Download A molecular probe for Basidiomycota: the spermidine

RESEARCH LETTER
A molecular probe for Basidiomycota : the spermidine
synthase-saccharopine dehydrogenase chimeric gene
Claudia G. León-Ramı́rez1, Laura Valdés-Santiago1, Eduardo Campos-Góngora1,2,
Lucila Ortiz-Castellanos1, Elva T. Aréchiga-Carvajal3 & José Ruiz-Herrera1
1
Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato,
Guanajuato, Mexico; 2Centro de Investigación en Nutrición y Salud Pública, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico;
and 3Departamento de Microbiologı́a e Inmunologı́a, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico
Correspondence: José Ruiz-Herrera,
Departamento de Ingenierı́a Genética,
Unidad Irapuato, Centro de Investigación y de
Estudios Avanzados del Instituto Politécnico
Nacional, Irapuato, Guanajuato, Mexico. Tel.:
152 462 623 9652; fax: 152 462 624 5849;
e-mail: [email protected]
Received 7 January 2010; revised 1 June 2010;
accepted 13 August 2010.
Final version published online 15 September
2010.
Abstract
By means of an in silico analysis, we demonstrated that a previously described
chimeric gene (Spe-Sdh) encoding spermidine synthase, a key enzyme involved in
the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved
in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature
to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene
by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity
of this procedure was tested using different fungal species. As expected, positive
results were obtained only with Basidiomycota species, whereas no amplification
was achieved with species belonging to other fungal phyla.
DOI:10.1111/j.1574-6968.2010.02099.x
MICROBIOLOGY LETTERS
Editor: Jan Dijksterhuis
Keywords
Basidiomycota; spermidine synthase;
saccharopine dehydrogenase; chimeric gene;
molecular probe.
Introduction
Traditional available methods to identify and taxonomically
describe fungal isolates are mainly based on morphological
characteristics. In the specific case of Basidiomycota, the
growth characteristics and/or pigmentation of the colonies
in different media were used to distinguish some species
(Dowson et al., 1988; Burgess et al., 1995). Other techniques
involve the use of selective inhibitors or indicator substrates
(Thorn et al., 1996). These methods have the disadvantages
of being time-consuming and may lack accuracy. On the
other hand, molecular methods have proved to be specific,
sensitive, and rapid (Gardes & Bruns, 1996; Prewitt et al.,
2008; Nicolotti et al., 2009). Amplification of ITS or Intergenic Spacer Regions of the rDNA sometimes combined
with restriction analyses have been used to identify mycorrhizal, wood decay, and rust Basidiomycota species (Gardes
& Bruns, 1993; Erland et al., 1994; Prewitt et al., 2008).
FEMS Microbiol Lett 312 (2010) 77–83
Detection of specific genes has also been used as molecular
markers, for example PCR analysis of genes encoding rRNA
and intron determination in CHS genes (genes encoding
chitin synthases) (Mehmann et al., 1994), or in Gpd, the
gene encoding glyceraldehyde-3-phosphate dehydrogenase
(Gardes et al., 1990; Mehmann et al., 1994; Kreuzinger et al.,
1996). Although in general, these molecular methods may
be useful in identifying some species, none of them cover
most Basidiomycota, and may even render false positives.
Previously, we reported the presence of a bifunctional
gene encoding spermidine synthase (Spe) and saccharopine
dehydrogenase (Sdh) in the Basidiomycota fungus Ustilago
maydis, confirming previous data from Cryptococcus neoformans (Kingsbury et al., 2004). This gene contains a 5 0 -region
encoding Spe, followed, without a termination signal or a
second initiation codon, by a 3 0 -region encoding Sdh
(Valdés-Santiago et al., 2009). Apparently, this chimeric
gene is specific to Basidiomycota, because in a preliminary
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78
search, it could not be identified in several Ascomycota
species. Spe catalyzes the transfer of the aminopropyl group
from decarboxylated S-adenosylmethionine to putrescine
during spermidine biosynthesis. Regarding lysine, it is
known that fungi synthesize it via their exclusive mechanism, the a-aminoadipate pathway (see Xu et al., 2006). Sdh,
also called saccharopine reductase, catalyzes the penultimate
step in this pathway (Bhattacharjee, 1992).
In the present work, we have performed an exhaustive
analysis for the presence of a homologous gene in those
Basidiomycota species whose genome has been sequenced, in
other fungal taxa, and in the rest of living organisms
reported in data banks. With the results obtained and the
experimental data of gene amplification by PCR in different
species, we propose the use of this gene as a molecular
marker for Basidiomycota in general.
Materials and methods
Strains used and growth conditions
Yarrowia lipolytica P01A was obtained from Claude Gaillardin
(INRA), Saccharomyces cerevisiae S288C was obtained from
American Type Culture Collection (ATCC 26108), Mucor
rouxii IM80 (ATCC 24905) was obtained from Salomón
Bartnicki-Garcia (University of California, Riverside), Rhizopus oryzae 2672 was obtained from CECT (Colección
Española de Cultivos Tipo), U. maydis FB2 was obtained
from Flora Banuett (California State University, Long Beach),
Coprinus cinereus UAMH4103 was obtained from University
of Alberta Microfungus Collection and Herbarium, Ustilago
hordei 8A was obtained from ATCC (90511); Ganoderma
lucidum, Ganoderma sp., Schizophyllum commune, Pleurotus
ostreatus, Rhizoctonia solani, Agaricus bisporus, Ustilago cynodontis, Tilletia foetida, and Bjerkandera adusta belong to the
collection from Laboratorio de Micologı́a (Universidad
Autónoma de Nuevo León, Monterrey, NL, Mexico).
Fungal strains were maintained in 50% glycerol at
70 1C. For propagation, strains were inoculated in liquid
YPG medium [yeast extract (Difco), 2%; peptone (Difco),
1%, and glucose (Merck), 1%] and incubated at 28 1C for
18 h under shaking conditions (150 r.p.m.).
Escherichia coli strain ElectroMAXTMDH10BTM (Invitrogen Life Technologies) was used for transformation with the
PCR-amplified products cloned previously in TOPOTM4
vector (Invitrogen). It was grown in Luria–Bertani (LB)
liquid medium (Becton Dickinson) at 37 1C or on plates of
the same medium containing 2% agar plus the antibiotics
required for plasmid selection.
C.G. León-Ramı́rez et al.
sterile distilled water. DNA was isolated following the glass
beads lysis protocol as described by Hoffman & Wriston
(1987).
In silico search of homologs of the chimeric
gene and their alignment
Searches for homologs were performed using as query the
whole sequence of the U. maydis chimeric gene (EMBL
accession number FN178523; Valdés-Santiago et al., 2009) at
the NCBI (http://www.ncbi.nlm.nih.gov), and The Joint
Genome Institute, U.S. Department of Energy (http://
genome.jgi-psf.org), using BLASTN, BLASTP or TBLASTN programs (Altschul et al., 1990). Sequence alignments of
putative chimeric Spe-Sdh gene fragments were performed
using CLUSTAL W (Thompson et al., 1994).
Degenerate primers design
Degenerate primers were designed at the most highly conserved
regions to the chimeric genes based on their alignment. The
sequences of these primers are the following: forward primer
(F-CHIM) 5 0 -CA(G/A)GA(G/A)ATGAT(C/T)GC (T/C/G) CA
(T/C)(C/T)T(C/T/G/A)CC-3 0 , and reverse primer (R-CHIM)
50 -(C/T)T(C/T/G/A)GG(C/G/A)T(A/C)(C/A)AA (G/A)TTCTC
(G/C/A/T)TGGTC(G/C/T/A)(T/C/G)C-3 0 .
PCR conditions
A standard protocol was performed for PCR amplification:
an initial denaturation at 94 1C for 5 min, followed by 35
cycles with the following program: DNA denaturation at
94 1C for 1 min; primer annealing at 60 1C for 1 min, DNA
extension at 72 1C for 2 min, with a final extension cycle at
72 1C for 10 min. The PCR reaction products were separated
by electrophoresis in 0.7% agarose gels and stained with
ethidium bromide.
Cloning and sequencing of PCR products
PCR products amplified with the degenerate primers described above were either cloned or not cloned into a
TOPOTM4 vector (Invitrogen) and sequenced using an ABI
PRISM Model 370 sequencer (Applied Biosystems). Cloned
fragments were sequenced from the vector with the universal primers, whereas not cloned fragments were sequenced using the degenerate primers.
Results and discussion
DNA isolation
Identification of homologs of the chimeric gene
Cells were grown as described above for 18 h, harvested
by centrifugation, and washed twice by centrifugation with
During the search of homologs as described in Methods, we
observed that only fungi belonging to Basidiomycota
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FEMS Microbiol Lett 312 (2010) 77–83
79
Chimeric gene in Basidiomycota
contained homologs of the Spd-Sdh chimeric gene. No
homologs were detected, not only in the rest of the fungal
groups but also in any other living organism.
Among the sequences identified, besides the U. maydis
gene described previously (Valdés-Santiago et al., 2009),
the ones that possessed NCBI accession numbers corresponded to the following species: Coprinus cinereus
(EAU83678), Malassezia globosa (EDP44132), Laccaria
bicolor (EDR1322), and Cryptococcus neoformans
(EAL19736). Other species possess homolog genes encoding
proteins with the identification number (protein ID)
from the Joint Genome Institute: Heterobasidion annosum
7(34012), Tremella mesenterica (74272), Sporobolomyces roseus (23418), Pleurotus ostreatus (172366), Postia
placenta (107976), Melampsora laricis populina (73077),
and Phanerochaete chrysosporium (9263). The Puccinia
graminis homolog corresponded to PGTG_06954 (Broad
Institute).
Alignment of chimeric genes with their
corresponding independent homologs
A comparison of the chimeric gene Spe-Sdh nucleotide
sequence from the Basidiomycota species reported in databases with the independent Spe and Sdh genes present in
species of Ascomycota (S. cerevisiae and Aspergillus fumigatus) revealed the presence of two distinct regions. The one
located at the 5 0 - region showed high homology with the
Spe genes, whereas the one present at the 3 0 -region was
homologous to the Sdh genes; both were linked through a
region of approximately 60 nucleotides without homology
(not shown). As expected, the alignment of amino acid
sequences encoded by these genes showed the same pattern
of homology, demonstrating the high preservation of the
gene in the Basidiomycota (not shown). With these data we
designed degenerate primers to be used for PCR amplification of the chimeric genes. The forward primer was selected
(a)
5′
3′
TTGGGCTACAAGTTCTCTTGGTCGTC
CTTGGATCAAAGTTCTCGTGGTCGTC
CTCGGCTACAAGTTCTCATGGTCGGC
CTCGGCTACAAGTTCTCGTGGTCGTC
CTCGGCTACAAATTCTCCTGGTCCTC
CTCGGCTACAAGTTCTCCTGGTCGTC
CTCGGGTACAAGTTCTCTTGGTCCCC
CTTGGCTACAAGTTCTCGTGGTCTTC
CTGGGCTACAAATTCTCGTGGTCTTC
TTAGGATACAAGTTCTCCTGGTCATC
* ** ** ** ***** ***** *
1 CAAGAGATGATCGCTCACTTGCC
2 CAAGAGATGATCGCTCACCTTCC
3 CAGGAGATGATCGCTCATCTACC
4 CAGGAAATGATTGCACACCTCCC
5 CAAGAGATGATCGCCCATCTCCC
6 6CAGGAGATGATTGCTCACCTTCC
7 CAGGAGATGATCACTCATCTTCC
8 CAGGAGATGATCGCCCATCTCCC
9 CAAGAGATGATCGCTCATTTGCC
10 CAGGAAATGATTGCGCATATACC
** ** ***** ** ** * **
(b)
Kb
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
M
2
1.6
1
Fig. 1. Amplification of a fragment from the Spe-Sdh chimeric gene by PCR. (a) Schematic representation of the multiple alignment of putative Spe-Sdh
chimeric genes reported in the GenBank. Black box, Spe region of the gene; speckled gray box, inter ORF region; white box, Sdh region of the
gene. Location of the sequences selected for degenerate primer design are schematically indicated. 1, Coprinus cinereus; 2, Sporobolomyces roseus; 3,
Postia placenta; 4, Malassezia globosa; 5, Laccaria bicolor; 6, Ustilago maydis; 7, Cryptococcus neoformans; 8, Phanerochaete chrysosporium; 9,
Puccinia graminis; 10, Melampsora laricis populina. (b) Gel electrophoresis of PCR products amplified with the degenerate primers from DNA of
different species. M, molecular marker; lane 1, Ustilago maydis; lane 2, Bjerkandera adusta; lane 3, Tilletia foetida; lane 4, Ustilago cynodontis; lane 5,
Ustilago hordei; lane 6, Rhizoctonia solani; lane 7, Schizophyllum commune; lane 8, C. cinereus; lane 9, Pleurotus ostreatus; lane 10, Ganoderma lucidum;
lane 11, Ganoderma sp.; lane 12, Agaricus bisporus; lane 13, Saccharomyces cerevisiae; lane 14, Yarrowia lipolytica; lane 15, Rhizopus oryzae; and lane 16,
Mucor rouxii.
FEMS Microbiol Lett 312 (2010) 77–83
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Published by Blackwell Publishing Ltd. All rights reserved
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C.G. León-Ramı́rez et al.
Fig. 2. Alignment of the sequences encoded by the fragments of the chimeric genes belonging to different Basidiomycota species amplified in this work.
80
2010 Federation of European Microbiological Societies
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FEMS Microbiol Lett 312 (2010) 77–83
81
Chimeric gene in Basidiomycota
at the 3 0 -end of the region with homology to Spe, and the
reverse primer was designed from the homologous region at
the 5 0 -end of the Sdh, in such a way that the amplification
fragment covered the nonhomologous region that separates
both coding regions (see Fig. 1a).
Specificity of degenerate primers, and cloning
and sequencing of the PCR products
The sequences of the fragments were deposited in GenBank, with the following accession numbers: Ustilago cynodontis, FN646089; Tilletia foetida, FN646090; Bjerkandera
adusta, FN646091; Rhizoctonia solani, FN822770; Schizophyllum commune, FN822771; Ustilago hordei, FN822772; Ustilago
maydis, FN822773; Coprinus cinerea, FN822774; Pleurotus
ostreatus, FN822775; Ganoderma lucidum, FN822776; Agaricus bisporus, FN827330; and Ganoderma sp., FN827329.
Phylogenetic analyses
Using the PCR conditions described above and the designed
degenerate primers, it was possible to amplify DNA fragments of the predicted size from genomic DNA of all the
Basidiomycota species tested (see Materials and methods),
whose genomes have been sequenced or not, that represented the three subphyla from Basidiomycota. The size of
the fragments (around 1300 bp) coincided with the expected
values. On the other hand, and as expected, no such
amplification occurred when DNA from Ascomycota or
Zygomycota species was used as template (Fig. 1b).
The PCR products corresponding to the Basidiomycota
species analyzed in this work were sequenced. Alignment of the
encoded sequences revealed their high conservation (Fig. 2).
Additionally, the encoded sequences of the amplified fragments from Basidiomycota species whose genomes had been
previously sequenced were compared with those existing in
their corresponding data banks. The results obtained confirmed the fidelity of the PCR amplification (Table 1). The
differences observed can be explained by the fact that
different isolates were used in these studies.
The sequences of the regions corresponding to the fragments
amplified by PCR from the Spe-Sdh genes obtained in this
Table 1. Comparison of chimeric Spe-Sdh sequences determined in this
work with corresponding sequences reported in GenBank
Species (ID)
Identity
Ustilago maydis
(http://www.ncbi.nlm.nih.gov/protein/71023471)w
Coprinus cinereus
(XP_001838101.1)w
Pleurotus ostreatus
(ID 114149)z
Agaricus bisporus
(ID 188516)z
Schizophyllum commune
(ID 257519)z
97% (357/367)
96% (356/369)
96% (397/410)
85% (308/360)
99% (407/409)
Percentage of amino acid identity (no. of identical residues/no. of
residues compared).
Sequences obtained from NCBI.
z
The Joint Genome Institute.
w
Puccinomycotina
Ustilaginomycotina
Fig. 3. Dendrogram of the sequences
corresponding to the amplified fragments of
the putative Spe-Sdh genes from species
representative of the three Basidiomycota
subphyla. Sequences of the fragments from
Ustilago cynodontis, Tilletia foetida, Bjerkandera
adusta, Agaricus bisporus, Rhizoctonia solani,
Schizophyllum commune, Ustilago hordei,
Ustilago maydis, Coprinus cinerea, Pleurotus
ostreatus, Ganoderma lucidum and Ganoderma
sp. were obtained in this work; the rest were
obtained from databases (see Materials and
methods).
FEMS Microbiol Lett 312 (2010) 77–83
Agaricomycotina
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c
58
Malassezia
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Ganoderma sp.
bisporus
Agaricus
adusta
Bjerkandera
lucidum
Ganoderma
ostreatus
Pleurotus
56
47
62
49
60
77
61
81
69
77
67
Coprinus
cinereus
58
57
57
54
77
63
63
64
62
81
64
62
81
79
58
Ustilago maydis 56
Ustilago hordei
commune
Schizophyllum
62
60
solani
62
Rhizoctonia
62
54
62
68
Tilletia foetida
cynodontis
Ustilago
mesenterica
Tremmella
annosum
Heterobasidion
larici-populina
Melampsora
graminis
Puccinia
65
62
Phanerochaete
chrysosporium
62
Laccaria bicolor
globosa
64
Postia placenta
roseus
100
55
46
57
49
54
51
69
67
56
72
73
72
51
57
57
56
56
57
100
placenta globosa
roseus
Sporobolomyces 100
chaete
Phanero-
73
66
78
65
79
74
57
57
80
62
62
63
59
82
62
60
77
100
bicolor
72
61
86
64
75
67
59
59
74
64
64
65
60
77
65
63
100
sporium
Malassezia Laccaria chryso-
Postia
myces
Sporobolo-
57
48
61
50
57
56
54
53
61
58
59
58
51
62
80
100
59
51
64
51
63
60
56
53
62
59
60
60
54
64
100
graminis populina
Puccinia sora larici-
MelampTremmella
Ustilago
Tilletia
74
66
78
66
78
71
61
60
79
65
66
66
61
100
58
48
61
51
59
52
52
53
61
58
57
57
100
60
49
65
52
60
53
91
94
63
97
95
100
60
48
64
52
60
53
96
91
62
94
100
annosum mesenterica cynodontis foetida
basidion
Hetero-
Table 2. Homology matrix of the Spe-Sdh gene fragments from different Basidiomycota species
60
48
65
52
59
52
90
97
62
100
solani
ctonia
Rhizo-
72
63
76
64
75
70
58
58
100
56
46
59
50
54
50
89
100
56
45
58
51
54
50
100
maydis
Ustilago Ustilago
commune hordei
phyllum
Schizo-
66
64
68
60
71
100
cinereus
Coprinus
erma
andera
Ganod- Bjerk-
70
62
78
62
100
84
56
65
100
73
61
100
ostreatusa lucidum adusta
Pleurotus
Gano-
61
100
100
bisporus sp.
Agaricus derma
82
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FEMS Microbiol Lett 312 (2010) 77–83
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Chimeric gene in Basidiomycota
study, and those reported in the databases, were used for the
construction of a phylogenetic tree. The results obtained
showed the phylogenetic relationship (Fig. 3) and similarity
(Table 2) among the fragments belonging to the different
species analyzed that represent the three subphyla from Basidiomycota: Puccinomycotina, Ustilaginomycotina, and Agaricomycotina, respectively. It may be also observed that the
dendrogram obtained (Fig. 3) coincides with the phylogenetic
division of the Basidiomycota subphyla, confirming the unique
and common origin of the chimeric gene in this phylum.
It is interesting to recall that the chimeric gene encoding
Spe and Sdh is specific to Basidiomycota, whereas biosynthetic Sdh genes from other non-Basidiomycota fungal
species exist in a free independent form. Additionally, the
catabolic Sdh gene may be chimeric with the gene encoding
lysine ketoglutarate reductase, which is the next enzyme
involved in the catabolism of lysine. In other organisms, the
catabolic Sdh gene may be bound to a motif that is related to
alanine dehydrogenase.
The reasons behind the appearance of the Spe-Sdh
chimeric gene are obscure, because there does not appear
to be a direct relationship between the metabolism of
polyamines and lysine. The event should have occurred in a
common ancestor of Basidiomycota, as it is present in all the
modern members of the phylum, and as hypothesized
previously (Valdés-Santiago et al., 2009), it is possible that
both genes remained associated throughout evolution, because the high cost of losing simultaneously the pathways
leading to the synthesis of different essential metabolites.
The results presented here indicate that, as mentioned
repeatedly, the Spe-Sdh chimeric gene is specific to Basidiomycota, being absent not only in any other fungal group but
also in any other eukaryotic taxa. Therefore, it is a specific
marker of the phylum Basidiomycota, and its detection
undoubtedly will be the most useful method for the validation of any isolate belonging to this phylum.
Acknowledgements
The present work was partially supported by Consejo Nacional
de Ciencia y Tecnologı́a (CONACYT), Mexico. L.V.S. is a
doctoral student supported by a fellowship from CONACYT.
L.O.C., E.T.A.C. and J.R.H. are National Investigators, Mexico.
References
Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215: 403–410.
Bhattacharjee JK (1992) Evolution of a-aminoadipate pathway
for the synthesis of lysine in fungi. Evolution of Metabolic
Function (Mortlock RP, ed), pp. 47–80. CRC Press, Boca
Raton, FL.
FEMS Microbiol Lett 312 (2010) 77–83
Burgess T, Malajczuk N & Dell B (1995) Variation in Pisolithus
based on basidiomycetes and basidiospore morphology,
culture characteristics and analysis of polypeptides using 1D
SDS-PAGE. Mycol Res 99: 1–13.
Dowson CG, Rayner ADM & Boddy L (1988) Inoculation of
mycelial cord-forming Basidiomycota into woodland soil and litter
II. Resourse capture and persistence. New Phytol 109: 343–349.
Erland S, Henrion B, Martin F, Glover LA & Alexander IJ (1994)
Identification of the ectomycorrhizal basidiomycete Tylospora
fibrillose Donk by RFLP analysis of the PCR-amplified ITS and
IGS regions of ribosomal DNA. New Phytol 126: 525–532.
Gardes M & Bruns TD (1993) ITS primers with enhanced
specificity for Basidiomycota – application to the identification
of mycorrhizae and rusts. Mol Ecol 2: 113–118.
Gardes M & Bruns TD (1996) ITS-RFLP Matching for
identification of fungi. Method Mol Biol 50: 177–186.
Gardes M, Fortin JA, Mueller GM & Kropp BR (1990) Restriction
fragment length polymorphism in the nuclear ribosomal DNA
of four Laccaria spp.: L. bicolor, L. laccata, L. proxima and
L. amethystine. Phytopathology 80: 1312–1317.
Hoffman CS & Wriston F (1987) A ten- minute DNA preparation
from yeast efficiently releases autonomous plasmids for
transformation of Escherichia coli. Gene 57: 267–272.
Kingsbury J, Yang Z, Ganous T, Cox G & McCuster J (2004) Novel
chimeric spermidine synthase-saccharopine dehydrogenase
gene (SPE-LYS9) in the human pathogen Cryptococcus
neoformans. Eukaryot Cell 3: 752–763.
Kreuzinger N, Podeu R, Gruber F, Göbl F & Kubicek CP (1996)
Identification of some ectomycorrhizal basidiomycetes by PCR
amplification of their gpd (glyceraldehyde-3-phosphate
dehydrogenase) genes. Appl Environ Microb 62: 3432–3438.
Mehmann B, Brunner I & Braus GH (1994) Nucleotide sequence
variation of chitin synthase genes among mycorrhizal fungi and its
potential use in taxonomy. Appl Environ Microb 60: 3105–3111.
Nicolotti G, Gonthier P, Guglielmo F & Garbelotto MM (2009) A
biomolecular method for the detection of wood decay fungi: a
focus in tree stability assessment. Arboriculture Urban Forestry
35: 14–19.
Prewitt ML, Diehl V, McElroy TC & Diehl WJ (2008) Comparation
of general fungal and basidiomycete-specific ITS primers for
identification of wood decay fungi. Forest Prod J 58: 66–71.
Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W:
improving the sensitivity of progressive multiple alignment
through sequence weighting, position specific gap penalties
and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
Thorn RG, Reddy CA, Harris D & Paul EA (1996) Isolation of
saprophytic basidiomycetes from soil. Appl Environ Microb 62:
4288–4292.
Valdés-Santiago L, Cervantes-Chávez JA & Ruiz-Herrera J (2009)
Ustilago maydis spermidine synthase is encoded by a chimeric
gene, required for morphogenesis, and indispensable for
survival in the host. FEMS Yeast Res 9: 923–935.
Xu H, Andi B, Qian J, West AH & Cook PF (2006) The
a-aminoadipate pathway for lysine biosynthesis in fungi. Cell
Biochem Biophys 46: 43–64.
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