The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.

A family of eukaryotic RNA binding proteins is defined by the conserved RNP motif. The poly(A) binding protein has four such motifs. We report on the isolation and structural characterization of several variant cDNA clones, as well as of a gene encoding this protein in Xenopus laevis embryos. Wild-type protein as well as truncated versions carrying isolated single motifs or artificial combinations of two and more such elements were characterized for their ability to bind specifically to RNA homopolymers. Three of the isolated repeats were functional in specific RNA binding, whereas the N-terminal RNP motif was non-functional. Combinatorial effects in RNA binding were measured with constructs carrying multiple repeats, which were not predictable from the activity of isolated domains.     

Xenopus laevis TRK-fused gene (TFG) is an SH3 domain binding protein highly expressed in the cement gland

TRK-fused gene (TFG) was originally identified in humans as the N-terminus of an oncogenic fusion protein TRK-T3, associated with papillary thyroid carcinoma. An amino-terminal coiled coil domain of TFG is responsible for mediating oligomerization of the TRK-T3 oncoprotein, resulting in constitutive activation of the TRK protein tyrosine kinase and oncogenesis. We have cloned the Xenopus laevis homologue of TFG and demonstrated that xTFG was highly expressed in the cement gland of tailbud embryos. Overexpression of xTFG2-136 (including the coiled coil domain) in early embryos, via mRNA microinjection as well as transgenic expression using the recently described restriction enzyme mediated integration (REMI) transgenesis, did not alter embryonic development or development of a functional cement gland, despite clear evidence that xTFG2-136 strongly interacted with endogenous xTFG. Finally, we have identified a potential SH3 binding motif in xTFG (and in TFG) and have demonstrated that xTFG selectively interacted with SH3 domains of Src, PLCgamma, and the p85 phosphatidylinositol 3-kinase subunit.     

Xenopus laevis FoxE1 is primarily expressed in the developing pituitary and thyroid

The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.     

Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)     

Pancreatic protein disulfide isomerase (XPDIp) is an early marker for the exocrine lineage of the developing pancreas in Xenopus laevis embryos

The pancreas develops from dorsal and ventral epithelial extensions at the foregut/midgut boundary in Xenopus embryos. Endocrine and exocrine specification is thought to occur from a pool of uniform precursor cells. While the genetic network controlling endocrine specification and differentiation has been the object of extensive investigations, the corresponding mechanism leading to the exocrine pancreas is much less understood. Here, we report on the identification and characterisation of a novel molecular marker for the early exocrine pancreas in Xenopus embryos. Xenopus pancreatic protein disulfide isomerase is expressed in both dorsal and ventral pancreatic buds. By whole mount in situ hybridization it is detected as early as stage 39 in the exocrine lineage of the developing pancreas; RT-PCR reveals onset of expression as early as stage 35/36.     

A detergent-activated tyrosinase from Xenopus laevis. II. Detergent activation and binding

Tyrosinase purified from Xenopus is enzymatically inactive in aqueous buffers but is activated for both of its substrates by exposure to a variety of anionic detergents. Cationic and nonionic detergents, as well as a variety of other agents are ineffective. This stimulation by detergents is observed at all stages of the purification (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12535-12541). Sodium dodecyl sulfate (NaDodSO4) is the most effective activator, and it was chosen for further characterization. Activation of both activities by NaDodSO4 is rapid and concentration dependent, resulting in maximal activity after 4 min at 1 mM NaDodSO4. NaDodSO4 treatment also results in both long and short term stabilization of the enzyme. The activation and stabilization are separable but stoichiometrically related. Both effects occur well below the critical micelle concentration suggesting that the interaction of NaDodSO4 monomers with the enzyme is involved in these processes. In support of this suggestion, the enzyme is shown to bind NaDodSO4 with high affinity, as determined by equilibrium dialysis. The isotherm for this binding correlates well with the requirement of NaDodSO4 for both activation and stabilization. All three effects are observable at 3 X 10(-5) M NaDodSO4 in the presence of 0.1 M sodium chloride. Activation and stabilization are maximal at 6 X 10(-4) M NaDodSO4, the critical micelle concentration of NaDodSO4 under these conditions.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.     

A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.     

Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.     

Two novel Xenopus frizzled genes expressed in developing heart and brain.

A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain.     

A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

A middle-affinity estrogen-specific binding protein in livers of vitellogenic and nonvitellogenic Xenopus laevis

Administration of estradiol-17β to male Xenopus laevis evokes massive liver synthesis of the egg yolk precursor protein, vitellogenin, and its cognate mRNA. Since previous experiments had implicated only a nuclear estrogen receptor in vitellogenesis, we examined Xenopus liver cytosol for other estrogen-binding proteins. Cytoplasmic extracts from unstimulated Xenopus liver contain high levels (approximately 500,000 sites/cell) of an estrogen-specific binding protein. This protein exhibits a K_d of approximately 4 × 10^?8M for estradiol-17β, binds estrogenic steroids only, and has a sedimentation coefficient in the range 2–3 S. It is not a classical estrogen receptor, as it does not translocate into the nucleus following estrogen administration. We discuss possible functions of this protein, which include a role in the ontogeny of the vitellogenic response, and in the cytoplasmic transport and storage of estrogen.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.     

The Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.