An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

A test for masked message: the template activity of messenger ribonucleoprotein particles isolated from sea urchine eggs

The hypothesis that the “masked message” of unfertilized eggs consists of nontranslatable mRNP particles was directly tested by in vitro translation of mRNPs in a system derived from wheat germ. Three classes of mRNPs were tested: particles prepared from sea urchin eggs in buffers containing 0.35 M K⁺, particles prepared from sea urchin eggs in 0.35 M Na⁺, and particles released with EDTA in 0.35 M K⁺ from polysomes of sea urchin embryos cultured in the presence of actinomycin D. The mRNA content of particles was monitored by determination of poly(A) content. The wheat germ system used is quantitatively stimulated by addition of mRNA derived from eggs or from any of the classes of mRNPs used. Particles prepared from eggs with Na⁺ or released from polysomes contain less protein than particles isolated from eggs in K⁺, and as expected these particles are fully translatable in vitro. Particles prepared from eggs in buffers containing 0.35 M K⁺ produce little or no stimulation in the in vitro system. That this lack of translation represents in vivo masking is indicated by several considerations: (1) The nontranslatable particles were prepared in 0.35 M K⁺ and 5 mM Mg²⁺, ion concentrations similar to those found in echinoderm eggs; (2) density and sedimentation rate characteristics of the particles are little changed by isolation; (3) RNA extracted from isolated particles is fully translatable; and (4) particles prepared from polysomes or under conditions which destabilize RNPs are translatable. These data support the masking hypothesis for the protein synthesis repression system of eggs.     

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V

Oxanine (O) is a deamination product derived from guanine with the nitrogen at the N¹ position substituted by oxygen. Cytosine, thymine, adenine, guanine as well as oxanine itself can be incorporated by Klenow Fragment to pair with oxanine in a DNA template with similar efficiency, indicating that oxanine in DNA may cause various mutations. As a nucleotide, deoxyoxanosine may substitute for deoxyguanosine to complete a primer extension reaction. Endonuclease V, an enzyme known for its enzymatic activity on uridine-, inosine- and xanthosine-containing DNA, can cleave oxanosine-containing DNA at the second phosphodiester bond 3′ to the lesion. Mg²⁺ or Mn²⁺, and to a small extent Co²⁺ or Ni²⁺, support the oxanosine-containing DNA cleavage activity. All four oxanosine-containing base pairs (A/O, T/O, C/O and G/O) were cleaved with similar efficiency. The cleavage of double-stranded oxanosine-containing DNA was ~6-fold less efficient than that of double-stranded inosine-containing DNA. Single-stranded oxanosine-containing DNA was cleaved with a lower efficiency as compared with double-stranded oxanosine-containing DNA. A metal ion enhances the binding of endonuclease V to double-stranded and single-stranded oxanosine-containing DNA 6- and 4-fold, respectively. Hypothetic models of oxanine-containing base pairs and deaminated base recognition mechanism are presented.     

Genome independent specificity of DNA-directed f Met-dipeptide synthesis

Under conditions which are optimal for RNA-directed polypeptide chain initiation in vitro, ribosomes bind to single-stranded DNA and synthesize fMet-dipeptides. The products directed by natural single-stranded or denatured double-stranded DNA from eight different organisms are strikingly similar: predominantly fMet-Thr, fMet-Val and fMet-^Leu_Ile. For four of these templates, there exists evidence that these dipeptides are not representative of those synthesized from the corresponding messenger RNAs. The observations are discussed in the light of results which indicate that DNA can serve accurately as a substitute message for binding of ribosomes and synthesis of polypeptides in. vitro.     

Polysome-dependent synthesis of embryonic proteins of Artemia salina during cell differentiation and analysis of heme-containing protein.

The polysomes isolated from the different developmental stages of Artemia salina have been translated in a homologous S-30 extract or pH 5 enzyme fraction as well as in a heterologous (wheat germ) pH 5 enzyme fraction. The specific template activity of the polysomes increases linearly up to a stage which is equivalent to about two-thirds of the whole preemergence period and thereafter remains rather constant until the nauplius stage. The specific template activity of the polysomes appears to be dependent on a source of in vitro systems, but not to be dependent on the size of the polysomes used. Moreover, the efficiency of polypeptide chain release from the polysomes in vitro also seems dependent on the developmental stages of the embryos, but in this case it is not influenced by the in vitro system employed. Analysis of the proteins synthesized in vitro by the polyacrylamide gel electrophoresis has revealed that the species and their relative amounts of the proteins are rather specific for each developmental stage analyzed.Heme-containing proteins from the nauplii have been partially purified and analyzed. This heme-protein complex contains two distinct polypeptide chains, MW > 100,000 and MW = 50,000–60,000 daltons, respectively, as the major species as judged by SDS-polyacrylamide gel electrophoresis. During preemergence development, no heme-protein complexes have been detected, but after hatching of the embryos, they have been detected.     

Adenovirus DNA replication in vitro: Origin and direction of daughter strand synthesis

We have characterized a soluble enzyme system from adenovirus-infected cells that is capable of replicating exogenously added adenovirus DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from purified adenovirus virions, is added as template. Under these conditions DNA replication starts at or near either end of the template. Daughter strand synthesis then proceeds in the 5′ to 3′ direction displacing the parental strand of the same polarity. Thus, the r daughter strand is synthesized from right to left on the conventional map of the adenovirus genome, and the l daughter strand is synthesized from left to right. This course of events is the same as that which occurs during adenovirus DNA replication in vivo. In contrast, when deproteinized adenovirus DNA is added to the in vitro system, the limited DNA synthesis that is observed appears to be due to a repair-like reaction. In particular, synthesis can begin at many sites within the template, and the synthetic product consists largely of short DNA chains that are covalently linked to template DNA strands.     

Inhibition of Leukaemia Virus Replication by Polyadenylic Acid

A PREVIOUS communication from this laboratory demonstrated that the DNA polymerase of the Rauscher leukaemia virus is strongly inhibited in vitro by unprimed, single stranded polyribonucleotides¹ as a result of competition between the polymers and the active template for the same enzyme binding site. This inhibition was apparently specific, since partially purified preparations of DNA polymerase from Escherichia coli and BALB/c mouse embryos were not inhibited in the same conditions. We attempted to determine therefore whether single stranded polyribonucleotides would have any effect on the activities of oncogenic RNA viruses in cultured cells.     

Timing, localization, and control of wheat germ agglutinin synthesis in developing wheat embryos

The lectin, wheat germ agglutinin (WGA), is synthesized de novo by developing wheat (Triticum aestivum, L.) embryos but is not synthesized or localized in developing endosperm as shown by radioimmunoassay. Young embryos removed from the grain and cultured on a defined medium germinate precociously and concomitantly cease WGA synthesis. In vitro precocious germination of young embryos is reversibly inhibited by low levels (1–100 μM) of the plant growth substance abscisic acid (ABA). Embryos inhibited from germinating by this growth regulator not only continue synthesizing WGA, but do so at an accelerated rate when compared with embryos left associated with the grain.     

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection.Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks.This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos.This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10³ TCID₅₀/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.     

Phosphorus-32, a Clinically Available Drug,Inhibits Cancer Growth by Inducing DNA Double-Strand Breakage

Radioisotopes that emit electrons (beta particles), such as radioiodine, can effectively kill target cells, including cancer cells. Aqueous ³²P[PO₄] is a pure beta-emitter that has been used for several decades to treat non-malignant human myeloproliferative diseases. ³²P[PO₄] was directly compared to a more powerful pure beta-emitter, the clinically important ⁹⁰Y isotope. In vitro, ³²P[PO₄] was more effective at killing cells than was the more powerful isotope ⁹⁰Y (P ≤ 0.001) and also caused substantially more double-stranded DNA breaks than did ⁹⁰Y. In vivo, a single low-dose intravenous dose of aqueous elemental ³²P significantly inhibited tumor growth in the syngeneic murine cancer model (P ≤ 0.001). This effect is exerted by direct incorporation into nascent DNA chains, resulting in double-stranded breakage, a unique mechanism not duplicatable by other, more powerful electron-emitting radioisotopes. ³²P[PO₄] should be considered for human clinical trials as a potential novel anti-cancer drug.     

The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients

A series of covalently closed bacteriophage PM2 DNA samples with varying degrees of superhelicity were prepared in vitro. The amount of bound ethidium per DNA nueleotide needed for the removal of all superhelical turns, v_c⁰, was determined for each sample by a number of methods. In order to evaluate the unwinding angle for the binding of one ethidium molecule to a DNA double helix, the pH dependence of the buoyant densities in CsCI of these samples was examined. A new calibration relating the change in buoyant density of a DNA to the fraction of bases titrated has been obtained, by measuring the buoyant densities of a number of catenanes (interlocked rings) containing both single-stranded and double-stranded λ DNA rings, at a pH such that the single-stranded DNA is fully titrated while the double-stranded DNA is not titrated. This calibration was used to obtain the pH dependence of the fraction of DNA bases titrated for the phage PM2 DNAs with differing extents of supercoiling. A simple theoretical analysis shows that in a restricted pH range close to pH_m, the melting pH of the DNA in the absence of the topological constraint associated with covalently closed double-stranded DNAs, the difference in the fraction of bases titrated at a certain pH between two covalently closed DNAs with different degrees of superhelicity is directly proportional to the difference in the v_c⁰ values of the DNAs. The unwinding angle per bound ethidium molecule can be obtained from the proportionality constant. In this way, it is not necessary to know precisely the actual pH value for either DNA, pH_e, at which the DNA is titrated to the extent that it contains no superhelical turns. The conclusion of the theoretical analysis and the experimental results is that the binding of an ethidium molecule to a double-stranded DNA unwinds the DNA helix by an angle φ_e = 26 °. The uncertainty in this value is estimated to be less than 10%. The new value for φ_e is approximately a factor of two larger than the value 12 °, which has been in use in the past decade. In the earlier alkaline titration results for polyoma DNA (Vinograd et al., 1968), which had been interpreted as supporting the 12 ° value, the calculation of φ_e was critically dependent on knowing pH_e. It is believed that pH_e was underestimated in the earlier work, resulting in a low φ_e value. Since the previous value φ_e = 12 ° has been widely used in the determination of the number of superhelical turns for many DNAs, and in measurements on the angular alterations of the DNA helix by the binding of a variety of small and large molecules and by solvent and temperature changes, the new value φ_e = 26 ° requires proportional adjustments of many previous results.     

Tryptophanyl-tRNA synthetase is found closely associated with and stimulates DNA polymerase α-like activity from wheat embryos

DNA polymerase α-like from wheat embryos is found to purify closely associated with a tryptophanyl-tRNA synthetase activity. No other aminoacyl-tRNA synthetases were present. A purified preparation of wheat tryptophanyl-tRNA synthetase free of polymerase activity was able to stimulate plant DNA polymerase of the α-like type, while the γ-like polymerase from wheat embryos was not affected by the enzyme. We have not been able to find a diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate binding activity associated to the polymerase-synthetase complex. We have also observed a specific inhibition by beef tRNA^Trp of DNA polymerase α-like activity, while other tRNAs will not change the enzyme activity.     

Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg²⁺, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(−) orientation is as efficient as the switch from G(−) to G(+).