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From each cluster a consensus sequence was generated by multiple alignment with Map.
DNA sequencing of the 353 pb fragment followed by multiple alignment with database sequences for Leishmania species indicated Le. infantum, the etiological agent of VL, as the infecting agent in both pool samples of Lu. longipalpis.
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The VlcpcA gene was identified in the sequenced sample of the cDNA library of V. longisporum by multiple alignments with its fungal counterparts (data not shown).
The remaining cases were validated by multiple alignments with sequences from other available genomes and it is clear that these were also cases with an incorrectly annotated start codons.
When the ability to degrade sugar was strain-dependent, all protein sequences corresponding to transporters or enzymes involved in the pathway were compared by multiple alignments with other orthologues.
Positions of promoter sequence, transcription start site, introns and exons were determined by NCBI- blast and by multiple alignments with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The in silico translated amino acid sequence derived from the BbKst gene was further analyzed for conserved domains and motifs using ProSite and Inter-Proscan.
This was confirmed by multiple alignments with proteins from this family (Fig. S2), and lip8 and lip11 contained the lipase-conserved catalytic triad of residues Glu, replacing Asp (residue 229 and 263 for lip8 and lip11, respectively), His (259 and 293, respectively) and the catalytic nucleophile Ser (138, 172), respectively, in the consensus pentapeptide GDSAG.
Multiple alignments were generated by the MULTALIN program (Corpet 1988).
Multiple sequence alignments were generated using Multiple Alignment with Fast Fourier Transform (22 ) in SeaView (23 ) and sequence diversity was calculated by using molecular evolutionary genetics analysis (MEGA) 4 (24 ).
Linker regions were determined by multiple alignments of fusion sequences with corresponding free-standing domains present in the closest relatives to organisms that lack the gene fusions.
The overall consensus binding motif was obtained by multiple alignments of the significant binding sequences with ClustalW, and it was visualized with SeqLogo program.
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