Microinjected Xenopus Oocytes Research Articles

Characterisation of cis-acting DNA sequences required for the expression of the chicken 5-aminolevulinate synthase gene in Xenopus oocytes

In this paper, we have constructed deletion and deletion-insertion mutations of the chicken 5-aminolevulinate synthase 5′ flanking region and examined the expression of these constructs in microinjected Xenopus oocytes. Utilising this assay, we have delimited the boundary of the 5′ flanking region required for expression to be 80 bp upstream from the transcription initiation site. A second major focus of this study has been to define the role of known putative cis-acting sequences in regulating 5-aminolevulinate synthase gene expression. Expression of the insertion-deletion mutants demonstrated that only a TATA box at position −28, and a single GC box at position −78 was necessary for expression of the 5-aminolevulinate synthase gene in Xenopus oocytes. This result is unusual in view of the current state of knowledge of the function of cis-acting sequence elements in transcriptional regulation.

Cis-acting elements involved in the regulated expression of a human hsp70 gene

Expression of reporter genes under the control of upstream sequences of a human hsp70 gene was examined in microinjected Xenopus oocytes, transfected monkey COS and human HeLa cells. The genes were strictly heat-regulated in all three cell systems. A 69 nucleotide long segment of hsp70 5′ non-transcribed sequence that included at least one functional heat shock regulation sequence was sufficient for heat-controlled expression in Xenopus and monkey cells but not in human HeLa cells. An additional segment of about 200 nucleotides in length was required for optimal activity. This segment contains two heat shock regulation elements, each of which appears to contribute to the overall activity in heat-treated human cells. Upstream non-transcribed sequences of the human hsp70 gene are capable of conferring heat regulation on a heterologous promoter. The potential roles in transcription regulation of a bending center in the TATA box region, a CCAAT-like sequence and some of many potential Sp1 binding sites in the hsp70 5′ non-transcribed region were investigated.

Biosynthesis of catalytically active rat testosterone 5 alpha-reductase in microinjected Xenopus oocytes: evidence for tissue-specific differences in translatable mRNA.

The enzyme 4-ene-3-ketosteroid-5 alpha-oxidoreductase [5 alpha-reductase; 3-oxo-5 alpha-steroid delta 4-dehydrogenase, 3-oxo-5 alpha-steroid: (acceptor) delta 4-oxidoreductase, EC 1.3.99.5] plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5 alpha-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. We developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of [3H]testosterone revealed the in ovo appearance of active 5 alpha-reductase. Polyadenylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1600- to 2000-base-pair fraction of hepatic poly(A)+ RNA. 5 alpha-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5 alpha-reductase mRNA was found to be a short-lived molecule (t1/2 = 2 hr), whereas its in ovo translatable 5 alpha-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5 alpha-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5 alpha-reductase activities in female rat liver, male rat liver, and prostate homogenates; the ratio of their specific activities was of 2500:630:1, respectively. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5 alpha-reductase expression in rat tissues.

Cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

Cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

5′-Flanking and 5′-proximal exon regions of the two Xenopus albumin genes : Deletion analysis of constitutive promoter function

The 5′-flanking regions and the first two exons of the 68kd and 74kd albumin genes of Xenopus laevis reveal extensive sequence homology between the two in the exon part, in the 5′-flanking region up to position −400 as well as in the first intron. Sequence comparisons of the Xenopus genes with either the albumin genes of the chicken and mammals or the mammalian alpha-fetoprotein genes reveals no homology in the 5′-flanking region but some conserved features in the first exon. The analysis of the chromatin structure demonstrates a DNase I hypersensitive region in the promoter of the 68kd albumin gene specific for hepatocytes that express the albumin gene. Deletion analysis of albumin-CAT fusion genes indicates that a 69 base-pair fragment extending from −50 to +19 of the 68kd albumin gene is sufficient for constitutive transcription in microinjected Xenopus oocytes. The addition of 5′-flanking sequences did not change the transcriptional activity. This is consistent with the sequence data that revealed no other promoter element in this region other than the TATA box. The absence of a CCAAT box distinguishes the Xenopus albumin genes from the mammalian albumin genes but is in agreement with the promoter structure of the alpha-fetoprotein genes.

Highly efficient β globin transcription in the absence of both a viral enhancer and erythroid factors

We have studied the transcription of the Xenopus major adult beta globin gene in microinjected Xenopus oocytes at various levels of injected template, with or without the SV40 enhancer. We find that enhancer-independent transcription is highly efficient, being only two orders of magnitude below the calculated in vivo rate. Linkage to the SV40 enhancer has very little stimulatory effect. We have also tested the effect of replication on transcription in the oocyte system where replication was induced by progesterone treatment followed by prick activation. We found that the presence of replicated templates did not stimulate expression of the Xenopus beta globin gene either in the presence or absence of the SV40 enhancer. In addition, we found that specificity of transcription, in cryptic of initiation at the cap site versus initiation at cryptic promoters upstream of the cap site, was dramatically improved by the injection of higher numbers of beta globin templates, by oocyte maturation and activation or by the presence of the SV40 enhancer.

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes complete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.

Properties of a U1 RNA enhancer-like sequence.

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells.

Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents.

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.

GLUCOSE REPRESSION OF AMYLASE GENE EXPRESSION IN DROSOPHILA MELANOGASTER

We have previously shown that dietary glucose can reduce amylase activity in both adults and larvae of Drosophila; this reduction in enzyme activity reflects a reduction in the quantity of amylase protein, rather than an inhibition of enzyme activity. Here, we report that we have now defined conditions in which the repressive effect of glucose can be greater than 100-fold. Moreover, this repression is partially counteracted by the addition of exogenous cyclic AMP. We also show that there is a direct correlation between changes in amylase activity and changes in the amount of translatable mRNA as assayed in microinjected Xenopus oocytes. This means that the glucose repression is occurring at a pretranslational stage.

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, α-fetoprotein, transferrin, cholinesterase, and α 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterease, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes.

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.

Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.

A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes.

The purified Escherichia coli-expressed human subgroup C adenovirus E1A 13S mRNA product induces expression from the adenovirus type 5 E3 promoter when injected into Xenopus oocytes. In the present communication, the E. coli-expressed E1A 13S and 12S mRNA products are shown to undergo a posttranslational modification in microinjected Xenopus oocytes, which causes a 2- to 4-kDa increase in apparent molecular size, identical to that occurring in HeLa cells expressing the E1A gene. The E. coli-expressed E1A proteins are similarly modified in vitro in a soluble rabbit reticulocyte lysate. The modified form of the E1A proteins preferentially localizes to the oocyte nucleus following cytoplasmic microinjection. The use of various deleted forms of E1A protein synthesized in E. coli shows that a first exon-encoded domain of E1A, residing between amino acid residues 23 and 120, is sufficient for the posttranslational modification and nuclear localization of E1A and also for the trans-activation of the E3 promoter by E1A in Xenopus oocytes. These results suggest that the posttranslational modification of E1A protein may be important for its function.

Transcription signals in embryonic Xenopus laevis U1 RNA genes.

A genomic clone of the most abundant U1 RNA genes from Xenopus laevis was isolated from erythrocyte DNA and sequenced. Two different U1 RNA genes, U1A and U1B, are encoded in an HindIII 1.5-kb fragment and both are expressed after microinjection in Xenopus oocytes. Deletions and site-directed mutagenesis of the clones revealed two promoter elements in the U1B gene; one, located 250-220 nucleotides upstream from the 5' terminus of mature U1 RNA, functions as an activator, yielding a 10-fold promotion of transcription; the other, located 60-50 nucleotides upstream of the cap site, functions as an essential element for promotion of transcription. The U1A gene contained only the latter element in the cloned fragment. Homologous sequences can be identified in several U RNA genes of X. laevis.

Involvement of cytotoxins in the immune response to viral infection

A human cytotoxin (CTX) with an Mr of 17,500 was purified to homogeneity from cytokine preparations by the use of a monoclonal antibody against that protein. Sendai virus and, to a lesser extent, the lectin phytohemagglutinin were found to induce effective production of that CTX in cultures of human peripheral-blood mononuclear cells, the first - by stimulating monocytes to produce the proteins, and the latter - by stimulating T cells. With both kinds of inducers, CTX production correlated to a marked increase in the cellular levels of mRNA for CTX, as quantitated by translation of that mRNA, to biologically active CTX, in microinjected Xenopus oocytes. Crude CTX preparations, as well as purified CTX, were found to be selectively cytotoxic to metabolically depressed and to virus infected target cells; they effectively killed cells which were treated with inhibitors of macromolecule synthesis, such as cycloheximide, or infected by viruses, such as VSV, but failed to exert a cytotoxic effect in the absence of such sensitizing treatments. IFN, most notably IFN-gamma, further potentiated destruction of virus-infected cells by the purified CTX, when applied on these cells at subantiviral concentrations, while uninfected cells remained resistant to CTX following treatment with IFN. Formation of the 17.5K CTX in response to viral infection and the selective cytotoxic effect of that protein on cells infected by viruses, indicate a role of CTX in the defense against viral infections.

Translation of mRNA for human lymphotoxin in microinjected Xenopus oocytes

Synthesis and secretion of biologically active human lymphotoxin (LT) can be detected in Xenopus laevis oocytes following their inoculation with poly(A +) RNA from human stimulated peripheral blood lymphocytes, but not in oocytes inoculated with RNA from unstimulated lymphocytes or from fibroblastoid cells. In size-fractionating mRNA of stimulated lymphocytes most LT activity is found to be coded for by RNA with an approximate sedimentation value of 19 S.

Eukaryotic transcription complexes.

Accurate transcription of eukaryotic genes in vitro is preceeded by the assembly of the template DNA into stable DNA-protein complexes. Such complexes have been reported for genes transcribed by each of the three eukaryotic RNA polymerases. DNAase I protection or footprint assays have yielded information as to the sites of protein factor binding. These sites correlate with many of the sequences which have been implicated as promoter elements through analysis of deletion mutants. Stable transcription complexes are also formed in microinjected Xenopus oocytes and such complexes can be shown to exist in vivo in nuclei and chromatin. The propagation of active transcription complexes may prove to be a crucial element in cellular differentiation.

Adenovirus E1a Gene Product Expressed at High Levels in Escherichia coli Is Functional

The human type C adenovirus E1a 13S messenger RNA encodes a gene product, that positively regulates the transcription of viral genes and certain cellular genes and is involved in the transformation of primary mammalian cells. The E1a gene product was expressed at high levels in Escherichia coli. In a Xenopus oocyte microinjection assay, the purified Escherichia coli-produced protein activated the E1a-responsive adenovirus E3 promoter and functioned as efficiently as the E1a gene itself.