Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. V. Inability of inhibitor to repress 5S RNA synthesis.

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos: VI. Occurrence in mature oocytes and unfertilized eggs

Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [³H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.     

RNA synthesis in nuclei isolated from early embryos of Xenopus laevis.

1. Rates of RNA synthesis in isolated Xenopus embryo nuclei decrease from blastula through gastrula and neurula stages to hatching tadpoles. 2. In blastula and gastrula nuclei, net synthesis of RNA continues for over 30 min, both in the presence of KCl at 0.4 M and in its absence. In nuclei from later stages, net synthesis continues for only about 10 min in the absence of KCl. 3. At low ionic strength, RNA synthesis in all nuclei is greater with optimum Mg-2+ (6 mM) than with optimum Mn-2+ (1 mM). At high ionic strength the reverse is true. 4. An unusual feature, which gradually disappears as development proceeds, is that curves relating RNA synthesis to KCl concentration show a peak at 0.1 M KCl. In blastula nuclei, RNA synthesis is more rapid at 0.1 M KCl than at 0.4 M. 5. This peak at low ionic strength is not observed in the presence of the initiation inhibitor rifamycin AF/013. It is concluded that the peak arises from initiation of RNA synthesis by an excess of RNA polymerases bound non-specifically to the isolated nuclei. The residual synthesis, representing elongation of chains that were initiated in vivo, still declines as development progresses. 6. In blastula nuclei, over half of the RNA synthesis is effected by polymerase II (inhibited by alpha-amanitin), the proportion remaining roughly constant with increasing ionic strength. In neurula nuclei, the proportion rises from about one-half to three-quarters. The initiation-dependent peak in blastula and gastrula nuclei is contributed by both alpha-amanitin-sensitive and alpha-amanitin-resistant enzymes.     

The nucleotide sequences of 5.8-S ribosomal RNA from Xenopus laevis and Xenopus borealis

1. The nucleotide sequence of 5.8-S rRNA from Xenopus laevis is given; it differs by a C in equilibrium U transition at position 140 from the 5.8-S rRNA of Xenopus borealis. 2. The sequence contains two completely modified and two partially modified residues. 3. Three different 5' nucleotides are found: pU-C-G (0.4) pC-G (0.2) and pG (0.4). 4. The 3' terminus is C not U as in all other 5.8-S sequences so far determined. 5. The X. laevis sequence differs from the mammalian and turtle sequences by five and six residue changes respectively. 6. A ribonuclease-resistant hairpin loop is a principle feature of secondary structure models proposed for this molecule. 7. Sequence heterogeneity may occur at one position at a very low level (approximately 0.01) in X. laevis 5.8-S rRNA, while none was detected in X. borealis or HeLa cell 5.8-S rRNA.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis

Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.     

More ribosomal spacer sequences from Xenopus laevis.

The base sequence analysis of a Xenopus laevis ribosomal DNA repeat (7) has been extended to cover almost the entire non-transcribed and external transcribed spacer. A compilation of these sequences is presented. All the repetitive and non-repetitive sequence elements of the spacer are identified and their evolution discussed. Comparison of the X.laevis and S.cerevisiae (25,26) ribosomal DNAs shows about 80% sequence conservation in the 18S gene but no sequence conservation, from the available data, in the external transcribed spacer. The sequence coding for the 3' terminus of the X.laevis 40S ribosomal precursor RNA is presented and its structural features analyzed.     

Ectopic germline cells in embryos of Xenopus laevis

Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.     

Transcriptional organization of the 5.8S ribosomal RNA cistron in Xenopus laevis ribosomal DNA.

Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.8S rRNA cistron lies within the transcribed region that links the 18S and 28S rRNA cistrons.