1. Design of degenerated primers from bioinformatics
online software for putative ACE-1 transcription
factor in Peniophora sp.
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,
Argentina.1annarhgen@yahoo.com.ar
Background
Cellulose‐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.
Materials and methods
Tables and figures
The aminoacid sequence of ACE‐1 from Phanerochaete
chrysosporium was used as reference contrasting it 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
against database published on BLASTp (Basic local degeneration percent.
Alignment Search Tool protein) (see figure 2).Then, the Minimus Maximus Optimus
selected similar sequences were aligned using T‐Coffee Tm 50 67 60
[5] and ClustalW2 [6] (see figure 3), in order to %CG 40 60 50
determine the score. The Prosite [7], was used to Fragment lenght 100
determinate if the conserved regions correspond with Primer lenght 18 24 20
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 Figure 1: The motifs of ACE‐1 related to copper‐fist DNA binding
domain found on PROSITE web site.
(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).
Results
It 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 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
picked up the primers (see figure 4 and 5). We used PLASTp. And the picture on the right is a capture of sequence alignment of selected aminoacid sequence with ClustalW.
wooble pairing to reduce the degeneration percent
(about 50%) of the primers, to which we included the
restriction site to EcoR1 enzyme.
Conclusion
These 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.
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..
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.
References
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