DNA fragment.
Approximately 10 different clones for each subclone (pGT1, pGT2, pCT1, and
pCT2) had an increasing number of nucleotides removed from their insert. Each
of the clones were tested for hybrïdization to either d(GT)s or d(CT)s probes.
Clones which failed to hybridize to either of the probes were discarded and the
last one in the ordered series of deletions Mich still was able to hybridize was
sequenced. The nucleotide sequence of each deletion clone is given in Figures:
16:(pdGT1), 1 7:(pdGT2) ,18:(pdCT1), 19:(pdCT2) and the location of primers
and simple sequence regions are indicated. In deletion clone pdGT1, a
d(GT/CA)- motif was identified, in pdGT2 a d(GT/CA)s (AîT)(T/A)(GT/CA)s and
a d(GA/CT)s motif, in pdCT1 a d(CTIGA)17 motif and in deletion clone pdCT2 a
CTiich sequenœ was identified. In order to study the feasibility of microsatellite
DNA as a polymorphic marker in isolates of A. flavipes, primers were designed
to amplify each simple sequence motif identified in isolate M. Primers were
based on DNA sequenœ flanking the simple sequence motifs (pdGT1, pdGT2,
pdCT1, pdCT2). (Figs. 16,17,18,19 and Table 12)
Amplification of simple sequence motifs
PCR was utilized to amplify four simple sequence motifs identified from
isolate #4 in eight different isolates of A. flavipes (Table 4). The PCR products
obtained from each amplification reaction were analyzed by agarose gel
eledrophoresis (Fig. 20A and Fig. ZIA). The expected produds were obtained
from isolate ATCC 16795 (our #4), and similar products were obtained in DNO of
the other isolates: ATCC 1 101 3 (#2) and ATCC 141 36 (#9). Faint bands were
obtained *th some of the other isolates (lane 1,5,6 Fig. 20A and lane 1 Fig.
21A) and some isolates did not amplify at all. Attempts were made to Vary the
PCR conditions, however, no prominent bands w r e observed for isolates #l ,
#3, #5, #6, #7,and #8.
Southem blotting of the agarose gel (Fig. 20A and 21 6 ) and probing with
d(GT)9 (Fig. 208) and d ( C n (Fig. 21 8) showed that only isolates #2, #4, and
#9 produced intense hybridization signals. In lane 2, figure 204 a PCR product
was visible, but the hybridization signal to the d(GT)s probe was absent (lane 2,
Fig. 208). This suggested the absence of the d(GT1CA) motif in isolate #2. In
another instance, isolate ül produced a faint band (lane 1 Fig. 21 B), but no
hybridization to the d ( G n probe was obsenred at that position (lane 1 Fig. 20A).
The PCR products from each amplification (isolates #2, W, and #9) were
subcloned into the BemHl site of pBluescript Ml 3 Ks + (Fig. 22). Plasmid
constnids containing PCR products inserts were named according to the strain
of A flavipes and the motif cloned, Table 1 3.
Cloned PCR products, pAf2GTI. pAf4GT1, pAfQGT1, pAfZGT2,
pAf4GT2, pAf9GT2, pAQCT1, pAf4CT1, pAfSCT1, pAf2CT2, pAf4CT2, pAf9CT2
were sequenœd and aligned respect to the motif amplified (GT1, GT2, CTl ,
or CT2). Alignment of the GT1 motifs (pAf2GT1, pAf4GT1, pAf9GT1 Fig. 23)
revealed numerous differences in the lengths of the d(GT1CA) arrays. An
d(GTICA)* anay in isolate #4 (pAf4GT1) in isolate #2 the array was decreased
to a d(GTICA)5 and in #9 to a d(GTlCA)=. The sequenœs flanking the arrays
in the three isolates w r e mostly homologous, with isolate #2 differing at 1
position, and isolate #4 at 4 positions (Fig. 23 boxed nucleotides).
Alignment of sequenœs from the GT2 locus (pAf2GT2, pAf4GT2,
pAf9GT2 Fig. 24) showed few polymorphisms in the simple sequence anay,
wi-th isolates #Pl and #9, having the same d(GT/CA)8 (AfT)(T/A)(GT)3 motif.
However, in isolate #2, the simple sequence array had been changed to
d(GTICA)e(GAICT)4. Examination of the flanking reg ion revealed that the
d(GT/CA) motif is embedded in a d(GNCT)-rich region, starting at position 69
and ending at position 183. The shortening of the array in isolate #2 is
accompanied by the generation of more d(GA/CT) repeats in place of the
d(GT1CA) repeats. Presumably, this conversion of d(GT1CA) to d(GAICT) is due
to the proximity of the d(GA/CT) arrays on both sides of the d(GT1CA) repeat
region. As with the other aligned locus GT1 (pAQGTf, pAf4GT1, pAf9GT1) the
fianking regions were mostly homologous with isolate #Z, differing at 5 positions
and isolate #9 at 1 position (Fig. 24, boxed nudeotides). In addition to the main
d(GT1CA) array a d(GA/CT). repeat (starting at position 172) was obsewed in
isolate #4 and #9 with isolate #2 having a d(GAICT)= . Thus, 2 different types of
motifs (d(GT/CA) and d(CT1GA)) in the cloned fragment from LGT2 displayed
length polymorphisms.
In the alignment sequences from the CTI locus (pAf2CT1, pAf4CT1,
pAf9CT1 Fig. 25) showed a nurnber of short d(CT/GA) arrays (starting at
positions 21 ?and 292) surrounding the larger d(CT/GA) (position 231-280) array.
Polymorphism is confined to the main d(CT/GA) array with isolate #2 having a
d(CTGA)21 repeat, isolate #4 a d(CT/GA)17, and isolate #9 a d(CTIGA)lr repeat
In addition, isolate #2 and #4 have a gap of 4 nucleotides starting at position
114. The flanking regions of the main d(CT1GA) array are highly homologous
with isolate #9 having only one difierence and isolate #9 having 3 differences
(Fig. 25, boxed nucleotides).
The alignment of sequences from the CT2 locus (pAf2CT2, pAf4CT2,
pAfSCT2 Fig. 26) reveals an abundance of very short CT arrays scattered
throughout the sequence, with the longest array (positions 96-1 07) consisting of
a d(CT/GA)s motif in isolate #2 and a d(CT/GA)5 in isolates #4 and #S. The three
sequences are highly hornologous, with isolate #2 differing at 3 positions (not
counting the main CT array), and isolate #4 at 2 positions and isolate #9 at 1
position (Fig. 26. boxed nucleotides). lsolate #S and iK4 have a 2 nucleotide gap
starting position 106, and isolate #9 with a gap starting at position 264. Cytosine
(C) and thymine (T) nucleotides represent 7356 of the total nucleotide
composition of the amplified region. The evidence fiom this data suggests that
limited polymorphism is associated *th these type of simple sequence
organization. Though this type of region is knociun as ayptic simple sequence, it
may be the remnant of a perfect simple sequenœ repeat (Tautz 19û6). The
occurrence of cryptic simple sequenœ has been thought to anse by point
mutations and DNA slippage events during replication which result in a disnipted
and less organized dinucleotide repeat structure (Tautz 1986).
Using the primen developed in these studies, PCR reactions were
wnducted using DNA fmm A. terreus ATCC 20542, and A. ve~sicolor ATCC
1 1730 (data not show). Attempts at arnplifying similar motifs from other species
using the primers designed for A. flavipes were unsuccessful. Thus the primers
used in these experiments may be spea'es-specific for certain isolates of A.
flavipes. although they do not amplify al1 isolates.
DNA sequence analysis of plasmid constructs of DNA from A. flevipes #4
The DNA sequenœ from pdGT1, pdGT2, pdCT1, and pdCT2 were used
to scxeen GenBank and EMBL (European Moleailar Biology Laboratory)
databases using the BlRCH (Biological Research Cornputer Hierarchy) program
at the University of Manitoba. The search did not reveal any significant
homologies to known DNA sequences. Although homology was found to simple
sequenœs, the fianking regions of the microsatellite motifs in A. flavipes were
rot homologous to the sequences in aie database search. To investigate if the
DNA sequence obtained from the various clones (pdGTi ,pdGT2, pdCT1 and
pdCT2) contained known DNA motifs, a search was perfomed using a database
of well established DNA motifis (Table 7). The search revealed open reading
frames neœssary for the sequences to be tramai-bed. Furthemore, lariat
motifs which were detected could represent RNA editing functions for these
sequences (introkexon splicing of mRNA, Benjamin Lewin, Genes V, 1994)
(Table 13).
Table 11. Abundance of various nucleotide motifs in the genome of S. cerevisiae
Motif Number of loci
d(GT1CA)g 92 d(GT1CA)s 114 d(GT/CA)7 1 47 d(GT1CA)e +195 Total .................................................................. -548
d(CT1GA)g 30 d(CT/GA)a 37 d(CTIGA)7 49 d(CTIGAl6 +a Total ................................................................... 180
d(ATKA)g 572 d(ATKA)s 769 d(ATKA)i 1022 d(ATKA)e +1384 Total.. ...................................................... .., ........ .3702
d(GC1CG)s O d(GC/CG)e O d(GCICG), O d(GClCG)6 O d(GC1CG)s 3 d(GC1CG)d +36 Total.. .................................................................. -39
Table 12. List of primers developed ftom sequenœ analysis to amplify simple sequence motifs in A. flavipes. . .
Name of primer. 5' to 3 DNA sequenceb
FpGTl primer
RpGTl primer
FpGT2 primer
RpGT2 primer GCATCCGCATrCCCCCRGTGTGATCCCAGAAC
FpCT1 primer
RpCT1 primer
FpCT2 primer
RpCT2 primer GCATCCCGRTrCCATCCRA~TCCAAXdLTGC
a R:reverse primer, F:foward primer. All were derived ftom sequence analysis of
A. flavipes clones pGT1, pGT2, pCT1, pCT2.
All primers vvere constnicted with 2 adjacent BamHl sites (GGATCCGGATCC)
at the 5' end.
Table 13. Name designation of cloned PCR products from isolates of A. falvi's.
- -
Name of clone Strain of A. flavipes PCR motif cloned
Table 14. Sequenœ analysis of pdGT1, pdGT2. pdCT1, and pdCT2
DNA Sequence Motif #Sites Nucleotide Position 9-3' pattern
pGTl sequence analysis TATA Box Lafiat Consensus Seq Translation lnit Seq
pGT2 sequence analysis Lariat Consensus Seq CCAAT Box Translation lnit Seq
pCT1 sequence analysis CCAAT Box Lariat Consensus Seq TATA Box
pCT2 sequence analysis CCAAT Box TATA Box Translation lnit Seq
TATA 502 YNYTRAY 439 RNNMTGG 12/1531195
YNYTRAY 541 CCAAT 9/587 RNNMTGG 232
CCAAT 326 YNYTRAY 55/193 TATA 2913111 13
CCAAT 6741681 TATA 2661563 RNNMTGG 881
Fig. 11. Autoradiogram of genomic DNA dot blots with 9 isolates of A. flavipes.
Hybridization was conducted 4 different oligonucleotide probes. A:d(GT)s,
B I~(CT)~ , C:d(AT)g and 0:d(GC)9. Lane number corresponds to the isolate
number (Table 4). Negative and positive controls are single spots in lane 1 of
each blot.
Fig. 12. RFLP analysis of 9 isolates of A. flavipes. Genomic DNA was digested
with BamHl and electrophoresed on 0.7% agarose gel. Hybridizations are with
A:d(GT)9 or B:d(CT)s DIG-labeled oligonucleotide probes. Lane numbers
correspond to isolate number (Table 4).
Fig. 13. Restriction digests and Southem blotting of lambda DNA clones. A:
lane 1, LGTl restriction digest and lane 2, Southem blot hybridization. 6: lane
1, LGT2 restriction digest and lane 2, Southem blot hybridization. Blots were
probed wïth d(GTb DIG-labeled oligonucleotide. Hybridization signals indicate
which band from the agarose gel hybridizes to the probe.
Fig. 14. Restriction digests and Southem blotting of lambda DNA clones. A:
lane 1, LCTI restriction digest and lane 2, southem blot analysis. 6: lane 1,
LCT2 restriction digest and lane 2, Southem blot analysis. Blots were probed
with d(CT)s DlG-labeled oligonucleotide. Hybridization signals indicate which
band from the agarose gel hybridizes to the probe.
Fig. 15. Restriction digests with BamHl of cloned lambda DNA fragments in
pBluescricpt Ks (+). Lane 1 :pGTl , 2:pGT2, 3:pCTI, and 4:pCT2.
Fig. 16. Nucleotide sequence of plasmid pdGT1, from A. flavipes #4. A
d(GTk simple sequenœ repeat is indicated in bold font. Arrows indicate
primer sequences chosen for PCR reactions in order to amplify the region
containing the simple sequence motif.
GAGAACACTAAGCAT TCCGAGTGGCAAGCGACGTAACCACGCTAAAAAAA CCCGATCGTCCACGCGTGAAGCGACCGAATCTGGATCGAAGGATCATCCC AGTGCAGGCGAAGAGGGTGTTTTGTTGATGCTATCTGGACCCAGGGTTTC CCCATCGGTGGATTCTGACAGGTACTGCGGTACCGGCGGTGATTTGTCCC TGGGACGGGCGTACCTTAACGGCGTGGGATGCGCCCTCTTGGGAGGCATT
CCTGTGTCGGCCCCTCGTACTACCCGTCCCGGCAGTCAGAGTCGTACTCG ATCCTCTTGGGAGAGAGGGAGAGAGCGCGACAGAGACCGAGAGAACTGGC - TATATTTGGCGG-3 '
Fig. 17. Nucleotide sequence of plasmid pdGT2, from A. flavipes #4. A
d(GT)&T(GTh simple sequenœ repeat is indicated in bold font Amiws indicate
primer sequences chosen for PCR readions in order to amplify the region
containing the simple sequence motif.
CGAGGGTGAATTCGAGGGTTTCCAGACAGTGGTTTTTTGGGAGAGATAGGCC A TGAAGGCGCTGAGGGTGTAGGGGGCGGGTGCCGTGTTGGCGAGCACCTCA TCGAGGGAGGGACGCATGGGGGCAGAACGCGCCCGGCGGGACGGTCAGACT GAGTGGCCGGGGGTCGGGACGGTCCATGTCGTCGTCCGACTCGGAGTCGGA
C
ATGCCAGAAGAGAAGCGGGGGGTGCAGGGTTTTCTTTTTGAGCATCGCGGT CGGG TGGGGAGTGAGAGGGGGGAGAGAG TGGG TG T A TGAGCGTGAGAGGG T
GAGAGAGT-TGGTGGTGT TGCTGCCTGTCACCAGGCGGAGG ACGAGAGTTTCGGAAGA TCGGTGGGAGTCAAGGGGCCGATCGGGGGGCGTG GGGACAAAGAAGGGATTCGTCGGATTGGCGCGCCTAAAGATAGGATCCT-3 '
Fig. 18. Nucleotide sequenœ of plasmid pdCT4, from A. flavipes #4. A
d(CT)i7 bp simple sequence repeat is indicated in bold font. Arows indicate
primer sequences chosen for PCR reactions in order to amplify the region
containing the simple sequence motif.
TCAAAAGCGTCTGGAACAGCGGCGATGGTTTCCCCTGGGTCTTCCCGTCTG ATCCGACTGTATATCTCGGGCGACCTGCCTCGAGGGTGGCGATCGCACTT~ GCTACCAAGTGCGGATACATTGCGGACGGCCGAAAGTCAAAACGCGTGTCG GAATCGATTGACTTTTTGCAGAGTTTGMTTTGGGGGCAGTTTTCTCATGA TGAGCGCGGGGAGGCGAACGACATACTTGGTGCAGGGATGGCGGCACGGTG TGGCATTTCCGCTGCGGATTGGACTGCGATTGATAAGGGCCTCACTCTCCA CTCTCCCTCTCTCCACTGCACTrCTCTCTCTCTCTrCTrC~TrCTCTICTCTCIY3 TCZGCTCTTCAACACCACACTCTCTCGTCCACACTCCTTCCACCCACACC CT TCCCGGTACGTACAGGCACCCCCCGGCCTGATTCATCCGCACGGCCAGG
Fig. 19. Nucleotide sequence of plasmid pdCT2. from A. flavipes #4. A d(CT)
rich simple sequenœ area is indicated in bold font. Arrows indicate primer
sequences chosen for PCR reactions in order to amplify the region containing
the simple sequence motif.
TGACGCCTAGGGGAGCACAGCTGATATCGGACGGCGAATCCGGATGGTGGC CACCGGCGCAGGCTGGAGGGATAGGGGTGTGTCCCATCCGATCGGGATTCC GCGTCTGCACGAGAAAGTCGCCGTGGCCGCTGCATGGCGATGGGATGGTCA TCATCGAGGGGATTTATGGTGGCACACTGGACCCTGGAGAGACCGTGTGGG TGGTTCGAT TATAAGGCGAGGATACCCATCTGGTCCAGTGCGTAGATACTC TTCTGGCGGACTGTACAGTACGGACTGACTGTACCGGTTG~TGCCCCTCG TCCCTCGTCTGACTGACACTCGTCTTTTCTTCTGTCGTTCCTCTCGACTTC
CGCCCCTCCCAAGATCGATCTCCACTCCGAACCAACGGTGGGTTCTTAATC TA TACTCAGGGCTGCTCTCCCACCATCTTCCTGTGGGCGAATGTCGCCTGT ATCACTTTTGCGCCCACCCTCTT TTACGCTGTCTCTATCTTGTGATCTTTT
TCATCGAGTCTCAAGCATGCCAACCATCCTACTGCCCTCGTCGGCCGCCGC CTTTGCGCCGCGGTCCTCGCCCAACGTGGTGCTGAGCACCCGCATCGAGCC CTGGCTCACGGCCACCCTCAAGCGAGTCAACCGGGTGAAGCGACCTCTCAA TAATGTCTCCCAGCACACCCGCTGTCTGACCGAGACCCTCTCCTCGCCCAA
Fig. 20. PCR amplification of d(GT/CA) type simple sequence motifs in 9
different isolates of A. tlavi's. Lanes 1 to 9 use primers to arnplify the GT1 motif
or the GT2 motif (underlined). A: Agarose gel electrophoresis of PCR products.
B: hybridization to d(GT)8 DIG-labeled oligonucleotide probe. Lanes 1 to 9
correspond to isolate numbers of A. flavipes (Table 4).
GTl Matif GT2 Mutif
Fig. 21. PCR amplification of d(CT1GA) type simple sequence motifs in 9
different isolates of A. flavipes- Lanes 1 to 9 use primers to amplify the CTl motif
or the CT2 motif (underlined). A: Agarose gel electrophoresis of PCR produds.
B: hybridization to d(GTk DIG-labeled oligonucleotide probe. Lanes 1 to 9
correspond to isolate numbers of A. flavipes (Table 4).
Fig. 22. Restriction digests wi-th BamHl of plasmid constnicts wntaining cloned
PCR products. Lane, 1 :pAf2GTl1 2:pAf4GTl, 3:pAf9GT1, 4:pAf2GT2,
5:pAf4GT2, 6:pAf9GT2, 7:pAf2CT1, 8:pAf4CTIt 9:pAf9CTl1 1 O:pAf2CT2,
1 tpAf4CT2, 12:pAf9CT2.
Fig. 23. DNA sequence alignment of cloned PCR products from pAf2GT1.
pAf4GT1, pAf9GT1. Dashed regions (-) indicate missing nucleotides. boxed
nucleotides indicate substitutions observed in campansons to the other two
sequences. and nucleotides in bold case indicate simple sequenœ motifs.
10 20 30 4 0 50 pAf2GTZ 5 ' -GTGAAGCGAC CGAATCTGGA TCGAAGGATC ATCCCAGTGC AGGCGAAGAG pAf4GT1 5 ' -GTGAAGCGAC CGAATCTGGA TCGAAGGATC ATCCCAGTGC AGGCGAAGAG pAfSGTf 5 '-GTGAAGCGAC CGAATCTGGA TCGAAGGATC ATCCCAGTGC AGGCGAAGAG
60 70 80 90 100 GGTGTTTTGT TGATGCTATC TGGACCCAGG GTTTCCCCATC GGTGGATTC GGTGTTTTGT TGATGCTATC TGGACCCAGG GTTTCCCCATC GGTGGATTC GGTGTTTTGT TGATGCTATC TGGACCCAGG GTTTCCCCATC GGTGGATTC
110 120 130 1 4 0 150 TGACAGGTAC TGCGGTACCG GCGGTGATTTG TCCCTGGGAC GGGCGTACC TGACAGGTAC TGCGGTACCG GCGGTGATTTG TCCCTGGGAC GGGCGTACC TGACAGGTAC TGCGGTACCG GCGGTGATTTG TCCCTGGGAC GGGCGTACC
1 60 1 70 180 190 200 TTAACGGCGT GGGATGCGCC CTCTTGGGAGG CATTGTGTGT GTGT---O- T TAACGGCGT GGGATGCGCC CTCT TGGGAGG CATTGTGTGT GTGTGTGTG TTAACGGCGT GGGATGCGCC CCCTTGGGAGG CATTGTGTGT GZGTGTCTG
2 60 2 70 280 290 300 ---------- ----- ACTGG GTACATCATCC CCTCCCTAGG TCGGCCCCT TL;TYITOTET W T A C T G G GTACATCATCC C C T C C C ~ TCGGCCCCT ~ ~ - - - - - - - ---O- ACTGG GTACATCATCC CCTCCCTAGG TCGGCCCCT
CGTACTACCC GTCCCGGCAGT C-GTCGTA ~ C G A T @ ' C TTGGGAGAG CGTACTACCC GTCCCGGCAGT CAGAGTCGTA GTCGATCGTC TTGGGAGAG
360 3 70 380 386 AGGGAGAGAG CGCGACAGAGA CCGAGAGAAC TGGCTA-3 ' AGGGAGAGAG CGCGACAGAGA CCGAGAGAAC TGGCTA-3 ' AGGGAGAGAG CGCGACAGAGA CCGAGAGAAC TGGCTA-3 '
Fig. 24. DNA sequenœ alignment of cloned PCR products from pAf2GT2,
pAf4GT2, pAf9GT2. Dashed regions (-) indicate missing nucleotides, boxed
nucleotides indicate substitutions observed in compafisons to the other tvvo
sequences, and nucleotides in bold case indicate simple sequence motifs.
10 20 30 40 50 pAfZGT25 ' - GGGAGTGTGA TGCCAGAAGA GAAGCGGGGG GTGCAGGGTT TTETTTTTGA - pAf4GT25 ' - GGGAGTGTGA TGCCAGAAGA GAAGCGGGGG GTGCAGGGTT TTGTTTTTGA p A f 9 G T 2 5 ' - GGGAGTGTGA TGCCAGAAGA GAAGCGGGGG GTGCAGGGTT TTGTTTTTGA
GCATCGCGGT CGGGTGGGGA GTGAGAGGGG GGAGAGAGTG GGTGTATGAG GCATCGCGGT CGGGTGGGGA GTGAGAGGGG GGAGAGAGTG GGTGTATGAG
110 120 130 140, 150 CGTGAGAGGG TGTGAGAGAG AGA- -----A f GAGAGAfGAG CGTGAGAGGG TGTGAGAGAG AGAGTGTGTG W G T G T A T-TGTGAG CGTGAGAGGG TGTGAGAGAG AGAGZGTGTG PGZGZGTOTA TGTGZ'GTGAG
TATGAGAGAG TGAGAGAGAG T- --AATGGTGG TGTTGCTGCC TATGAGAGAG TGAGAGAGAG T- AGAATGGTGG TGTTGCTGCC
210 220 230 240 250 TGTCACCAGG CGGAGGACGA GAGTTTCGGA A A T C G G T ~ AGTCAAGGGG
;-I
TGTCACCAGG CGGAGGACGA GAGTTTCGGA AATCGGTGGG AGTCAAGGGG TGTCACCAGG CGGAGGACGA GAGTTTCGGA AATCGGTGGG AGTCAAGGGG
CCGATCGGGG GGCGTGGGGA CAAAGAAGGG ATTCTCGGAT TGG-3 ' CGGATCGGGG GGCGTGGGGA CAAAGAAGGG ATTCTCGGAT TGG-3 '
Fig. 25. DNA sequence alignment of cloned PCR products from pAf2CT1,
pAf4CT1, pAf9CT1. Dashed regions (-) indicate missing nucleotides, boxed
nucleotides indicate substitutions observed in cornparisons to the other tvuo
sequences, and nucleotides in bold case indicate simple sequence motifs.
10 20 30 p A f Z C T 1 5 ' -TGCTACCAAG TGCGGATACA TTGCGGACGG p A f 4 C T l S ' -TGCTACCAAG TGCGGATACA TTGCGGACGG p A f 9 C T l S ' -TGCTACCAAG TGCGGATACA TTGCGGACGG
60 70 80
40 50 CCGAAAGTCA AA-CACGCGT CCGAAAGTCA AAACACGCGT CCGAAAGTCA AA-CACGCGT
90 100 GAATTTGGGG GCAGTTTTCT GAATTTGGGG GCAGTTTTCT GAATTTGGGG GCAGTTTTCT
GTCGGAATCG ATTGACTTTT TGCAGAGTTT GTCGGAATCG GTCGGAATCG
ATTGACTTTT TGCAGAGTTT ATTGACTTTT TGCAGAGTTT
110 CATGATG* CATGATGAGC CATGATGAGC
120 130 WC---- GGG AGGCGAACGA 140 150
CATACTTGGT GCAGGGATGG CATACTTGGT GCAGGGATGG CATACTTGGT ~AGGGATGG
GCG---- GGG AGGCGAACGA GCGTGCGGGG AGGCGAACGA
170 180 TGGCATTTCC GCTGCGGATT
190 200 GGACTGCGAT TGATAAGGGC GGACTGCGAT TGATAAGGGC GGACTGCGAT TGATAAGGGC
1 60 CGGCACGGTG CGGCACGGTG TGGCATTTCC GCTGCGGATT CGGCACGGTG TGGCATTTCC GCTGCGGATT
210 CTGACTCTCC ACTCTCCCTCT CTCCACTGC CTGACTCTCC CTGACTCTCC
ACTCTCCCTCT CTCCACTGC
290 300 TCAACACCAC ACTCTCTCGT TCAACACCAC ACTCTCTCGT TCAACACCAC ACTCTCTCGT
310 CCACACTCCT CCACACTCCT CCACACTCCT
Fig. 26. DNA sequence alignment of cloned PCR products from pAf2CT2,
pAf4CT2, pAfSCT2. Dashed regions (-) indicate missing nucleotides, boxed
nucleotides indicate substitutions obsewed in cornparisons to the other hAlo
sequences, and nucleotides in bold case indicate simple sequence motifs.
10 20 pAf2CT2 5 ' CTGTACAGTA CGGACTGACT pAf4CT2 5 ' CTGTACAGTA CGGACTGACT pAf9CT2 5 ' CTGTACAGTA CGGACTGACT
TGACTGACAC TGACTGACAC
CCTTTTTTCT CCTTTTTTCT
210 CCTCCCAGGA C C T C C C C ~ A CCTCCCAGGA
260 ACTCAGGGCT ACFCAGGGCT ACTCAGGGCT
31 O CACTTTTGCG CACTTTTGCG CACTT TTGCG
3 60 TTCGCATATT TTCGCATATT TTCGCATATT
TCGTCTTTTC TCGTCTTTTC TCGTCTTT TC
320 CCCACCCTCT CCCACCCTCT CCCACCCTCT
30 40 GTACCGGTTG GCTGCCCCTC GTACCGGTTG GCTGCCCCTC GTACCGGTTG GCTGCCCCTC
80 90 TTCTGTCGTT CCTCTC~CT TTCTGTCGTT CCTCTCGACT TTCTGTCGTT C-CGACT
CGTTTCTTCTC CGTTTCTTCTC
230 CTCCGAACCAA CTCCGAACCAA CTCCGAACCAA
280 CATCTTCCTGT CATCZTTCCTGT CATCTTCCTGT
330 TTTACGCTGTC TTTACGCTGTC TTTACGCTGTC
J I U J I &
GGATATTGGA T-3' GGATATTGGA T-3 ' GGATATTGGA T-3'
CGGTGGGTT CGGTGGGTT CGGTGGGTT
290 GGGCGAATGT GGGCGAATGT GGGCGAATGT
340 TCTATCTTGT TCTATCTTGT TiCTATCTTGT
50 GTCCCTCGTC GTCCCTCGTC GTCCCTCGTC
100 TCCACCTCTC TCCACCTCTC TCCACCTCTC
150 CTCCCCTCCGA CTCCCTCCGA CTCCCTCCGA
200 CGCGTTCGCC CGCGT TCGCC CGCGTTCGCC
250 CTTAATCTAT CTTAATCTAT
300 CGCCTGGAT
CGCCTGGAT
350 GATCTTTTC GATCTTTTC GATCTT TTC
DISCUSSION
Four main discoveries ernerge frorn the experiments reported here. First,
hybridization of d ( G n and d ( C n probes to genomic DNA dot blots (Fig. 1A
and 1 B) of nine isolates of A. flavipes showed that d(GT1CA) and d(CT/GA)
motifs are present in their genomes. DifFerences in intensities of hybridization
signals may support the notion that d(GT/CA) and d(CT/GA) arrays are
hypervariable. Attempts to identify the presence of d(ATKA) and d(GC/CG)
motifs by hybridization with d(AT)g and d(GC)g probes were unsuccessful.
Similar results have been reported previously and have been attributed to the
self annealing of these types of oligonucleotide probes (Lagercrantz et al- 1993).
In an attempt to cirwmvent this problem, the strigency conditions during
hybridization and blot washing were changed so that d(ATKA) and d(GC1CG) - motifs could be identified, but no visible hybridization signals to genomic DNA
were observed.
Second, screening a genomic library of A. tlavipes isolate #rl (ATCC
19795) d(GT)s and d(CT)@ probes showed that the number of d(GT/CA)
motifs is predominant over d(CT/GA) repeats. Abundance between these hm
types of repeats in A. flavipes was found similar to that of S. cemvisiae (Table
1 1). Early work on the amount of simple sequence in S. cemvisiae amved at
the estimate that d(GT1CA) is about 30 times more abundant than d(CTIAG)
(Lagercrantz et al. 1993). These estimates were obtained by slot blot
hybridizations using d(GT)lo and d(CT)ro probes. ln cornparison to Lageraantz's
wo* results of the database search presented for yeast indicate that d(GT/CA)
is about 3 tintes more abundant Vian the d(CT1GA) simple sequence (Table 11 ).
Furthemore, d(AT/AT) is the most abundant type of microsatellite followed by
d(GT/CA), d(CT1GA) and d(GC/CG) respectively. In contrast to fungi, studies in
plants, (rapeseed, wheat and Norway spnice), show that d(CT/GA) is more
abundant than d(GT/CA) motifs (Lagercrantz et al. 1993).
Third, RFLP studies using (GT)* and (CTk probes showed the high
degree of polymorphism that exist in d(CT/GA) and d(CT/GA) motifs (Fig. 12).
Few are shared behiueen the isolates. If this high degree of polymorphisrn is
attributed to simple sequenœs, it suggests that BamHl sites are highly
polymorphic and that probing of genomic digests abundant interspersed
elements such as d(GT)s or d(CT)9 can be successful in demonstrating these
polymorphisms. Furtheme, the appearance of prominent hybridization bands
may indicate the presence of repetitive gene families of simple sequence motifs.
Similar data using simple sequences as probes in RFLP analysis was obtained
for the filamentous fungi Trichodema, Penicillium, Atxula, Candida, Phoma and
different species of Aspergillus (A. niger, A. furnigaius, A. temus, A flavus, A.
niduans) (Meyer el al. 1991, Lieadeldt et el. 1992). Results obtained by Meyer
(1 991 )and Lieckfeldt (1 992) also showed the usefulness of simple sequences in
RFLP studies to bnng more transparency into various taxonomic problems,
especially at the level of related species and strains.
Fourth, no previous research in the fbngi has demonstrated the use of
PCR-amplified microsatellite for strain identification. Although data obtained
from amplified microsatellites in other organisms show that these sequences are
useful as molewlar markers, no research has been conduded in fungi.
Al ignment of microsatell ite loci (GTI , GT2, CT1, CT2) in isoiates #2, #4, and #9
demonstrates that simple sequenœ arrays are hypervariable. However, many
isolates (#il #3, #5, #6, #7, #û) did not amplify these loci. The problem of
selective amplification among isolates #2, X4 and #9 can be attributed to
variation in sequences flanking the simple sequenœ motifs. In some instances
gaps and substitutions were noticed in the alignments in the loci studied (Fig.
23. 24, 25, and 26). Although these differences rnay have inhibited the primers
from amplifying a partiwlar locus (isolates 11, #3, #SI #6, #7, and #û, Fig. 20
and 21), attempts to Vary the annealing temperatures during PCR were not
successful to arnplify the microsatellite loci in these isolates. To assist in
understanding why only some of the isolates generated PCR products.
biochemical characteristics were compared among the isolates (Table 4).
lsolates #2, and #4 produœ imiprimine and transfomi sesquiterpene lactone
constunolide. but #1 produœs imiprimine too and does not generate a PCR-
amplified product. It would be interesting to know if isolate #9 shared any other
specific characteristic with #2 and #4. Although RFLP and RAPD analysis of
the isolates showed few common banding patterns, many similarities may have
grouped the three isolates vuhich amplified the simple sequenœ loci. It is
diffiwlt however, to asses an additional cornmon factor be-n the isolates
which may help explain the selective amplification of the simple sequenœ motifs
in isolates #2, #4, and #9.
The largest locus was a d(GTICA)* bp repeat in isolate #4 (Fig. 16).
Repeats of this sire are rare and none have previously been reported in fungi.
Furthemore, this locus proved to be the most polyrnorphic, with isolate #2
containing a d(GT/CA)s array and isolate #9 containing a
d(GTICA)28(NT)(OTICA). array. In the GT2 locus, the tnincation of a d(GT1CA)
array and the concomitant increase in an adjacent d(AGKC) dinucleotide repeat
in pAf2GT2 (Fig. 24) is observed. This type of apparent transformation of a
simple sequence into another has not previously been reported. Thus. simple
sequences may allow another type of variability, the conversion of one
microsatel lite to another,
In pdCT1 a d(CT/GA)17 bp array in isolate #4 was identified (Fig. 1 8).
This locus, as with pdGTl (Fig. 16), showed a similar degree of length
polymorphism. When the CT1 loci were aligned, isolate #2 the contained a
d(CTIGA)21 motif and isolate #9 a d(CT/GA),8 .
In contrast to the pure microsatellite motifs identified, a cryptic simple
sequenœ was also identified (isolate #4, Fig. 19). A CTiich array of dispersed
d(CTIGA) dinucleotieds was amplified in isolates #2, #4, #9 and the sequences
aligned. Little variation in length was observed, but a d(CTIGA)s array identified
in #2 wes reduœd to a d(CTIGAls in isolates #4 and #9 (Fig. 26).
Attempts were made to amplify the microsatellite motifs in other species of
Aspergillus. Using the primers developed in these studies, PCR readions w r e
conducted using DNA from A. temus ATCC 20542, and A. vemicofor ATCC
1 1730. No PCR produds were generated using DNA from these species of
Aspergillus. These results may indicate that the primers developed from isolate
#4 are specific for certain isolates of A. flavipes.
The reason for microsatellite sequenœ instability has remained a matter
of discussion. However, in vif10 studies have show that slipped-strand
mispairing of the newly replicated strand during the replication proœss may
result in a change in the number of tandem arrays (Tautr 1986), although no
example of this occurrence has been demonstrated in vivo for fungi. Results
shown here indicate that a DNA slippage event may have brought about an
increase and decrease in DNA simple sequence length. Furthemore, the
generation of different motifs may arise by the expansion of sequenœs flanking
certain motifs. As in the case we have illustrated, part of the d(GTICA) motif of
pAf2GT2 has been removed and an adjacent d(GA/CT) array has been
expanded (Fig. 24).
In summary, several polymorphic simple sequenœ regions have been
identified in the genome of A- flav@es #4, and these regions can be arnplified in
different isolates of A. flaijoes. DNA sequenœ analysis of the simple sequenœ
loci revealed different patterns of simple sequenœ. Alignment of sequenœs
from isolates #2, #4 and #9 showed that varying amounts of simple sequence
for each locus (GTl . GT2 or CTl ) was produœd (Fig. 23,24, and 25). These
studies show the potential of using microsatellite DNA sequenœs to identify
different isolates of A. flavipes.
CONCLUSIONS
The objective of this wwk was to identify hypervariable DNA sequences
in A s ~ l l u s flavipes and Pythium uitïmum. Hypervariable DNA sequences
were used to differentiate isolates of P. ultimum and A. flavipes- Emphasis was
given to microsatellite DNA as a source of hypervariable DNA and its use as a
molecular marker, Research also focused on the abundance, and distribution of
different types of microsatellite DNA present in the genomes of these fungi. In
addition to these studies, the RAPD (random amplified polymorphic DNA)
approach was utilized to dmerentiate isolates of A. flavi,pes.
Studies first focused on P. ultimum because a microsatellite motif had
been previously discovered (Belkhiri 1996) and many isolates of this fungus
were availabie. To detennine the abundance of d(GT1CA) and d(CT1GA)
microsatellite arrays, a genomic library w s wnstnicted and probed wi d(GT)g
and d(CTl9 oligonucleotides. Results from probing the genomic library (P.
ultimum 8R471) indicated that d(GT/CA) arrays w r e more abundant than
d(CT1GA) arrays. To detemine whether d(GT1CA) microsatellite motifs are
polymorphic a Iibrary clone which hybridized to the d(GT)g probe was subcloned
and sequenœd. The DNA sequenœ revealed a number of d(GT/CA) motifs.
with the largest being a d(GT/CA)ll. Oligonudeotides flanking the motifs m r e
synthesized and used as primen in the PCR (polymerase chain reaction)
reaction to amplify this array in different isolates of P. uitimum. This locus was
successfully amplifid in al1 25 isolates of P. ulümum. Different size PCR
produds wre obsewed for most isolates. In addition, the primen did not
generate PCR products from several other species. To detemine if the
d(CAIGT)rl locus was responsible for the generation of PCR products with
different sizes, several PCR products were cloned and sequenced. Alignment of
the sequenœs showed that PCR site differences resulted from changes in the
number of d(GT/CA) repeats in the d(GT/CA)t locus M i l e the regions flanking
this motif were unchanged. The significance of these studies is Wfold: (1 ) this
is the first method involving the use of microsatellite DNA sequences for
differentiating isolates of P. ullimum, (2) P. ultimum is an important plant
pathogen and the use of a species-specific markers can aid in detemination of a
causative infective agent.
Having established a method for the isolation and characterization of
microsatellite sequenœs in P- u/tÏmum, sirniIar studies w r e undertaken with A.
flavipes. However, first the RAPD (rapid amplified polymorphic DNA) technique
was tried because it had been shown to be a simple and inexpensive way to
generate polymorphisms as an aid in fingerprinting isolates of many organisms.
A. flavipes is a pharmaceutically important fungus capable of producing
numerous compounds, and also an important aid for studying metabolisrn of
various compounds (Elmarakby et al. 1987). At the present time no information
on the use of DNA markers is available for this fungus. The results generated by
the use of RAPDs showed unique profiles for each isolate. The procedure was
optimized and the variables studied in order to achieve reproducibility. Hovuever,
the conditions and methodology to generate specific RAPD profiles for each
isolate of A. ffaMpes were important factors in the successful application of Mis
technique. The studies presented show that the RAPD technique is an
inexpensive way to generate pol ymorphisms and the technique proved highl y
successful in differentiating al1 isolates of A. flevipes studied.
Although the RAPD method is promising, its use is not almys
successful, and as shown in this work, it requires stringent conditions for
reproducible results to be achieved, especially the temperature profile. PCR
amplified microsatellite loci on the other hand, have been show to be easily
reproduœd, and require less stringent conditions than RAPDs do. Furthemore.
microsatellite loci might be a more definitive source of polymorphisrns since they
involve the amplification of a specific locus which is highly variable (Weber
I W O ) . Wth this in mind we first detemined whether microsatellite motifs were
present in the genome of A. flavipes, and then set about to investigate if these
sequenœs are suitable as polymorphic markers for strain identification.
The abundance of d(GT1CA) and d(CT1GA) motifs was detemined.
Results show that d(GT/CA) arrays are more abundant Vian d(CT1GA) arrays.
The abundanœ of each of these motifs was also detemined for yeast from the
available complete genome sequenœ. These results also indicate that
d(GT1CA) arrays are more abundant than d(CTIGA) arrays. These results. in
conjundion with the data obtained from P. ulamum, show that d(GT1CA) motifs
are more abundant than d(CT/GA) motifs in these three species. However. in
yeast. the most abundant simple sequenœ w s d(TA/Al) and the least
abundant was d(GC/CG). Attempts to detennine the abundanœ of these latter
motifs in P. ultimum and A, flavipes w r e unsuccessful due to problems
associated with using d ( A n and d(GC)g probes.
To investigate the use of microsatellite DNA for differentiating isolates of
A. flavipes, four simple sequence loci were isolated fram strain #4 (ATCC
16795). T w loci containing d(GT/CA) motifs (GTl and GT2) and two loci
containing d(CT/GA) motifs (CT1 and CT2) were cloned and sequenœd.
Oligonucleotides flanking these motifs were synthesized and used as primen in
the PCR readion to amplify these loci in different isolates of A. flavipes (Table
4). Only an additional tvvo isolates (#2, and #9) together with #4 produced PCR
product. Each microsatellite locus was sequenced and aligned. Length
polymorphisms corresponding to simple sequence regions were obsewed in loci
GTI , GT2, and CT1. Of particular interest was the isolation of a d(GT/CA)a
motif (pdGT1, Fig. 16). This locus showed the greatest length variation among
the 4 loci discovered (GT1 ,GT2,CTl ,CT2). Another interesting observation was
the apparent conversion of a d(CT/GA) repeat into a d(CT/GA) repeat in isolate
#2 (pAf2GT2, Fig. 24). In addition to the pure unintempted microsatellite loci, a
cryptic simple sequence (locus CTZ) (pdCT2, F ig. 19) was isolated. This motif
showad little polymorphism M e n the sequenœ was aligned with that of isolates
#2 and #9 ( pAf2CT2, pAf4CT2, pAf9CT2, Fig. 26). The reg ions fianking al1 of
these loci w r e homologous. howver, and in some loci several gaps were
noticed. The presenœ of gaps flanking the microsatellite arrays in the other
isolates (#i ,#3, #5, #6, #7, #8) may be the reason M y these loci did not
produœ a PCR product.
The significance of these studies is twofold. First, very little information
conceming the use of simple sequence polymorphisms or abundance of simple
sequenœ loci is available for fungi. Second, identification and classification of
lower eukaryotes, as well as filamentous fungi, has proven to be very dificuit
M e n based exclusively on differences in rnorphology, growth characteristics or
biochemical markers. Especially in the case of imperfed fungi, the number of
useful phenotypic traits is finite. DNA fingerprinting methods used in this
research could be helpful tools in overcoming these problems.
Nearly all research in DNA fingerprinting œnten on the hurnan and
animal models and only more recently on plant and fungal systems. These
models are of primary economic interest, and they serve as examples for
investigation of ather eukaryotes. Microsatellite sequences. RAPD, RFLP, and
PCR based DNA fingerprinting techniques have only recently been employed in
DNA fingerprinting of lower eukaryotes such as fungi. Not only do these types
of fingerprinting techniques aid in differentiating isolates of a partiwlar
organism. they have also recently been used (microsatellite DNA fingerprinting),
to Klentify and assist in explaining the causative agent in many human genetic
diseases such as Fragile-X syndrome and Huntington's disease, which are
attributed to simple sequenœ instabilities (Caskey et al. 1 992). In conclusion,
the use of microsatellite DNA sequenœs to detemine abundanœ and as a
source of molewlar markers, extends ouf knowledge of important diagnostic
tools for strain identification in fungi. F utthemore, the presence, abundance,
and the polymorphic behaviour of simple sequences contributes to out cuvent
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