Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.     

A detergent-citric acid technique for isolating nuclear and cytoplasmic fractions containing undegraded RNA from cells of Xenopus laevis.

A simple technique is described for fractionating cells of Xenopus laevis using Triton X-100 and citric acid. Cells are treated for 1 min with a hypotonic buffer containing 0.5% Triton X-100 and RNAase inhibitors. The cells are then disrupted by adding citric acid to a final concentration of 1% for 2 min and the nuclei are pelleted by centrifugation. The detergent-citric acid fractionation technique has been used to obtain pure nuclear and cytoplasmic fractions containing undegraded RNA from an epithelial cell line of Xenopus laevis. The procedure is very gentle, eliminates RNAase activity, and can be performed in about 5 min. The detergent-citric acid technique can be easily scaled up or down and has also been used successfully with mammalian and insect cell lines.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.     

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Effects of various ammonium salts, amines, polyamines and alpha-methylornithine on rRNA synthesis in neurula cells of Xenopus laevis and Xenopus borealis

Xenopus neurula cells were cultured in a medium that contained ammonium salts, amines, polyamines or alpha-methylornithine, and their rRNA synthesis was examined. All the ammonium salts and amines, but not polyamines, were strong and selective inhibitors of rRNA synthesis at 1.25-5.0 mM. alpha-Methylornithine did not inhibit rRNA synthesis, although it inhibited ornithine decarboxylase, an enzyme claimed to be a direct stimulator of rRNA synthesis. During the treatment ammonium ions and monomethylamines were accumulated within the treated cells. However, monomethylamines did not induce the accumulation of ammonium ions, and vice versa. Ammonium salts and amines also selectively inhibited rRNA synthesis in Xenopus borealis neurula cells.     

Albumin is encoded by 2 messenger RNAs in Xenopus laevis

A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis. Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants. Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322. Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences. This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes. Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis. Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level. The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.     

Presence of high molecular weight glycans on the surface of germ and embryonic cells of Xenopus laevis

Xenopus laevis male germ cells, fertilized eggs and gastrula cells were labelled with 3H labelled sodium borohydride reduction after galactose oxidase treatment. After pronase digestion, the bulk of the label is carried by high molecular weight glycans (greater than or equal to 6,000 D). The high molecular weight of these labelled glycans and their susceptibility to degradation by endo-beta-galactosidase suggest that they may be related to the polylactosaminoglycans.     

Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells.

HnRNP proteins have been implicated in most stages of cellular mRNA metabolism, including processing, nucleocytoplasmic transport, stability, and localization. Several hnRNP proteins are also known to participate in key early developmental decisions. In order to facilitate functional studies of these pre-mRNA- and mRNA-binding proteins in a vertebrate organism amenable to developmental studies and experimental manipulation, we identified and purified the major hnRNP proteins and isolated the hnRNP complex from Xenopus laevis oocytes and somatic cells. Using affinity chromatography and immunological methods, we isolated a family of >15 abundant single-stranded nucleic acid-binding proteins, which range in apparent molecular weight from approximately 20 kDa to >150 kDa, and with isoelectric points from <5 to >8. Monoclonal antibodies revealed that a subset of these proteins are major hnRNP proteins in both oocytes and somatic cells in culture, and include proteins related to human hnRNP A2/B1/B2 and hnRNP K. UV crosslinking in living cells demonstrated that these proteins bind poly(A)+ RNA in vivo. Immunopurification using a monoclonal antibodyto X. aevishnRNPA2 resulted in the isolation of RNP complexes that contain a specific subset of single-stranded nucleic acid-binding proteins. The protein composition of complexes isolated from somatic cells and from oocyte germinal vesicles was similar, suggesting that the overall properties and functions of hnRNP proteins in these two cell types are comparable. These findings, together with the novel probes generated here, will also facilitate studies of the function of vertebrate RNA-binding proteins using the well characterized X. laevis oocyte and early embryo as experimental systems.