DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function.     

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.
The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.     

Experimental analysis of lens-forming capacity in Xenopus borealis larvae

Previously, the only anuran amphibians known to have the capacity to regenerate a lens after lentectomy were Xenopus laevis and Xenopus tropicalis. This regeneration process occurs during the larval life through transdifferentiation of the outer cornea promoted by inductive factors produced by the retina and accumulated inside the vitreous chamber. However, the capacity of X. tropicalis to regenerate a lens is much lower than that of X. laevis. This study demonstrates that Xenopus borealis, a species more closely related to X. laevis than to X. tropicalis, is not able to regenerate a lens after lentectomy. Nevertheless, some morphological modifications corresponding to the first stages of lens regeneration in X. laevis were observed in the outer cornea of X. borealis. This suggested that in X borealis the regeneration process was blocked at early stages. Results from histological analysis of X. borealis and X. laevis lentectomized eyes and from implantation of outer cornea fragments into the vitreous and anterior chambers demonstrated that: (i) in X. borealis eye, the lens-forming competence in the outer cornea and inductive factors in the vitreous chamber are both present, (ii) no inhibiting factors are present in the anterior chamber, the environment where lens regeneration begins, (iii) the inability of X. borealis to regenerate a lens after lentectomy is due to an inhibiting action exerted by the inner cornea on the spreading of the retinal factor from the vitreous chamber towards the outer cornea. This mechanical inhibition is assured by two distinctive features of X. borealis eye in comparison with X. laevis eye: (i) a weaker and slower response to the retinal inducer by the outer cornea; (ii) a stronger and faster healing of the inner cornea. Unlike X. tropicalis and similar to X. laevis, in X. borealis the competence to respond to the retinal factor is not restricted to the corneal epithelium but also extends to the pericorneal epidermis.     

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.     

Immunological relationship between oocyte nuclear proteins of Xenopus laevis and X. borealis

Monoclonal antibodies (mABs) have been raised against oocyte nuclear proteins of Xenopus laevis and X. borealis and have been screened for species specificity. Although about 40% of all germinal vesicle polypeptides differ between the two species as judged by two-dimensional gel analysis, most mABs react with polypeptides of both species. Biochemical analysis of the antigens by immunoblotting revealed that a homologue of each antigen of one species could be detected in the other species, despite frequent differences in molecular structure. Nevertheless, five strictly species-specific mABs have been identified. All five are directed against the same acidic polypeptide B3 of X. borealis, which is a structurally altered homologue of the protein N1, previously described in X. laevis germinal vesicles. In oocyte nuclei of hybrids between X. laevis females and X. borealis males, polypeptide of both species appear to be accumulated equivalently. Exceptions to this rule are most easily explained by differences between individuals and by the loss of certain alleles resulting from the cross.