An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Ectopic expression of Xenopus noggin RNA induces complete secondary body axes in embryos of the direct developing frog Eleutherodactylus coqui

We tested the effects of noggin RNA from Xenopus laevis on axis induction in embryos of a direct developing frog, Eleutherodactylus coqui. We microinjected noggin RNA into one blastomere of 4-cell embryos at the site close to the animal pole, and found that overexpression of noggin RNA is not only sufficient to induce additional axes but also induces heads with eyes. We also injected noggin RNA into 8-cell or 16-cell embryos in various sites, including the marginal zone, above the marginal zone, and the vegetal pole, and found the formation of a complete secondary axis in all three types of injection. These effects of X. laevis noggin RNA on the E. coqui embryo are remarkably different from those found in the X. laevis embryo itself. It has been shown previously that overexpression of noggin RNA on the ventral side of the normal X. laevis embryo induces only a partial axis, with no head structures. We show here that the failure of noggin induction of a complete axis when overexpressed on the ventral side of the X. laevis embryos is not due to an insufficient amount of RNA injected. Also, the failure is unlikely due to inhibition from the primary axis since noggin RNA can induce duplicated head structures on opposite sides of UV-irradiated X. laevis embryos. There appear to be fundamental differences in the responses of E. coqui and X. laevis embryos to exogenous noggin RNA. We propose that these differences stem from an alteration in cytoplasmic arrangements that occurred during evolution of this large egg. Received: 26 July 1999 / Accepted: 1 September 1999     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNA^Phe, tRNA^Tyr, tRNA ₁ ^Met , tRNA^Asn, tRNA^Ala, tRNA^Leu, and tRNA^Lys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.We respectfully dedicate this paper to Prof. H. Bauer on the occasion of his 80th birthday.     

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.     

Biosynthesis of the neurofilament heavy subunit in Xenopus oocytes microinjected with rat brain poly(A)+ RNA

In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)⁺ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethanesulphonyl fluoride - PBS phosphate buffered saline - BSA bovine serum albumin     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

Nucleolar targeting of 5S RNA in Xenopus laevis oocytes: Somatic-type nucleotide substitutions enhance nucleolar localization

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, ³²P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, ³²P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of ³⁵S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule. J. Cell. Biochem. 69:490–505, 1998. © 1998 Wiley-Liss, Inc.     

A quantitative method for the measurement of cellular guanosine triphosphate pool specific activities

Studies on quantitation of RNA synthesis in eucaryotic cells have frequently used adenosine as the radioactively labeled precursor, largely because of the convenience of the firefly luciferin-luciferase assay in measuring ATP pool specific activity (1,2). This could result in some difficulties if the addition of poly(A) to the 3′ OH end of RNA represents a significant portion of total incorporation, as is the case in sea-urchin embryos (3). In addition, in some cases, the ATP pool may be large enough to prevent the use of adenosine as an effective labeling agent. Hence, a simple and sensitive method for the determination of the specific activity of the other nucleic acid precursor pools would be of value.Although the crystalline luciferase is specific for ATP, extracts of firefly lanterns most commonly used for quantitating ATP (4–9) also exhibit activity with other ribonucleoside triphosphates, adenosine tetraphosphate, ADP, and the deoxyribonucleoside triphosphates. This activity is due to the presence of contaminating enzymes such as nucleoside 5′-diphosphate kinase and adenylate kinase which catalyze the formation of ATP from these nucleotides and trace amounts of ADP, also present in the extracts (10–13). Recently, Manandhar and Van Dyke (14) have reported a procedure for quantitating picomole levels of GTP with a crude extract of firefly lanterns. In the present study, we have adapted their procedure to develop an assay for GTP pool specific activity in Xenopus laevis oocytes microinjected with [8-³H]GTP. Our assay may be extended to the analysis of any nucleoside triphosphate pool, provided that an adequate chromatography system is available for the separation of the extracted nucleotides.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Studies on the function of TM20, a transmembrane protein present in cereal embryos

The possible function of the maize transmembrane protein TM20 in hormone transport has been investigated using immunological methods and by microinjection of TM20 cRNA in Xenopus oocytes. The existence of a similar gene in rice indicates that the overall structure of the deduced protein is conserved between these two cereals. An antibody raised against a conserved motif, in a loop between two transmembrane domains, locates the protein (TM20) in differentiating provascular cells in maize embryo. The protein has a polarized distribution within the cell in the most differentiated stages of development. Xenopus laevis oocytes microinjected with TM20 appear to modify transport activities across the plasma membrane. These results are discussed in relation to other transport proteins that influence plant development.     

Lineage-Specific Tandem Repeats Riding on a Transposable Element of MITE in <Emphasis Type="Italic">Xenopus</Emphasis> Evolution: A New Mechanism for Creating Simple Sequence Repeats

Xstir is a repetitive DNA sequence element that is extremely amplified as a common component of two different structures: a tandem repeat (Xstir array) and a MITE (miniature inverted-repeat transposable element) in the genome of Xenopus laevis. To elucidate the origin and evolutionary history of Xstir-related sequences, we investigated their species specificity among three Xenopus species (X. laevis, X. borealis, and X. tropicalis). Analyses by sequence alignment and digestion with restriction enzymes of genomic Xstir-related sequences revealed that the MITE (Xmix MITE) was well conserved among the three Xenopus species, with small lineage-specific differences. On the other hand, the tandem repeat element (tropXstir) in X. tropicalis was different from the Xstir that X. laevis and X. borealis have in common. Both sequences of Xstir and tropXstir were, however, different segments of the Xmix MITE. The results suggest that these tandem repeats were formed by partial tandem duplication of the MITE internal sequence in each lineage of X. tropicalis and of X. borealis/X. laevis after their branching. A molecular mechanism for creating and elongating the tandem repeats from the MITE is proposed.Reviewing Editor: Dr. Jerzy Jurka     

Studies of microbial toxins in Xenopus laevis oocytes

Xenopus laevis oocytes have been incubated or microinjected with cholera and diphtheria holotoxins or their respective isolated fragments A and B. Effects on progesterone-induced maturation, protein synthesis and cAMP levels were observed. Xenopus laevis oocytes were highly susceptible to cholera toxin upon incubation as evidenced by the increase of cAMP (two-fold increase in cAMP with 0.1 nM cholera toxin) and the blockade of progesterone-induced maturation. When isolated cholera toxin fragments A or B were incubated with oocytes, no activity could be detected. However, microinjection of cholera toxin fragment A into oocyte was able to mimic the effects of incubated holotoxin. Microinjection of cholera toxin B fragment was only effective at very high concentrations, probably due to trace contaminations by the A fragment. On the other hand, Xenopus laevis oocytes were very resistant to diphtheria toxin action upon incubation, a result attributable to lack of specific membrane receptors since, after microinjection of diphtheria toxin A fragment into oocytes, inhibition of protein synthesis was demonstrated. By simultaneous microinjection of highly radioactive adenine-labelled NAD and diphtheria toxin fragment A into oocytes, radioactive ADP ribosylation of the elongation factor 2 (EF₂) was observed. It is proposed that Xenopus laevis oocytes provide a new experimental approach for studying the mechanisms of action of microbial toxins.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Simulated microgravity induce glutathione antioxidant pathwayin Xenopus laevis embryos

Space flights cause a number of patho-physiological changes. Oxidative damage has been demonstrated in astronauts after space flights. Oxidative stress is due to an imbalance between production of oxidant and antioxidative defence. In embryos of Xenopus laevis, the glutathione system is an inducible antioxidant defence. For this reason, we investigated the effect of gravity deprivation on endogenous antioxidant enzymes in X. laevis embryos developed for 6 days in a Random Positioning Machine. The results show that glutathione content and the activity of antioxidant enzymes increase in RPM embryos, suggesting the presence of a protective mechanism. An induction of antioxidant defence might play an important role for animals to adapt to micro-gravitational stress, possibly during actual space flights.     

Autoradiographic distributions of 3H-putrescine and 3H-uridine in a Xenopus liver cell line

Summary The distributions of ³H-putrescine and ³H-uridine were studied autoradiographically in cultured Xenopus laevis liver cells. Biochemical assay showed that at 4 h 10%, and at 24 h 30 % of the putrescine label was recovered as spermidine. Grain counts per unit surface area in light microscopic autoradiographs indicate that the ³H-polyamines show a similar intranuclear accumulation as ³H-uridine with a definite association with the nucleolus. The time course is different, however since ³H-polyamines continue to accumulate in the nucleus, while ³H-uridine reaches a peak nuclear concentration within 30 min and drops to one-half after 24 h. No instance of grains overlying mitotic figures was observed. These findings indicate an association of ³H-polyamines with nuclear and nucleolar RNA.Supported by US-PHS Grant No. NS-07934     

Hormonal effects on RNA synthesis by stage 6 oocytes of Xenopus laevis.

RNA synthesis has been studied in “large” oocytes of Xenopus laevis, both as a function of time after injection of females with human chorionic gonadotropin (HCG) and in relation to the induction of maturation with progesterone in vitro. Rates of RNA synthesis were measured by analyzing the kinetics of incorporation of exogenous [³H]guanosine, and microinjected [³H]- or [¹⁴C]GTP, into acid-precipitable material, coupled with measurements of precursor pool specific activity. The kinetics of incorporation into RNA of injected precursor are biphasic, indicating the synthesis of both stable and unstable RNA species. Estimates of the total rate of synthesis (stable and unstable) were derived from fitting a linear function to data over the first 60–90 min, while a linear function fit to the data beyond 90 min represented largely the synthesis of stable RNA species.Exposure of oocytes to progesterone had no effect on initial synthetic rates, but maturing oocytes synthesized stable RNA at 1.4–1.6 times the rate in control oocytes. A comparison of data obtained with oocytes from unstimulated (no prior HCG treatment) and HCG-stimulated females indicated that HCG has no substantial effect on rates of RNA synthesis. The significance of continued RNA synthesis in large full grown oocytes is discussed.     

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)⁺ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).