Smelt was the likely beneficiary of an antifreeze gene laterally transferred between fishes

Abstract

Background: Type II antifreeze protein (AFP) from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved.

Results: Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes.

Conclusions: These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between 'higher' eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

Figure 1

Annotation of the smelt BAC clone. Exons are indicated with blue boxes, introns by bent lines and genes encoded by the complementary strand are shown below the line. Start and stop codons are indicated by green and red bars, respectively (or bent bars for incomplete genes). Genes are named using HGNC compatible names (http://www.genenames.org/) with those that do not share conserved synteny with humans denoted with the suffix ‘a’. The locally duplicated copies of RBP3 are denoted RBP3-1 and −2. Two splice variants are predicted for MAPK8. Note that small exons cannot be resolved at this scale. The annotated sequence has been deposited under GenBank Accession JQ514278 and the genes are described in Additional file 4.

Figure 2

Alignment of the cDNA sequences of smelt and herring with their deduced protein sequences. Polymorphisms found in >5% of the Atlantic herring ESTs are shown below the herring sequence (GenBank S65819.1). Differences relative to the smelt sequence (GenBank M96154.1) are highlighted yellow (missense) or gray (silent or non-coding). The only significant polymorphism in smelt is a single base insertion, indicated with an open triangle. The signal peptide and polyadenylation signal are in lower case font. The eleven polymorphisms in the herring ESTs that match the smelt cDNA are in red font. Sequences have been trimmed to remove linker sequences and slight variations in the site of polyadenylation.

Figure 3

Mutational analysis of smelt and herring genes. Ratio of missense changes per missense site to silent changes per silent site (d_N/d_S) between orthologs (closed circles), paralogs (open circles) and the AFP gene (closed square) found in herring and smelt.

Figure 4

Apollo visualization of the annotation of the corresponding syntenic regions of other fishes to that of the BAC region containing theAFPin smelt. Features are denoted as in Figure 1. Brown hash marks or parallelograms across the scale line indicate the location and extent (if known) of gaps in the sequence. Genes indicated with orange and purple labels are discontiguous between smelt and the other species. Some repetitive DNAs, such as retrogenes or transposable elements (TE) are indicated with dark yellow boxes and aberrant coding sequence by a yellow arrowhead. Fine details, such as short introns, cannot be resolved at this scale.

Figure 5

Maximum-likelihood bootstrap consensus tree derived from an alignment of a portion of the RBP3-2 proteins of various fish. The names of type II AFP-producing fish are in bold. The scale bar represents 5% divergence. Bootstrap values (500 trials) are indicated at the nodes. The split between Euteleostei and Otocephala occurred over 200 mya [17,18].

Figure 6

VISTA comparisons between various fish sequences. Percent identity is scored over a 100 bp window with a bottom cut-off of 50%. Matches above 70% are colored pink. A) Comparison of the ARHGAP22 orthologs of five fish to that of smelt. Exons in smelt are numbered and indicated by blue rectangles. Ensembl accession numbers for these genes are as follows; fugu (ENSTRUG00000017136), spotted pufferfish (ENSTNIG00000004993), zebrafish (ENSDARG00000076434), stickleback (ENSGACG00000005702), medaka (ENSORLG00000002387). B) Comparison as in A but between the sequences of the two pufferfish. C) Comparison of the AFP sequences of herring (GenBank DQ003023.1) and smelt (GenBank DQ004949.1). Exons are color coded with brown for non-coding, pale green for signal peptide encoding and blue for mature protein encoding sequence. D) Comparison of the intergenic region between the FRMPD2 and GDF10 genes using the shuffle-LAGAN alignment. The AFP exons are indicated as in C.