BLAST analysis of these 62 clusters, using a representative contig consensus sequence, showed that 14.5% have no significant alignment, 29% align to hypothetical proteins and 14.5% align to other nematode ESTs; there are various other alignments (42%) to known C. elegans proteins but, again, with no unifying theme; there are no alignments to ribosomal proteins.
Further details of these data are shown in Table 3. BLAST analysis of the 37 clusters with a greater than two-fold difference in expression in the L1 stage, compared with the iL3 stage, shows that three have no significant alignment; seven have a significant alignment to hypothetical proteins from C. elegans and the remaining 27 have significant alignment to known C. elegans proteins (Table 4).
We designed and implemented an efficient method for aligning putative coding DNA sequences, which builds expressive alignments between hypothetical nucleotide sequences obtained by back-translating the proteins, that can provide some information about the common ancestral sequence, if such a sequence exists.
The high proportion of iL3-up genes with significant alignment to hypothetical proteins, or to uncharacterised ESTs, or with no significant alignment at all, suggests that the transcriptional profile of these infective stages involves genes whose role and function remain to be elucidated in nematodes (including C. elegans) and other organisms.
The alignments and hypothetical secondary structures were used to search for additional homologs in the RefSeq25 database [ 80] along with metagenome sequences from acid mine drainage [ 82], soil and whale fall [ 83], human gut [ 84, 85], mouse gut [ 86], gutless sea worms [ 87], sludge [ 88], Global Ocean Survey scaffolds [ 12, 13], other marine sequences [ 89] and termite hindgut [ 90].
BLAST analysis of the 62 clusters with a greater than two-fold difference in expression in the iL3 stage, compared with the L1 stage, shows that nine have no significant alignment, 25 have a significant alignment to known C. elegans proteins and 17 have a significant alignment to hypothetical proteins from C. elegans.
In this alignment the hypothetical positions of a magnesium binding site and site of double stranded DNA binding in the ICP-8 protein are shown, potentially orienting a bound magnesium ion towards the bound DNA strand to permit, for example, magnesium dependent strand exchange typical of DBP proteins such as ICP-8.
In addition, 19% align to hypothetical proteins and 7.5% have no significant alignment.
In contrast to known protein families, peptides from hypothetical proteins have very distinct alignment patterns, allowing them to be easily filtered.
By comparison, the alignment between the hypothetical TFIID-31 domain in APITD1 and the TFIID-31 domain in TAFII31 was only 57%.
