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on both strands (as described in Martínez-Agüero et al
.,
2006) in order to confirm their
identity as a MHC locus throughout a BLAST homology search (Altschul et al,
.
1990). The
remaining individuals were assayed for their DQB polymorphism by applying a previously
described PCR protocol and single strand conformational polymorphism (SSCP) methods
(Orita et al
.,
1989) following previously reported protocols (Nigenda-Morales et al
.,
2008).
After electrophoresis, the SSCP gel was silver stained using the protocol of Basam et al
.,
(1991) or incubated for 30 min in darkness with SYBR Gold© 0.5X (Molecular Probes,
Invitrogen) and visualized by UV illumination for SSCP scoring. All PCR products
displaying unique SSCP patterns were cloned using a TOPO TA cloning kit (Invitrogen), and
5 positive clones per sample were sequenced on both strands using the BigDye Terminator
Cycle Sequencing Ready Reaction Kit and an ABI 373 automated sequencer (Macrogen Inc.,
Seoul, Korea).
Unique sequences were aligned using Clustal X (Thompson et al
.,
1997) and corrected by
hand, and later translated using BioEdit 7.0.5.2 (Hall, 1999). Amino acid sequence divergence
was estimated by means of the MEGA 4.0 program by using different algorithms (Kumar et
al
.,
2004).
R
ESULTS
We successfully obtained amplification of DQB exon 2 from all individuals analyzed.
Eighteen different alleles were identified. None of the sequences had shown in/del mutations
and no more than two different sequences for each animal were obtained, thus suggesting that
only one locus was amplified. The nucleotide sequences were translated on the three possible
reading frames and we obtained just one open reading frame (Table 1) and 16 different
alleles, all of them with 57 amino acids. We highlighted the binding peptide sites in red, and
the lateral chains of the PBR in green (Table 1).
Three major groups of alleles were found by comparing the similarity of the sequences
between them and with a consensus sequence using a median-joining algorithm (Bandelt et
al., 1999):
Inge
DQB1-01, with eight different peptides,
Inge
DQB1-02, with five peptides,
and
Inge
DQB1-03, with three different peptides. The third group was the most divergent one.
Fifteen animals were homozygous and 45 were heterozygous; Table 2 shows the results
for each river and basin indicating the alleles and number of copies of each allele found in the
different populations. The Bolivian population showed nine different alleles. Seven animals
were homozygous and 16 were heterozygous; the alleles
Inge
DQB1-0104, -0107, and -0205
were only found in the Mamoré River population. At the Orinoco basin, nine different alleles
were found; these specimens yielded the major levels of homozygosity; five animals were
homozygous and five were heterozygous; the alleles
Inge
DQB1-0302 and -0303 were only
found in the Inirida River. Finally, in the upper Amazon we found 11 different alleles, two of
these,
Inge
DQB1-0105 and -0202, were only in the Peruvian population. In this basin, three
homozygous and 24 heterozygous animals were determined.
The amino acid sequences have 17 polymorphic sites (Table 1), most of them on the PBR
(peptide binding region) and the variability at the amino acid level is 29.8% (17/57). These
peptide mutations were analyzed using PAM 1 and Blossum 80 similarity matrices trying to
identify the most important changes for the structure of the peptide comparing the alleles with
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