Microinjected Xenopus Oocytes Research Articles

Competition between Xenopus satellite I sequences and Pol III genes for stable transcription complex formation.

We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans. Transcription of a tRNA gene is also precluded by satellite I DNA. The Xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the satellite promoter is largely temperature independent over the range of 0-20 degrees, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants.

Synthetic leader peptide modulates secretion of proteins from microinjected Xenopus oocytes.

To investigate the role of the leader peptide in modulating secretion from living cells, we injected a synthetic peptide into Xenopus oocytes. The peptide consisted of the NH2-terminal leader sequence of mouse immunoglobulin light chain precursor. We found that the leader peptide has two different roles in regulating secretion from the oocytes. First, it competitively inhibits the synthesis of secretory and membrane proteins but not of cytoplasmic proteins. The inhibition occurs both with oocyte proteins and with proteins directed by coinjected myeloma mRNA. The inhibition reaches a maximum 2 hr after injection and decays within 3 hr. It appears to be mediated through the cell membrane, because 125I-labeled leader peptide segregates into the membrane fraction of microinjected oocytes simultaneously with the interference with methionine incorporation. A second role of the microinjected leader peptide is to induce a rapid acceleration in the rate of export of secretory proteins from the oocyte. The maximal enhancement effect is obtained upon injection of 50 ng of leader peptide per oocyte. It is not merely due to the small size, negative charge, or hydrophobicity of the peptide, because enhanced secretion does not occur when glucagon, poly-L-glutamic acid, or Triton X-100 is injected. Furthermore, immunoreaction of the peptide with specific antibodies prior to microinjection prevents the accelerated export. Our observations indicate that in Xenopus oocytes, the leader peptide is involved in both translocation and later step(s) in the secretory pathway.

Microinjection of vesicular stomatitis virus ribonucleoprotein into animal cells yields infectious virus

Microinjection of purified transcriptionally active ribonucleoprotein (RNP) complex of vesicular stomatitis virus in vero cells resulted in the production and release of virus. Compared to the release of virus by cells treated with RNP in the presence of DEAE-dextran, the microinjection technique was highly efficient. Microinjection in Xenopus oocytes also resulted in initiation of infection as shown by the synthesis of virus-specific proteins in the cell cytoplasm. It was further observed that RNP stripped of L protein but containing residual NS protein was capable of initiating virus production or protein synthesis when microinjected in vero cells or in oocytes, respectively. Since L and NS proteins are essential for in vitro transcription by RNP, these results suggest that a trace amount of L protein may remain bound to the RNP and a host factor may stimulate residual L activity in vivo.

Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.

HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.

The recruitment of membrane-bound mRNAs for translation in microinjected Xenopus oocytes.

Xenopus oocytes were injected with several heterologous mRNAs to investigate specific message recruitment onto polysomes. When large amounts of mRNA such as ovalbumin, which is translated on membrane-bound polysomes, are injected, most of the stable message accumulates in a postpolysomal supernatant. Conversely, when similar amounts of nonmembrane-bound mRNAs such as those which code for adenovirus protein are injected, most of the messages are found in a polysome pellet. Nonpolysomal mRNAs such as zein, ovalbumin, and lysozyme can be recruited onto membrane-bound polysomes by injected rough endoplasmic reticulum (RER). Only RER, but not Golgi apparatus, plasma membrane, or salt-washed RER has such recruiting ability. In addition, the nontranslating messengers can be recruited by injecting the salt wash of the RER. Since most nontranslating mRNAs sediment to regions less than the 80 S monosome, the step at which translational regulation takes place is probably initiation rather than elongation. The mechanism by which recruitment might occur is discussed.

Analysis of expression of adenovirus DNA (fragments) by microinjection in Xenopus oocytes : Independent synthesis of minor early region 2 proteins

Injection of whole adenovirus DNA into Xenopus oocytes results in the synthesis of large amounts of the early region 2A DNA-binding protein (E2A-DBP) and smaller amounts of polypeptide IX. The lack of synthesis of any functional messenger RNAs transcribed from the major late promotor at 16.3 map units is remarkable. Cleavage of the adenovirus DNA outside the E2A gene proper by restriction enzymes decreases synthesis of the DBP to about 10% of the amount produced after injection of intact DNA. On the other hand, presence of the terminal (Bellett) protein on the injected template enhances DBP synthesis considerably. Experiments with injected DNA restriction fragments, as well as reconstructed genes cloned into pBR322, indicate that efficient synthesis of DBP in oocytes requires the presence of either or both of the two main promoters from which the E2A gene is transcribed plus an intact 3′ end of the gene. In the absence of any known promotor, 100-fold lower amounts of otherwise normal DBP are produced. Unlike in a regular infection, synthesis of DBP in oocytes does not require the product of the E1A gene. The same series of experiments also demonstrates that the DBP, a phosphoprotein, is the substrate of a cellular rather than a virus-encoded protein kinase. Two minor E2A proteins, although colinear with the major DBP, are synthesized independently. Synthesis of a 44,000 M r protein, probably corresponding to the carboxy-terminal 360 amino acid residues of the DBP, is not decreased after injection of “promotorless” E2A genes. Unlike the 44,000 M r protein, production of a 67,000 M r protein (carboxy-terminal 483 amino acid residues) by one DNA-construct is probably directed by a T-A-T-A-A-A-T-A sequence in the vector DNA.

Messenger RNA from human brain gliomas and meningiomas directs the synthesis of cholinesterase in microinjected Xenopus oocytes

Messenger RNA from human brain gliomas and meningiomas directs the synthesis of cholinesterase in microinjected Xenopus oocytes

Synthesis of vaccinia virus thymidine kinase in microinjected Xenopus oocytes

Messenger RNA was extracted from vaccinia virus (VV) -infected thymidine kinase (tk)-deficient cells early in infection and introduced into the cytoplasm of Xenopus laevis oocytes by microinjection. Extracts prepared from these oocytes after 24 hr incubation exhibited a thymidine kinase activity that was more than 250-fold greater than the endogenous oocyte level. Messenger RNA extracted late during VV infection, or from cells infected with a tk − mutant of VV, failed to direct the increase in tk activity. The enzyme synthesized in oocytes appeared to be identical to authentic VV tk from infected human tk − cells by the criteria of thermal inactivation kinetics, sedimentation velocity, and electrophoretic mobility under nondenaturing conditions. Moreover, tk synthesis was specifically arrested if, prior to microinjection, the early VV mRNA was hybridized to a recombinant plasmid that contained the VV tk gene. However, attempts to detect the synthesis of VV tk in Xenopus oocytes which had been injected intranuclearly with VV DNA fragments containing the tk gene were uniformly unsuccessful.

Multiple HLA-DR antigens: detection with monoclonal antibodies and translation in vitro.

L203 and L227 are mouse monoclonal antibodies specific for human HLA-DR antigens. Their reactivity with multiple, structurally different, HLA-DR antigens was examined by using radioiodinated human lymphoblastoid cells and cell lines, heterozygous or homozygous at the HLA-D locus. Antibody L203 precipitates DR antigens composed of 34,000 Mr heavy chains (alpha), and 28,000-29,000 Mr light chains (beta). Antibody L227 recognizes two other species of DR antigens. Their heavy chains have similar molecular weights of 34,000 but their light chains are different, one (beta) being 28,000-29,000 Mr and the other (beta') 23,000-25,000 Mr. Whereas L203 is directed against a combinatorial determinant generated by both DR chains, L227 is able to recognize each of the two light chains alone and reprecipitate them after their separate elution from sodium dodecyl sulfate gels. Poly(A)-containing mRNA was isolated from Raji cells and translated in vitro by microinjection in Xenopus oocytes. The oocytes translated and fully assembled all the DR molecules recognized by L203 and L227 antibodies.

Increase in the translatable mRNA for acetylcholine receptor during embryonic development of Torpedo ocellata electric organ

The availability of large quantities of the nicotinic acetylcholine receptor (AChR) in the highly specialized electroplates of Torpedo permitted detailed studies of its structure [l-3], as well as preparation of specific antibodies against the receptor in its native and in its denatured forms [4-61. Anti-AChR antibodies have also been used to immunoprecipitate all 4 nascent chains of the newly synthesized AChR, translated by Torpedo mRNA in vitro [7,8] and in ovo, in microinjected Xenopus oocytes [9]. The immunoprecipitation of newly synthesized AChR subunits with anti-AChR antibodies of various specificities provides a means to determine which of the receptor antigenic sites already exists in the nascent chains. Here, we used antibodies raised against the native and denatured AChR and have demonstrated that newly synthesized AChR subunits probably do not possess the final spatial. conformation of the ‘adult’ receptor, but rather appear to be closer in structure to the denatured receptor. Differentiation of electroplates within the embryonic electric organ is accompanied by a steep increase in cholinergic constituents such as AChR and acetylcholinesterase (AChE) [IO]. Moreover, messenger RNA from the embryonic electric organ of Torpedo ocellata induces the production of catalytically active AChE in microinjected Xenopus oocytes [ 111. Following their maturation, the electroplates lose most of their ribosomes [ 121 and their capacity for protein synthesis is hence greatly reduced. Several mechanisms can explain the differentiationinduced accumulation of AChR in the electric organ. The elevation in AChR level might be preceded and/ or accompanied by a selective increase in its specific mRNA species (AChR mRNA). Alternatively, AChR production could be a transient phenomenon, characteristic of electroplate maturation stage. In both cases, the regulation of AChR expression might involve variation in the rate of proteolytic degradation of the receptor itself. In order to distinguish between these possibilities, we determined the level of AChR mRNA and compared it to the concentration of the receptor protein at 3 stages during electroplate differentiation: the electroblast stage (I); the stage when the celk acquire a columnar shape (II); and when morphological appearance of electroplates is displayed (III). Our findings indicate that the maturation stage of Torpedo ocellata electroplates is accompanied by a burst in AChR synthesis, and that following this stage, the mRNA species directing the synthesis of AChR represents a constant fraction out of total poly(A)containing mRNA.

Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.

A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.

Yeast mitochondrial tRNATrp injected with E. coli activating enzyme into Xenopus oocytes suppresses UGA termination.

It is now clear that the genetic code used in mitochondria differs in a number of ways from the standard code1–3, the most prominent being the use of the opal codon UGA to specify tryptophan4–7. This change in the mitochondrial genetic code is accommodated by the presence of an anticodon U*CA in mitochondrial (mt) tRNATrp from yeast8 and Neurospora crassa1. Furthermore, the translation of a mtDNA-encoded mRNA has been achieved in a eukaryotic cell-free system9: the mRNA of subunit II of yeast cytochrome c oxidase, which contains UGA codons in its reading frame, can be translated into a full-length protein if the system is supplemented with the Schizosaccharomyces pombe10 cytoplasmic UGA suppressor tRNASer. It remains to be demonstrated whether a mitochondrial tRNA would be able to function in cytoplasmic protein synthesis. In the case of mt tRNATrp, this would cause the production of readthrough proteins due to the suppression of the UGA termination codon present in certain cytoplasmic mRNAs. Using the Xenopus oocyte microinjection system11–12, we show here that the mt tRNATrp from Saccharomyces cerevisiae, when injected together with rabbit globin mRNA, suppresses UGA termination with high efficiency, thus leading to the production of a β-globin-related readthrough protein of molecular weight (Mr) 18,500. However, the suppressor activity of this tRNA within the oocyte cytoplasm is strictly dependent on the co-injection of an exogenous (Escherichia coli) acylating enzyme which is needed to charge the mt tRNATrp in vivo. The absence of an endogenous enzyme capable of acylating the yeast mt tRNATrp suggests that there is a biological barrier for the activity of a mt tRNA in the cytoplasm if a tRNA exchange between the two cellular compartments occurred.

Easy-to-use equipment for the accurate microinjection of nanoliter volumes into the nuclei of amphibian oocytes

Microinjection of Xenopus oocytes has become a valuable technique for studying many aspects of control of eucaryotic gene expression. Previously published methods for controlling and monitoring the volume of sample injected into oocytes are time consuming and tedious and require considerable expertise to obtain reproducible results. The construction and manner of usage of micropipets are the most critical parts of a successful injection technique. Methods for the construction of micropipets and ways to assess the accuracy and precision of the injection of nanoliter volumes into amphibian oocytes are described. Construction of pipets is done almost entirely using instruments that are relatively inexpensive and allow for a high degree of reproducibility. Micropipets made in this manner have several advantages: (1) They inflict little damage on the oocytes during the injection process; greater than 95% of the oocytes survive after microinjection. (2) Oocytes do not require defolliculation prior to microinjection. (3) One micropipet can be used to inject as many as 100 oocytes before needing to be refilled with sample and without acquiring a blockage. Using the microinjection techniques described, the accuracy of sample delivery was within 9% of the mean and the precision increased with increasing volume: the standard deviations of volumes in the 5-nl range were within 9% of the mean and volumes in the range 10–50 nl were within 4–6% of the mean.

Secreted proteins in the medium of microinjected Xenopus oocytes are degraded by oocyte proteases

Secreted proteins in the medium of microinjected Xenopus oocytes are degraded by oocyte proteases

RNA processing in microinjected Xenopus oocytes : Sequential addition of base modifications in a spliced transfer RNA

Cloned yeast tRNA Tyr genes are transcribed and processed after micro-injection into Xenopus laevis oocyte nuclei (De Robertis & Olson, 1979; Melton et al., 1980) . The early transcript, about 104 nucleotides long, has a 5′ leader, two or three U residues at the 3′ end and an intervening sequence. The termini are then matured, giving rise to a molecule 92 nucleotides long, which is subsequently spliced to produce a 78 nucleotides long tRNA by a mechanism similar to that found in yeast. Here we analyse the base modifications in these three RNA molecules to test whether the extra RNA segments, and especially the intervening sequence, influence the introduction of base modifications into tRNA. The 104, 92 and 78 RNAs were isolated from gels and analysed by two-dimensional chromatography after complete digestion with nuclease P 1 into 5′-mononucleotides (direct labelling) or ribonuclease T 2 into 3′-mononucleotides (nearest neighbour labelling). By analysing oocytes labelled separately with [α- 32P]ATP, -GTP, -UTP or -CTP it was possible to examine all the base modifications added by the oocyte. The results show that the tRNA Tyr base modifications occur in a sequential order: (1) the early 104 precursor already has five base modifications on it, including all the modifications in the TΨ loop; (2) 11 additional modifications only become apparent in the 92 precursor; and (3) finally, two base modifications, Q base and isopentenyladenosine, are introduced in the anticodon loop only after splicing of the intervening sequence. No modifications were observed in the 5′ leader, 3′ trailer or intervening sequences. Some base modifications added by the oocyte are not normally found in yeast (e.g. Q base), and it seems as if the Xenopus oocyte processes this yeast tRNA as one of its own tRNAs. However, all the modifications normally present in yeast were added by the oocyte, with the exception of a Gm. All base modifications were introduced in a sequential fashion. The splicing pathway found in vitro with yeast extracts (Knapp et al., 1979 ; Peebles et al., 1979) , which has an unusual 3′ phosphate-ended intermediate, was confirmed in vivo in the frog oocyte by analysing the nearest neighbour labelling of the isopentenyladenosine that is located adjacent to the splicing point.

Microinjection of Xenopus oocytes : An automated device for volume control in the nanoliter range

Injection of Xenopus oocytes is a technique widely used in studies of gene expression for both qualitative and quantitative analysis of DNA and mRNA. Published methods for controlling and assessing volumes transferred into oocytes are tedious, time consuming, and require considerable expertise. We describe the construction of an apparatus which automatically controls nanoliter injection volumes. For injection volumes from 50 to 10 nl, the mean volume injected into 10 oocytes was within 10% of the expected value and often better, and even for 1-nl injections was within 20% of the expected value. The precision of volume dispensing into individual oocytes decreased with decreasing volume; the standard deviation was about 10–15% of the mean for volumes in the 30- to 50-nl range and about 30–40% of the mean for volumes in the 1- to 2-nl range.