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1.
Antisense oligodeoxyribonucleotide-directed cleavage of maternal mRNA in Xenopus oocytes and embryos 总被引:10,自引:0,他引:10
We have investigated the effect of specific antisense oligodeoxynucleotides (oligos) on endogenous histone H4 mRNA in Xenopus oocytes, eggs and embryos. In unfertilised eggs and non-matured oocytes, one 20-mer oligo (H4-1) mediated the RNAse H-like cleavage of up to 95% of H4 mRNA (which included polysomal mRNA), and cleavage was still obtained when the size of the oligo was reduced to a 10-mer; no cleavage was observed with 6- and 8-mers. The residual uncleaved mRNA appeared to be completely inaccessible to H4-1 since a second injection caused no further cleavage. A second 20-mer (H4-2) directed against a different region of H4 mRNA was much less effective (less than 5% cleavage). In fertilised embryos, injections of H4-1 and an oligo directed against the localised Vg1 mRNA caused less cleavage than in oocytes and also showed signs of inducing localised, non-specific mRNA cleavage. However we have been able to prepare fertilised embryos devoid of Vg1 mRNA by maturing and fertilising oligo-injected oocytes in vitro. 相似文献
2.
Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. 总被引:33,自引:14,他引:19
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C Cazenave C A Stein N Loreau N T Thuong L M Neckers C Subasinghe C Hlne J S Cohen J J Toulm 《Nucleic acids research》1989,17(11):4255-4273
We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides. 相似文献
3.
The Xenopus Vg1 gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-beta family of small secreted growth factors. We have raised antibodies to recombinant Vg1 protein and used them to show that Vg1 protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vg1 protein is synthesized until the gastrula stage. The embryonically synthesized Vg1 protein is present only in vegetal cells of an early blastula. We find that Vg1 protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vg1 protein is cleaved to give a small peptide of M(r) = approximately 17 x 10(3). These results support the proposal that the Vg1 protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo. 相似文献
4.
Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity. 总被引:3,自引:0,他引:3
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We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier. 相似文献
5.
Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog. 相似文献
6.
7.
Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides 总被引:15,自引:0,他引:15
Agrawal S 《Biochimica et biophysica acta》1999,1489(1):53-68
The antisense approach is conceptually simple and elegant; to design an inhibitor of a specific mRNA, one needs only to know the sequence of the targeted mRNA and an appropriately modified complementary oligonucleotide. Of the many analogs of oligodeoxynucleotides explored as antisense agents, phosphorothioate analogs have been studied the most extensively. The use of phosphorothioate oligodeoxynucleotides as antisense agents in various studies have shown promising results. However, they have also indicated that quite often, biological effects observed could be solely or partly non-specific in nature. It is becoming clear that not all phosphorothioate oligodeoxynucleotides of varying length and base composition are the same, and important consideration should be given to maintain antisense mechanisms while identifying effective antisense oligonucleotides. In this review, I have summarized the progress made in my laboratory in understanding the specificity and mechanism of actions of phosphorothioate oligonucleotides and the rationale for designing second-generation mixed-backbone oligonucleotides. 相似文献
8.
In an effort to understand how polarity is established in Xenopus oocytes, we have analyzed the process of localization of the maternal mRNA, Vg1. In fully grown oocytes, Vg1 mRNA is tightly localized at the vegetal cortex. Biochemical fractionation shows that the mRNA is preferentially associated with a detergent-insoluble subcellular fraction. The use of cytoskeletal inhibitors suggests that (1) microtubules are involved in the translocation of the message to the vegetal hemisphere and (2) microfilaments are important for the anchoring of the message at the cortex. Furthermore, immunohistochemistry reveals that a cytoplasmic microtubule array exists during translocation. These results suggest a role for the cytoskeleton in localizing information in the oocyte. 相似文献
9.
Translation of Vg1 mRNA is repressed in Xenopus oocytes until it is localized to the vegetal cortex. Localization and translational repression are mediated by separate elements in the 3'UTR of the mRNA. VgRBP71 binds to the 3' end of the localization element and stimulates cleavage at an adjacent polyadenylation signal. The protein has an RNA strand-separation activity that likely underlies this event. Polyadenylation occurs at this site in Vg1 mRNA with the consequence of removing the downstream translational repressor element. Ectopic expression of VgRBP71 in stage II oocytes results in cleavage of the mRNA and premature expression of Vg1 protein. These results support a model in which VgRBP71 activates translation of Vg1 mRNA by promoting the removal of a cis-acting repressor element. 相似文献
10.
The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex. 相似文献
11.
12.
Jasmani S Ohira T Jayasankar V Tsutsui N Aida K Wilder MN 《Journal of experimental zoology. Part A, Comparative experimental biology》2004,301(4):334-343
In situ hybridization and immunohistochemical techniques were used to investigate the dynamics of vitellogenin (Vg) mRNA expression and Vg uptake during ovarian maturation in the hepatopancreas and ovary at differing stages of ovarian maturation in both intact and eyestalk ablated female Macrobrachium rosenbergii. In the hepatopancreas of intact animals, Vg mRNA expression was detected faintly two days after ecdysis, and signals showed a gradual increase as the molt cycle advanced to the premolt stages, but decreased at the late premolt stage. Vg mRNA was detected in the R-cells of the hepatopancreas, indicating that these cells are responsible for synthesizing Vg. No Vg mRNA expression was observed in the ovary. Immunohistochemistry results for the hepatopancreas showed a pattern of staining intensity similar to that of in situ hybridization. Increases in the accumulation of yolk protein in the oocytes occurred concomitantly with increasing Vg mRNA expression. In eyestalk ablated animals, Vg mRNA expression and Vg uptake showed similar but accelerated patterns to those of intact animals. This study has confirmed on the cellular level previous results that Vg synthesis is intrinsically correlated to ovarian maturation and the molt cycle in M. rosenbergii. 相似文献
13.
Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1. 总被引:4,自引:3,他引:1
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Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development. 相似文献
14.
A 340 nucleotide element within the 3' untranslated region of Vg1 mRNA determines its localization to the vegetal cortex of Xenopus oocytes. To identify protein factors that bind to this region, we screened a cDNA expression library with an RNA probe containing this sequence. Five independent isolates encoded a protein (designated Prrp for proline-rich RNA binding protein) having two RNP domains followed by multiple polyproline segments. Prrp and Vg1 mRNAs are co-localized to the vegetal cortex of stage IV oocytes, substantiating an interaction between the two in vivo. Prrp also associates with VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. The proline-rich domain of Prrp interacts with profilin, a protein that promotes actin polymerization. Prrp can also associate with the EVH1 domain of Mena, another microfilament-associated protein. Since the anchoring of Vg1 mRNA to the vegetal cortex is actin dependent, one function of Prrp may be to facilitate local actin polymerization, representing a novel function for an RNA binding protein. 相似文献
15.
Allison R Czaplinski K Git A Adegbenro E Stennard F Houliston E Standart N 《RNA (New York, N.Y.)》2004,10(11):1751-1763
Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development. 相似文献
16.
Compagno D Lampe JN Bourget C Kutyavin IV Yurchenko L Lukhtanov EA Gorn VV Gamper HB Toulmé JJ 《The Journal of biological chemistry》1999,274(12):8191-8198
Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs. 相似文献
17.
Kaneshiro K Miyauchi M Tanigawa Y Ikenishi K Komiya T 《Biochemical and biophysical research communications》2007,355(4):902-906
Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed. 相似文献
18.
Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents. 总被引:34,自引:25,他引:9
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The effects of anti-messenger oligodeoxynucleotides, covalently linked to an intercalating agent, on translation of rabbit beta-globin mRNA, were investigated both in wheat germ extract and in microinjected Xenopus oocytes. A specific inhibition of beta-globin synthesis was observed in both expression systems with a modified 11-mer covalently linked to an acridine derivative. In injected oocytes a more efficient block was observed with this modified oligonucleotide than with its unsubstituted homolog. This was ascribed to stacking interactions of the intercalating agent with base pairs which provide an additional stabilization of the [mRNA/DNA] hybrid. We demonstrated that in wheat germ extract, the modified and unmodified oligonucleotides behaved similarly due to the presence of a high RNaseH activity. RNaseH was also present, although to a lesser extent, in the oocyte cytoplasm. This anti-messenger DNA-induced degradation of target mRNA resulted in amplified efficiency of hybrid-arrested translation. This additional mechanism might provide anti-sense DNAs with an advantage over anti-sense RNAs. 相似文献
19.
Marcin Cieslak Jolanta Niewiarowska Magdalena Nawrot Maria Koziolkiewicz Wojciech J Stec Czeslaw S Cierniewski 《The Journal of biological chemistry》2002,277(9):6779-6787
A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo. 相似文献
20.
Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis. 相似文献