[Frontiers in Bioscience 14, 177-191, January 1, 2009]
The fate of duplicated immunity genes in the dodecaploid Xenopus ruwenzoriensis Louis Du Pasquier1, Melanie Wilson2, Benedicte Sammut3

1University of Basel, Department of Zoology and Evolutionary Biology University of Basel Vesalgasse 1 CH-4051 Basel Switzerland, 2University of Mississippi Medical Center, Department of Microbiology University of Mississippi Medical Center2500 North State StreetJackson, MS 39216-4505, 3 Washington University School of medicine in St-Louis Department of bone and mineral diseases 660 South Euclid Avenue, Campus Box 8301 St. Louis, MO 63110

FIGURES

Figure 1. Four species of clawed toads. Silurana (Xenopus) tropicalis (2n = 20 chromosomes), Xenopus laevis (2n = 36 chromosomes, Xenopus wittei (2n = 72) and Xenopus ruwenzoriensis (2n=108)

Figure 2. Xenopus ruwenzoriensis metaphase (left) stained with immunoglobulin VH1 probe. Compare with Xenopus laevis (right).

Figure 3. Immunoglobulin JH, Cmu, VH5 segments segregation in haploid progenies of X. ruwenzoriensis. Left: southern blot analysis three parental females P 1, 2, 3 were used. "r" indicates recombinants. (VH5 was the family with the highest percentage of recombination with respect to Cmu (see text). Numbers 1,2, 3 etc. correspond to sibs from each parent. Right: interpretation of the results for p1 and p3. The figures on the chromosomes correspond to the classification of the band on the right part of the panel. a b, a'b' ruwenzoriensis 1n (each contains 54 chromosomes) contents. Due to inbreeding in our colony and to the fact that our original X. ruwenzoriensis came all from the same narrow spot near Bundibugyo in Ouganda, the two animals used for segregation of haploid tadpoles were both homozygous at one of the pair of Ig carrying chromosome . Due to recombination the locus could be homozygous in some places and heterozygous in other as shown on figure for the relatively simple VH 5 family (r arrows). Southern blots have been monitored by densitometry to monitor homozygosity or heterozygosity at the constant region gene locus. The thick bands correspond to homozygosity.

Figure 4. In situ hybridization with MHC Class Ib probes and double stain with Class II beta.

Figure 5. Segregation of Class Ia (left) and Class II beta (center) ruwenzoriensis genes in (ff) X (ruwenzoriensis) hybrids with chromosome attribution (right) fitting with the segregations patterns. Bands were given number in function of their molecular size starting from bottom to top. Due to the type of cross all individual possess the f haplotype only polymorphic ruwenzoriensis haplotypes segregate. Ruwenzoriensis parent was homozygous at one of the Class Ia loci (band 3, I3 in the chromosome diagram) and at one of the Class II beta loci (band 4, II4 in the chromosome diagram). See also table 2.

Figure 6. Alignment of Xenopus laevis and ruwenzoriensis Class II beta amino acid sequences. Predictions of domains, helices and strands have been superimposed. DAB, DBB, DCB sequences are from X.laevis , XERU, from X. ruwenzoriensis.

Figure 7. Phylogenetic relationships between X. laevis and X. ruwenzoriensis Class II beta chain loci DNA sequences (same origin as in the alignment of Fig 6). All sequences start in with O are from X. laevis DAB, DBB and DCB products (alleles) sequences starting witrh Xeru are from Xenopus ruwenzoriensis. Xeru 53, 614, 57, 410, 419 are homologous to DBB laevis sequences, whereas XeRU 22, 51, 28, 55, 422, 61 cannot be firmly reattached to either DAB or DCB lineages. Clustal alignment and neighbour joining tree were drawn with the genebee cluster algorithm found at http//:www.genebee.msu.su:genebee.html.

Figure 8. Model of duplications to explain the Class II beta situation in X. ruwenzoriensis and laevis.

Figure 9. Morphology and immuno-staining of TOUF cells grown in vitro