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Design of degenerated primers from bioinformatics online software for putative ACE-1 transcription
factor in Peniophora sp.
Materials
and
methodsThe aminoacid
sequence of ACE‐1 from Phanerochaete
chrysosporium
was
used
as
reference
contrasting
it
against
database
published
on
BLASTp
(Basic
local
Alignment Search Tool protein) (see figure 2).Then, the
selected similar sequences were aligned using T‐Coffee
[5]
and
ClustalW2
[6]
(see
figure
3),
in
order
to
determine
the
score.
The
Prosite
[7],
was
used
to
determinate
if
the
conserved
regions
correspond
with
functional
sites
of
protein.
In
order
to
design
degenerated
primers,
the
highly
conserved
region
was
selected,
the
selected
aminoacid
sequence
was
decoded
with
Expasy
[8],
then
we
search
the
ORF
of
the
aminoacid
sequence
with
ORF
finder
from
NCBI
(see figure 7), and the primers picked up were analized
with Primer3 [9] (see table 1 and table 2) and Fast PCR
software.
The
sequence
fragment
obtained
was
analyzed
again to
confirm
its
validity
in
silica.
Then
we
added
a
restriction
site
to
Eco‐R1
enzymes
for
cloning
the
fragment
in
the
future,
the
obtained
primers
were
analyzed with NebCutter
(see figure 6).
ResultsIt was found a conserved domain about 50 aminoacids
from
about
80‐90%
of
similarity
on
alignment
with
T‐
coffee
and
Clustalw2.
It
could
be
observed
that
the
conserved
motif
codifies
a
copper‐fist
DNA
binding
domain (see figure 1). The score obtained with T‐Coffee
was
higher
than
the
score
given
by
clustalw2,
so
we
decided
to
use
the
first
software.
At
this
region
we
picked
up
the
primers
(see
figure
4
and
5).
We
used
wooble
pairing
to
reduce
the
degeneration
percent
(about
50%)
of
the
primers,
to
which
we
included
the
restriction site to EcoR1 enzyme.
ConclusionThese
results
suggest
that
the
conserved
domain
we
found must be due to function similarities of the ACE‐1
with
related
proteins
and
the
difference
must
be
aminoacid
synonymous
substitutions.
From
these
results
we
concluded
that
the
use
of
online
software
has
facilitated
and
has
accelerated
investigations
on
molecular biology research nowadays.
Minimus Maximus Optimus
Tm 50 67 60
%CG 40 60 50
Fragment lenght 100
Primer lenght 18 24 20
Acknowledgements:The authors thank the Experimental Mycology Department at the University of Buenos Aires and the Culture Collection of
the Faculty of Forestry Eldorado of the Universidad Nacional
de Misiones for the kind fungi supply. Part of
the
experimental
work
was
funded
by
the
Fundación
Banco
Río and
Secretaría
de
Ciencia y Tecnología de la Universidad Nacional de Misiones, through
the
respectively
grants
for
innovation projects. M.I. Fonseca has a fellowship for doctoral studies of CONICET, Argentina.
References1. Fonseca MI, Shimizu
E, Zapata PD, Villalba LL: Copper
inducing
effect
on
laccase
production
of
white
rot
fungi native
from
Misiones (Argentina), Enzyme and Microbial technology 2010, 46:534‐539. 2. Piontek
K; Antorini
M, Choinowski
T: Crystal
Structure
of
a Laccase
from
the
Fungus
Trametes
versicolor
at
1.90‐Å
Resolution
Containing a Full Complement of Coppers. The Journal of Biological 2002, 40 (277): 37663–37669. 3. Polanco
R, Canessa
P, Rivas A, Larrondo
LF, Lobos S, Vicuña R: Cloning and functional characterization of the gene encoding the
transcription factor Ace1 in the Basidiomycete
Phanerochaete
chrysosporium. Biol
Res 2006, 39: 641–648. 4. Álvarez JM, Canessa
P, Rodrigo A, Polanco R, Santibáñez PA: Expression
of
genes encoding
laccase
and
manganese‐dependent
peroxidase
in the fungus Ceriporiopsis
subvermispora
is mediated by an ACE1‐like copper‐fist transcription factor. Fungal Genetics and
Biology
2009, 46: 104–111. 5. T‐
Coffee Multiple sequence alignment [http://www.ebi.ac.uk/Tools/t‐coffee/index.html?] 6. ClustalW2
Multiple
sequence
alignment
[http://www.ebi.ac.uk/Tools/clustalw2/index.html?] 7. Database of protein domains, families and functional sites. [http://www.expasy.ch/prosite/] 8.
Translate
tools
[http://www.expasy.ch/tools/dna.html] 9. WWW Primer tools
[http://biotools.umassmed.edu/bioapps/primer3_www.cgi]
BackgroundCellulose‐paper industries have high environmental impact, that is why there are efforts
to
reduce
pollution. In
this
sense
white
rot
fungi
such
as
Peniophora
sp
(BAFC
633) present enzymes with wide degradative
capacity, like Laccase
(Lac) [1], with potential to alleviate environmental problems. Several cupper‐binding sites in Lac have
been discovered [2] and the presence of DNA sites coding transcription factor in response to Cu+2
(ACE‐1) in Phanerochaete
chrysosporium
(Genbank
accession number
ABF60559.1)
has
been
demonstrated
[3],
which
could
activate
the
laccase
gene
transcription
[4].
The
objective
of
this
study
was
to
define
conserved
regions
using
bioinformatics tools and to design degenerated primers to amplify a segment of a putative region coding ACE‐1 in Peniophora
sp. genome.
Key
words: degenerated
primer, score, ORF, motif, ACE‐1, transcription
factor, Phanerochaete
chrysosporium ,
Peniophora
sp.
Table
1:
Parameters
adjusted
for
searching
the
primers
on
Primer3 and
Fast PCR software. Table
2:
Sequence
of
forward
and
reverse primers
wich
were
picked
up and
their
respective
degeneration
percent.
Figure 1:
The
motifs
of
ACE‐1 related
to
copper‐fist DNA binding
domain found
on
PROSITE web
site.
Figures 2 and
3:
The
picture
on
the
left
is
a capture of
results
from
aminoacid
sequence
of
ACE‐1 from P
chrysosporium
contrasting against Basidiomycete
database of
PLASTp. And the picture on the right is a capture of sequence alignment of selected aminoacid
sequence with ClustalW.
Figures 4 and
5: The
picture
on
the
left
is
a capture of
characteristics
of
the
primers
picked
up with
Primer3 softwares. and
the
picture
on
the
right
is
a capture of
characteristics
of
the
primers
pricked
up with
Fast PCR.
Figure 6 and
7: The
picture
on
the
left
is
a capture of
the
result
from cuting
the
primer (with
restriction
enzime
site) with
NEBcutter. And
the
picture
on
the
right
is
a capture of
result
from
searching
of
the
ORF of
the
aminoacid
sequence
of
ACE‐1..
Ana Belén Ramos Hryb1(1), María Isabel Fonseca(1), Laura Villalba(1), Pedro Darío Zapata(1).
(1)Laboratory
of
Molecular Biotechnology, FCEQyN, Posadas, 3300, Misiones,
Tables
and
figures
