To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

The cyclic nonapeptides, oxytocin and vasopressin, are neurohypophysial hormones that regulate many significant physiological processes related especially to reproduction and osmoregulation. In this study, we characterized an oxytocin-related peptide cDNA from a urochordate, Styela plicata, thought to be a sister group to vertebrates. Sequence analysis of the deduced precursor polypeptide revealed that the precursor is composed of three segments: a signal peptide, an oxytocin-like sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin/vasopressin family precursors. However, unlike other members of this family, the tunicate oxytocin-like peptide (CYISDCPNSRFWST-NH2) is a tetradecapeptide. We termed this peptide Styela oxytocin-related peptide (SOP). Furthermore, analyses of mass spectrometry, in situ hybridization, and immunohistochemistry demonstrated production of mature SOP in the cerebral ganglion. To elucidate the physiological action of SOP, we kept the tunicate for 2 d under the three different concentrations of seawater, 60, 100, and 130%, and measured the expression levels of SOP mRNA in the cerebral ganglion. The greatest expression of SOP mRNA was observed in the 60% seawater. In 60% seawater, but not in 100 or 130%, the tunicate mostly closed the atrial and branchial siphons. Therefore, we investigated the contractile effects of SOP on the siphons in vitro. SOP caused contractions in both siphons in a dose-dependent manner. Taken together, these results suggest that SOP acts to prevent the influx of a low concentration of seawater into the body and thus play an important role in osmoregulation.

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During embryogenesis of the ascidian Halocynthia roretzi, 42 unicellular striated muscle cells are formed in the tail of the tadpole larva. Isolation of cDNA clones demonstrated that multiple genes for larval muscle actin are expressed in this process. Among them, at least five muscle actin genes (HrMA2, HrMA4a, HrMA4b, HrMA5, and HrMA6) form a cluster (HrMA2/4 cluster) within about 30 kb of the genome. The 5' flanking sequences of the five actin genes resemble each other. When constructs in which 184 bp of the 5' flanking region of each of these genes fused with lacZ were introduced into fertilized eggs, the reporter gene was expressed in muscle cells of the tailbud embryo, suggesting that the 5' flanking region of each cluster gene has promoter activity. In addition, a pair of muscle actin genes, HrMA1a and HrMA1b (HrMA1 pair), was isolated from a genomic region different from that of the HrMA2/4 cluster. The HrMA1a and HrMA1b are linked in a head-to-head arrangement on opposite strands and share a 340-bp 5' flanking sequence containing two symmetrically located TATA boxes. HrMA1a showed basically the same expression pattern as that of HrMA4a. When constructs in which the shared upstream region of HrMA1 pair fused with lacZ in either direction were microinjected into eggs, the reporter gene was expressed in muscle cells of the larval tail, suggesting a bidirectional promoter that regulates muscle-specific transcription of the HrMA1 pair. The tandem cluster of HrMA2/4 genes and the bidirectional promoter of the HrMA1 pair could expedite utilization of muscle-specific trans-acting factors. The organization of genes in the genome may play an important role in the synthesis of a large amount of actins during the process of rapid differentiation.

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocyntbia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial p-galactosidase gene (lac-Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5'flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 pgiml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 pgiml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocyntbia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5'flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.