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PubMed1983

Histone gene number and organisation in Xenopus: Xenopus borealis has a homogeneous major cluster.

Using a Xenopus laevis H4 cDNA clone as a probe we have determined that the numbers of H4 histone genes in Xenopus laevis and Xenopus borealis are approximately the same. These numbers are dependent on the hybridization stringency and we measure about 90 H4 genes per haploid genome after a 60 degrees C wash in 3 X SSC. Using histone probes from both Xenopus and sea urchin we have studied the genomic organization of histone genes in these two species. In all of the X.borealis individuals analyzed about 70% of the histone genes were present in a very homogeneous major cluster. These genes are present in the order H1, H2B, H2A, H4 and H3, and the minimum length of the repeated unit is 16kb. In contrast, the histone gene clusters in X.laevis showed considerable sequence variation. However two major cluster types with different gene orders seem to be present in most individuals. The differences in histone gene organization seen in species of Xenopus suggest that even in closely related vertebrates the major histone gene clusters are quite fluid structures in evolutionary terms.

Animals↗
PubMed1985

Individual Xenopus histone genes are replication-independent in oocytes and replication-dependent in Xenopus or mouse somatic cells.

We have assessed the response of many histone H3 mRNAs and an H1C mRNA in Xenopus tissue culture cells after treatment with the DNA synthesis inhibitor hydroxyurea. The amount of the histone mRNAs falls rapidly in response to the inhibitor. This response is prevented by cycloheximide. Cloned Xenopus histone genes were transfected into mouse cells and a cell line was obtained in which the Xenopus genes were actively expressed giving rise to mRNA with correct 5'-termini. The Xenopus genes were correctly regulated at the level of mRNA amounts in the mouse cell line. Nuclear microinjection experiments with Xenopus oocytes and S1 nuclease analysis of normal ovary RNA showed that the H1C gene, and probably also two H3 genes, which are replication-dependent in somatic cells are expressed in oocytes and are therefore replication-independent in this cell type. The same promoters are used in both replication-dependent and independent expression.

Amino Acid Sequence↗
PubMed1984

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. VII. Inhibition of 40s pre-rRNA synthesis in Xenopus neurula cells.

A fraction containing acid-soluble materials (PCA-extract) from Xenopus early blastulae preferentially inhibits the incorporation of [3H]uridine into 18S and 28S rRNA in Xenopus neurula cells. Pulse- laneling experiments revealed that whereas tubercidin, a known inhibitor of rRNA processing, produced a marked accumulation of the label in 40S pre-rRNA, Xenopus inhibitor suppressed labeling of the pre-rRNA. When tubercidin was added to cells whose activity for rRNA synthesis had been lowered by the Xenopus inhibitor, there was still an accumulation of label in the pre-rRNA. These results indicate that Xenopus inhibitor suppresses the synthesis of rRNA at transcription rather than at processing.

Animals↗
PubMed2001

Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis.

Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole stages. genesis 30:110--113, 2001.

Animals↗
PubMed1994

Molecular cloning of cDNA for XTCAD-1, a novel Xenopus cadherin, and its expression in adult tissues and embryos of Xenopus laevis.

We have isolated from a Xenopus tailbud cDNA library a novel cadherin cDNA, denoted as XTCAD-1, which contained an open reading frame including the entire coding region. XTCAD-1 codes for 714 amino acids (molecular mass: 96 kDa), which include five characteristic extracellular cadherin motifs, a single putative transmembrane domain, and a cytoplasmic domain. In each domain, XTCAD-1 shared extensive homologies with other cadherins, and was related to EP-, E-, and P-cadherins more closely than to N- and M-cadherins. In adult Xenopus, XTCAD-1 mRNA was strongly expressed in intestine/stomach, kidney and skin, which are respectively derived from endoderm, mesoderm, and ectoderm. In Xenopus embryogenesis, expression of XTCAD-1 mRNA was first detected at blastula stage, and the level of the expression increased gradually during gastrula stage, reached a peak at tailbud stage and then decreased slightly at tadpole stage. These results suggest that in Xenopus laevis XTCAD-1 plays an important role in the maintenance of adult tissues that contain epithelial cells abundantly and also in morphogenesis in early embryonic development.

Amino Acid Sequence↗
PubMed2004

Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis.

The Tol2 transposable element from the medaka fish belong to the hAT family of transposons. In the previous studies, we have identified an autonomous member of this element, which encodes a fully functional transposase, and have shown that it can catalyze transposition in the zebrafish germ lineage. To date, the Tol2 element is the only natural transposon in vertebrates from which an autonomous member has been identified. We report here transposase-dependent excision of the Tol2 element in Xenopus laevis and Xenopus (Silurana) tropicalis embryos. We coinjected a plasmid DNA containing a nonautonomous Tol2 element and the transposase mRNA synthesized in vitro into two-cell-stage embryos, and analyzed DNA extracted from the injected embryos by polymerase chain reaction (PCR). We demonstrated that the Tol2 element could be excised from the plasmid DNA in both X. laevis and X. tropicalis only when it was coinjected with the transposase mRNA. In most cases, a complete loss of the Tol2 sequence was accompanied by addition of a short DNA sequence to the target sequence, indicating that transposase-dependent excision occurred. While these footprints were characteristic to those created upon excision of transposons of the hAT family, the additional bases found in Xenopus were longer and their structures were more complicated than those detected upon excision in zebrafish. This may reflect differences in the activities of host factors involved in either transposition, repair, or both between fish and frog. Our present study suggests that the Tol2 transposon system should be used as a novel genetic tool to develop transgenesis and mutagenesis methods in Xenopus.

Animals↗
PubMed2000

Primary structure requirements for Xenopus nodal-related 3 and a comparison with regions required by Xenopus nodal-related 2.

Transforming growth factor-beta superfamily members play important roles in the early development of animals. Activin and the Xenopus nodal related proteins 1, 2, and 4 induce muscle actin from Xenopus ectodermal explants, whereas the bone morphogenetic proteins 4 and 7 induce ectoderm to differentiate as epidermis. Bone morphogenetic proteins are antagonized by soluble binding proteins such as noggin and chordin, which leads to expression of neural cell adhesion molecule in animal caps. The transforming growth factor-beta superfamily member Xenopus nodal-related 3 also induces the neural cell adhesion molecule through inhibition of bone morphogenetic proteins. Therefore, whereas Xenopus nodal-related 2 and 3 share a high amount of sequence homology, they lead to very different cell fates. This study investigates the functional domains that distinguish the activities of these two factors. It was found that mutually exclusive regions of nodal-related 2 and 3 were required for activity. The central region of the mature domain is required for nodal-related 2 to induce muscle actin, whereas the N- and C-terminal ends of the mature domain are required for nodal-related 3 to induce neural cell adhesion molecule. These results help to define the minimal domains required for the unique activities of these factors.

Actins↗
PubMed2005

Characterization of the cDNA and amino acid sequences of Xenopus Metaxin 3, and relationship to Xenopus Metaxins 1 and 2.

The cDNA and protein structures of Xenopus metaxin 3, along with those of Xenopus metaxins 1 and 2, have been characterized. A protein of 309 amino acid residues is encoded by X. laevis metaxin 3 (XMTX3) cDNA. In comparison, the cDNA of X. laevis metaxin 1 (XMTX1) specifies a protein of 320 residues, while the metaxin 2 cDNAs of X. laevis (XMTX2) and X. tropicalis (SMTX2) both specify proteins of 274 amino acids. Aligning the amino acid sequences of XMTX3 and XMTX1 showed 39% identities; 22% identities were found for XMTX3 and XMTX2. However, 55% amino acid identities were revealed in aligning the XMTX3 and zebrafish metaxin 3 sequences. The construction of a phylogenetic tree gave further evidence for the existence of three distinct groups of metaxin genes and their common ancestry. Two conserved protein domains are present in each of the Xenopus metaxins: a glutathione S-transferase (GST) domain and a thioredoxin-like domain. The protein secondary structure predicted for the Xenopus metaxins is dominated by regions of alpha helix which alternate with regions that are neither alpha helix nor beta strand.

Amino Acid Sequence↗
PubMed1998

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes.

Animals↗
PubMed1995

G alpha-16 complements the signal transduction cascade of chemotactic receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) in Xenopus laevis oocytes: G alpha-16 couples to chemotactic receptors in Xenopus oocytes.

The human leukocyte chemoattractant receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) are important members of the superfamily of G-protein coupled receptors (GPCR). Uniquely among the GPCR, these two receptors cannot be expressed in a functionally active form in the oocytes of the frog Xenopus laevis, but require substitution of total RNA of the myelomonocytic U-937 or HL-60 cell lines, respectively. Recently, it was reported that the C5a-R may couple to the alpha subunit of G-16. We have tested this G-protein for its ability to complement the signal transduction cascade of the C5a-R and fMLF-R in Xenopus oocytes. Injection of cRNA for the C5a-R in combination with G alpha-16 led to expression of a functional C5a-R as measured by ligand-induced whole cell current. In contrast to a previous report, the fMLF-R exhibited some residual functional activity when transiently expressed in Xenopus oocytes the extent of which could, however, substantially be increased by coexpression of G alpha-16. Thus, G alpha-16 complements the signal transduction cascade of both receptors in Xenopus laevis oocytes and is most likely the complementing factor present in the U-937 and HL-60 cell lines.

Animals↗
PubMed1998

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells.

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues.

We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes. The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively. The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca. 65% and ca. 62% identity, respectively) and mouse (ca. 65% and ca. 60%), and show a certain degree of homology to Drosophila (ca. 27% with Ku70), Caenorhabditis elegans (ca. 20% with Ku80) and Saccharomyces cerevisiae (ca. 23% and ca. 19%). Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested. A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.

Amino Acid Sequence↗
PubMed1983

Replication and expression of Xenopus laevis globin genes injected into fertilized Xenopus eggs.

Cloned Xenopus adult alpha 1- and beta 1-globin genes were injected into fertilized Xenopus eggs, and the eggs were allowed to develop into swimming tadpoles. The injected DNA replicated during early Xenopus development but did not become methylated de novo. When DNA was modified with Hpa II methylase before injection, methylation was maintained during replication. Although the Xenopus adult globin genes are not normally expressed until metamorphosis, both the unmethylated and in vitro methylated adult alpha- and beta-globin genes were transcribed at low levels, but from the correct promoters, during early development.

Animals↗
PubMed2001

Two novel Xenopus homologs of mammalian LP(A1)/EDG-2 function as lysophosphatidic acid receptors in Xenopus oocytes and mammalian cells.

Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlp(A1)-1 and xlp(A1)-2, encoding LP(A1) homologs (approximately 90% amino acid sequence identity with mammalian LP(A1)). Both xlp(A1)-1 and xlp(A1)-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlp(A1)-1 and xlp(A1)-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp(A1)-1 or xlp(A1)-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLP(A1)-1 and XLP(A1)-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.

Adenylate Cyclase Toxin↗
PubMed2004

Isolation of desmosomes from the epidermis of Xenopus laevis and immunochemical characterization of the Xenopus desmosomal cadherins.

Here we report a new method of isolating epidermal desmosomes from Xenopus laevis, and a major constituent of desmosomes designated as Xenopus desmogleins (XDsg). Isolation of desmosomes from Xenopus laevis epidermis was carried out by a two step-incubation with different concentrations of NP-40. After discontinuous sucrose gradient centrifugation at 30,000 g for 60 min, a pure desmosomal fraction was obtained at 30%/40% interface. In the SDS-PAGE of isolated desmosomes, at least 12 bands (XDB1 to XDB12) were observed over a 75 kD region. Among them, three bands (XDB3, XDB7, XDB8; estimated MW 175, 124, and 112 kD respectively) were recognized as glycoproteins based on ConA binding. Monospecific polyclonal antibody against XDB3 cross-reacted with bovine Dsgs and vis-a-vis anti-bovine Dsgs with XDB3. By contrast, monospecific antibody against bovine Dsc a/b did not cross-react with either XDB7 or XDB8. Heterogeneous molecular constituents of desmosomal adhesion molecule, which have been observed among different bovine tissues, were confirmed in a phylogenetically different animal, Xenopus laevis. Combined results with other evidence could suggest an alternative system for desmosome-mediated cell adhesion.

Animals↗
PubMed2006

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis.

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.

Activins↗
PubMed1987

Differential transcription of Xenopus oocyte and somatic-type 5 S genes in a Xenopus oocyte extract.

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.

Animals↗
PubMed2004

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis.

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.

Animals↗