Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

Differential expression of the TFF-peptides xP1 and xP4 in the gastrointestinal tract of Xenopus laevis

TFF-peptides (formerly P-domain peptides, trefoil factors) represent major secretory products of the mammalian gastrointestinal tract. A molecular cloning approach revealed the existence of two TFF-peptides, xP1 and xP4, also in the stomach of Xenopus laevis. Here, the localization of these two peptides by Western blot analysis as well as immunohistochemistry is presented. xP1 is found predominantly in the surface mucous cells of the stomach, whereas xP4 is mainly localized to a specific population of goblet cells in the esophagus, to mucous neck cells of the stomach, and to closely resembling cells in antral glands. xP4 in the esophagus and in the stomach differ by their N-glycosylation patterns. Compared to mammalian TFF-peptides, xP1 obviously represents the frog homologue of human TFF1 (formerly pS2) and xP4 seems to be the amphibian equivalent of human TFF2 (formerly hSP).     

The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.     

Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.     

Lipase H,a new member of the triglyceride lipase family synthesized by the intestine

We report here the molecular cloning of a novel member of the triglyceride lipase family, a 2.4-kb cDNA encoding human lipase H (LIPH) and the mouse ortholog (Liph). The human LIPH cDNA encodes a 451-amino-acid protein with a lipase domain. Mouse Liph shows 85% amino acid identity and 75% nucleotide identity to human LIPH. Human LIPH exhibits 47% identity with phosphatidylserine-specific phospholipase A1 (PS-PLA1) and 46% identity with endothelial lipase (LIPG) and lipoprotein lipase (LPL). LIPH is localized on human chromosome 3q27-q28. Northern blot analysis revealed specific expression of LIPH mRNA in intestine, lung, and pancreas. Lipase H protein was also detected in human intestine. Lipase H is a secreted protein with an apparent molecular weight of 63 kDa. Although several lipid substrates were tested, the lipid substrate of LIPG was not identified. Like the other members of this gene family, LIPH may be involved in lipid and energy metabolism.     

Raver2, a new member of the hnRNP family

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo-cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.     

The role of growth factors in embryonic induction in Xenopus laevis.

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of cDNA encoding a new peptide C-terminal alpha-amidating enzyme having a putative membrane-spanning domain from Xenopus laevis skin

A cDNA clone encoding a precursor of a peptide C-terminal alpha-amidating enzyme (AE-I) from Xenopus laevis skin was recently isolated and sequenced in our laboratory. In this study, by using the restriction fragment of this clone as a hybridization probe, we have identified the cDNA encoding another new peptide C-terminal alpha-amidating enzyme (tentatively named AE-II) distinct from AE-I. The cDNA encodes a polypeptide of 875 amino acid residues, which contains a region extensively homologous to AE-I precursor at N-terminus. The encoded protein characteristically has a putative membrane-spanning domain near C-terminus. Our results indicate that C-terminal alpha-amide formation of peptides in Xenopus skin is regulated by at least two distinct alpha-amidating enzymes.     

Overexpression of camello, a member of a novel protein family, reduces blastomere adhesion and inhibits gastrulation in Xenopus laevis

Vertebrate gastrulation involves complex coordinated movements of cells and cell layers to establish the axial structures and the general body plan. Adhesion molecules and the components of extracellular matrix were shown to be involved in this process. However, other participating molecules and detailed mechanisms of the control of gastrulation movements remain largely unknown. Here, we describe a novel Xenopus gene camello (Xcml) which is expressed in the suprablastoporal zone of gastrulating embryos. Injection of Xcml RNA into dorsovegetal blastomeres retards or inhibits gastrulation movements. Database searches revealed a family of mammalian mRNAs encoding polypeptides highly similar to Xcml protein. Characteristic features of the camello family include the presence of the central hydrophobic domain and the N-acetyltransferase consensus motifs in the C-terminal part, as well as functional similarity to Xcml revealed by overexpression studies in Xenopus embryos. Xcml expression results in the decrease of cell adhesion as demonstrated by the microscopic analysis and the blastomere aggregation assay. Cell fractionation and confocal microscopy data suggest that Xcml protein is localized in the secretory pathway. We propose that Xcml may fine tune the gastrulation movements by modifying the cell surface and possibly extracellular matrix proteins passing through the secretory pathway.     

Unresponsive, a new behavioral mutant in Xenopus laevis

Abstract. A new recessive nonlethal behavioral mutant, unresponsive (ur), was recovered from a wild-caught Xenopus laevis female by gynogenesis and inbreeding. Mutant embryos do not move until they are three days old, just before feeding begins, in contrast to normal embryos which begin movements at one day, during tailbud stage. Recovery of mutant embryos is complete but slow, requiring another nine days. Grafting analysis suggests that sensory neuron function is normal in homozygous mutant embryos, but that both motorneurons and their target muscles derived from somites are affected by the mutation. Either muscle or motorneurons of unresponsive embryos can participate in normal movements during early development, prior to the stage at which intact mutant embryos recover, in chimerae with normal tissue. Failure of mutant muscle to respond normally to acetylcholine, along with the behavior of chimerae, suggests that mutant embryos do not move because they do not form functional neuromuscular junctions during early development and that the component process of neuromuscular junction formation affected by this mutation is normally performed by both nerve and muscle. during embryogenesis due to a defect in the muscle cells [5]. Armstrong and collaborators [I] have used the immobile mutant to demonstrate that the cholinergic stimulation- induced loss of gap junctions during development does not depend on muscle contraction. In this report, we examine a new mutant of Xenopus laevis, unresponsive (ur), which does not move voluntarily or in response to stimulation until just prior to the feeding stage, after which it recovers.     

Characteristics of immunoglobulin synthesized early in the development of Xenopus laevis.

A radioimmune assay has been used to detect the onset of immunoglobulin synthesis during development. In Xenopus laevis, immunoglobulin is first produced at stage 35 of embryonic development or about the time of emergence of the embryo from its jelly coat. At all embryonic stages measured, beginning at stage 35, both 19 and 7 S immunoglobulin are found. These immunoglobulins contain heavy and light chains identical in size to adult Xenopus IgM. These observations suggest the presence of both cell-associated and circulating IgM in Xenopus embryos.     

B-raf, a new member of the raf family, is activated by DNA rearrangement.

Complementary DNA clones of a putative transforming gene were isolated from NIH 3T3 cells transformed with human Ewing sarcoma DNA. The gene was termed B-raf because it is related to but distinct from c-raf and A-raf. It appears that substitution in the amino-terminal portion of the normal B-raf protein confers transforming activity to the gene.     

Cloning and expression of xP1-L,a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H⁺/K⁺-ATPase β subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.