In the phylogenetic tree, the cDNA clone of XLIG4 is placed in the ephrin-A3 group. This clone has a highest peptide sequence homology to murine ephrin-A3 (71.9%).
To support the hypothesis that the full length XLIG4 sequence is a Xenopus homologue of the ephrin-A3 group, the following BestFit sequence analyses were carried out. First, the peptide sequence of the XLIG4 cDNA sequence was compared with all cloned ephrins (see Table 4), to indicate the relatedness of this cDNA to them. Second, a similar analyses with mouse ephrin-A5 (Table 5) and chick ephrin-A2 were carried out, to indicate the relatedness of other ephrins to all cloned ephrins. Third, the sequence identities between Xephrin-Al (Table 6) and all other ephrins was carried out, also as a basis to discuss relatedness between ephrins.
Table 4 shows that the XLIG4 cDNA sequence has its highest sequence identity with murine ephrin-A3 which is 71.9%. This is closely followed by an sequence identity with
THE CLONING OF
XENO PU SEPHRINS
EPHRIN GROUP mEphrin-AS PROTEIN IDENTITY %
M X R H C Z ephrin-A1 5&5 53.1 48.4 54.1 - - ephrin-A2 62.9 - - - 67.5 62.9 ephrin-A3 54.8 59.8 - 53.3 - - ephrin-A4 49.3 - - 48.7 - - ephrin-A5 100 - 99.1 99.1 90.8 76.9 T a b le 5
Peptide Identities between murine ephrin-A5 and all other cloned ephrin-A ligands
m-mouse, x-Xenopus, r-rat, h-human, c-chick and z-zebrafish
EPHRIN GROUP xEphrin-A1 PROTEIN IDENTITY % M X R H C Z ephrin-A1 56.3 100 58.6 57.5 - - ephrin-A2 50.0 - - - 46.9 56.3 ephrin-A3 46.9 50.0 - 48.5 - - ephrin-A4 45.2 - - 48.2 - - ephrin-A5 53.1 - 5Z3 52.3 52.6 49.0 T a b le 6
Peptide Identities between Xenopus ephrin-A l and all other cloned ephrin-A ligands
THE CLONING OF
XENO PU SEPHRINS
human ephrin-A3 of 71.8%. Both the ephrin-A2 and ephrin-A5 groups had a highest peptide identity with the XLIG4 cDNA sequence of 60.6% and 59.9% respectively. The lowest peptide identity was found between full length XLIG4 sequence and the ephrin- A4 group (46.9%). The findings of this analysis indicate that the XLIG4 cDNA sequence has greatest homology to the ephrin-A3 group.
The sequence analysis can be taken further by comparing the sequence identities between recognised orthologues within a specific ephrin group. In support of the percentage sequence identities of full length XLIG4 sequence with its prospective homologue mouse ephrin-A 3 ,1 found that a sequence identity of 73.0% was present between chick ephrin- A5 and zebrafish ephrin-A5 and second, a 76.9% sequence identity was present between zebrafish ephrin-A5 and murine ephrin-A5. Additionally, the sequence identity between chick ephrin-A2 and murine ephrin-A2 is 75%.
Higher sequence identities do exist such as those shown in Table 5, where BestFit comparisons have been constructed between murine ephrin-A5 and all cloned ephrins. Murine ephrin-A5 was chosen since clones of all the known ephrin groups have been isolated from the mouse and second, since the ephrin-A5 group has representative clones from the most species. This ephrin is 99.1% identical to rat and human ephrin-A5.
Lower sequence identities were identified within the same ephrin group when Xenopus
ephrin-Al (the only other cloned Xenopus ephrin), was also compared with all the other cloned ephrins (Table 6). This ephrin had a highest sequence identity of 58.6% with rat ephrin-Al. This percentage is less than some sequence identities revealed between different ephrin groups such as murine ephrin-A5 with all the cloned ephrin-A2 members (Table 5).
The data from Tables 4, 5 and 6 indicate that within the same ephrin group, sequence identities can range between 99.1% (ephrin-A5 group) and 58.6% (ephrin-Al group). These data also indicate that sequence identities between members of the same group are not always very high (Table 6). Additionally, the sequence comparisons show that sequence identities between individual ephrins of different ephrin groups may also be similar. For example, the sequence identity between Xenopus ephrin-Al and rat ephrin-
A1 is 58.6% (Table 6) and between mouse ephrin-A5 and mouse ephrin-A3 is 54.8% (Table 5).
Therefore, the sequence identities found between the full length XLIG4 sequence and the ephrin-A3 group support the proposition that XLIG4 cDNA sequence represents a
Xenopus ephrin-A3. 3.3.3 Pseudotetraploidy
Due to the presence of XLIGl from the PCR screen, in addition to XLIG4, it is possible that this clone is another Xenopus homologue of the ephrin-A3 family. Sequence comparisons indicate that the XLIGl class has greater sequence homology to the ephrin- A3 group that the XLIG4 class (see Table 3).This presence of a second ephrin-A3 gene would be due to pseudotetraploidy in Xenopus.
The cloning of the XLIG3 class is another potential example of pseudotetraploidy. The only other Xenopus ephrin to have a published sequence is Xenopus ephrin-Al (Weinstein et ah, 1996), which has a significantly lower homology to other ephrin-Al members than the XLIG3 class (see Table 3). Therefore, XLIG3 may represent the second Xenopus ephrin-Al gene.
Presently, five ligands of the ephrin A-class have been cloned across five species. Assuming all these five groups are represented in Xenopus, one would expect 10 ephrins to be eventually cloned. The same is true of zebrafish where already two forms of ephrin- A5 have been identified (Brennan, unpublished data). If XLIG5 does represent a new ephrin group, it would increase the number of ephrin groups to six. Therefore, 12 ephrins would be expected to be found in Xenopus.
In summary, the XLIG class sequences suggest they have homology to existing ephrin groups. Only when longer cDNA's of XLIGl, XLIG2, XLIG3 and XLIG5 are cloned will their homologues be known. The nucleotide and protein sequence identities of the XLIG4 cDNA sequence to murine ephrin-A3, and comparisons with all cloned ephrins, strongly suggests that this XLIG4 cDNA is a Xenopus homologue of ephrin-A3 and therefore, the XLIG4 cDNA sequence will now be referred to as Xephrin-A3.