Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.     

Lipid composition of developing Xenopus laevis embryos.

The total lipid content, amount of phospholipid, proportions of major polar and neutral lipid classes, and the overall fatty acid composition were examined in Xenopus laevis embryos. No obvious differences were observed in any of the parameters between fertilization and hatching or between eggs produced by different females. The average lipid content per egg was 113 mug, 31.6 mug of which was phospholipid. The major phospholipids were phosphatidylcholine and sphingomyelin. The major fatty acids were palmitic and oleic acids, but polyunsaturated fatty acids were also present in substantial amounts. The results suggest that significant de novo synthesis of lipids does not occur until after hatching.     

A role in DNA binding for the linker sequences of the first three zinc fingers of TFIIIA.

Zinc fingers of the TFIIIA type are connected by short linker sequences between the structural units. Structural investigations by 2D NMR in solution and by X-ray crystallographic analyses of complexes with DNA point to a passive role for the linkers. We have therefore investigated the influence of the linker sequence on DNA binding using as a model the first three fingers of the protein TFIIIA. Insertion of certain heterologous linkers abolishes binding, and replacement of individual amino acids can reduce binding by factors of up to twenty-four.     

Changes in the polysome content of developing Xenopus laevis embryos

A method for preparing polysomes from all embryonic stages of Xenopus laevis is described. In the oocyte only about 1–2% of the total ribosomes are present in polysomes, the remainder being a developmental reserve. Upon conversion to an egg the polysome content rises by up to 3-fold, and by about a further 2-fold after fertilization. There is only a small further increase during cleavage, but by the tailbud stage, when organogenesis begins, there is a more rapid rise. Most of the ribosomes are incorporated into polysomes by stage 42, shortly before feeding begins.At very early stages, the changes in polysome content seem to mirror the changes in protein synthesis. At later stages the polysome contents reported here provide the only available guide to changes in the rate of protein synthesis. Judged by polysome content, the stage 42 tadpole seems to make protein about 20 times faster than the unfertilized egg, though it contains very few more ribosomes. The relationship between polysome content and the synthesis of various types of RNA is discussed.     

31P nuclear-magnetic-resonance studies on the developing embryos of Xenopus laevis.

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5-4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (approximately 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their alpha, beta and gamma phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +/- 0.2.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

Overexpression of wild-type and dominant negative mutant vimentin subunits in developing Xenopus embryos.

Vimentin belongs to the diverse multigene family of intermediate filament proteins, each member of which is expressed in a tissue-specific and developmentally regulated pattern. The existence of vimentin filaments has been documented in oocytes, eggs, and early embryos of Xenopus laevis, but the role of these cytoskeletal components remains unknown. To investigate the functions of vimentin during early development in Xenopus, we induced the overexpression of wild-type and deletion mutant subunits in most of the cells of embryos by injecting synthetic RNA into fertilized eggs. Wild-type vimentin subunits, as well as subunits lacking most of the amino-terminal head piece, assembled into normal appearing filaments in vivo. Deletion mutants of the fourth alpha-helical rod domain were assembly incompetent and dominantly inhibited the polymerization of wild-type subunits when both types of subunit were co-expressed in cells. Expression of at least a tenfold excess of wild-type or mutant subunits within cells of embryos did not lead to any detectable morphological or developmental abnormalities, suggesting that the presence and proper regulation of vimentin expression is not essential during the initial stages of embryogenesis in Xenopus.     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

Ultraweak emissions of the developing Xenopus laevis eggs and embryos

We measured ultraweak emissions of the Xenopus laevis eggs and embryos during normal development and under the influence of stress factors in a spectral range of 250 to 800 nm using a photomultiplier. The registered emissions were analyzed by several basic characteristics: mean intensity, histograms, kurtosis, linear trends, and Fourier spectra. We followed relationships between these parameters and developmental stage, as well as the number of individuals in optic contact with each other. The ultraweak emissions did not differ from the background at all developmental stages according to the mean intensity. But Fourier analysis revealed the reliable presence of a number of spectral lines of ultraweak emission, predominantly in the ranges of 10-20 and 30-40 Hz, in the embryos at developmental stages 2 to 11. The intensity of ultraweak emissions reliably decreased within the first 10 min after egg activation and fertilization, as well as in the case of optic interaction between groups of embryos. Sharp cooling, increase in osmotic medium pressure, and transfer in a Ca(2+)- and Mg(2+)-free medium induced a short term (approximately 1-5 min) increase in the mean intensity of ultraweak emission. We studied specific features of ultraweak emissions from different parts of the embryo. The intensity of emission from the animal part of early blastula exceeded those from the vegetal area and entire embryo. Separated fragments of the lateral ectoderm at the neurula stage had higher mean intensities of ultraweak emission than intact embryos at the same developmental stages.