Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.     

Evidence for a nuclease in Saccharomyces cerevisiae that affects the cell-free translation of natural messenger RNA.

A postpolysomal extract of $\text{Saccharomyces}\text{cerevisiae}$, treated with micrococcal nuclease to remove endogenous mRNAs, translates exogenous natural and synthetic mRNA templates actively and accurately at 20°C. When the temperature of incubation is 30°C or higher, protein synthesis with yeast poly(A)⁺ mRNA is markedly reduced, but synthesis of polyphenyl-alanine with poly (U) is only slightly affected. The protein synthesizing activity of the extract is decreased 50% in 30 minutes at 37°C, while the ability of yeast mRNA to template for protein synthesis is decreased 50% in 5 to 7 minutes when it is incubated with the postpolysomal fraction at 37°C. The release of radioactivity from isotopically-labeled yeast mRNA, into the acid-soluble form, is also much greater at 37°C than at 20°C. Thus, at the elevated temperatures, the loss of mRNA templating activity and RNA hydrolysis occur more rapidly than the loss of activity of the translational apparatus. The evidence suggests that the failure of the extract to catalyze translation at 30°C or higher, as compared to 20°C, is due to a temperature-stimulated nuclease that degrades mRNA.     

Separation of plasma membrane markers by glycerol-induced blistering of muscle cells

Glycerol (50%, w/w) was found to cause blistering of chick primary myoblast and fibroblast plasma membranes and extensive blistering of 5–6-day-old-myotube plasma membranes in tissue culture. The tips of myoblasts and fibroblasts appeared to be the most sensitive portion of the plasma membrane to the blistering effect of glycerol. The glycerol-induced blistering of myotubes was reduced and delayed by brief EDTA pretreatment.Glycerol treatment (50, 15 and 8% sequentially) of myotubes was used to remove plasma membrane blisters and a plasma membrane-enriched fraction was isolated from these blisters using a modified Dextran T500-polyethylene-glycol 6000 aqueous two-phase polymer system. This fraction was found to be enriched 4.1-fold for 5′-nucleotidase activity, but not for other putative plasma membrane markers, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity or $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$ binding material. Autoradiographs of $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$, glycerol-treated (50%, w/w) myotubes showed the plasma membrane blisters to be devoid of reduced silver grains.5′-Nucleotidase was shown to be an ectoenzyme on myoblasts and 5-day-old myotubes and the total cellular activity was present on the cell surface. During the period of myoblast fusion and myotube formation, cell surface activity decreased to a low level while total cellular activity was elevated.     

The effect of beta-naphthoflavone on rabbit liver protein synthesis, and on the induction of cytochrome P-450 LM4 mRNA

A single injection of β-naphthoflavone dispersed in corn oil causes significant changes in rabbit liver polysome and polysomal poly(A+)mRNA driven $\text{in vitro}$ protein synthesis. The changes occur between 6–18 hr and 30–36 hr after the injection. Our data indicate that the first effect is due to the β-naphthoflavone and the second effect is due to the oil vehicle. $\text{In vitro}$ translation of rabbit liver polysomes obtained from treated rabbits followed by specific immunoprecipition and gel electrophoresis, showed that maximal levels of translatable cytochrome P-450 LM₄ occurred 18–24 hr after β-naphthoflavone treatment.     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

Translation of bacteriophage T4 mRNA into active lysozyme by a wheat germ cell-free system.

T4 bacteriophage mRNA for lysozyme was extracted from T4 phage infected $\text{E. coli}$ cells, partially purified by column chromatography, and translated in a heterologous cell-free protein synthesizing system prepared from wheat germ. The translation product was confirmed by SDS polyacrylamide gel electrophoresis and enzymatic activity — bacteriolysis as tested with $\text{Micrococcus luteus}$. The specific activity of the enzyme prepared was 660 U/mg.     

Sub-cellular localization of vesicular stomatitis virus messenger RNAs.

Vesicular stomatitis virus (VSV) messenger RNAs (mRNAs) appear to be compartmentalized within the infected HeLa cells. Analysis by polyacrylamide gel electrophoresis in formamide of the RNA associated with the membrane bound polyribosomes from VSV-infected cytoplasmic extracts shows predominantly one size class of VSV mRNA, which is absent from the remaining cytoplasm. These results are consistent with the mRNA for the viral glycoprotein being exclusively associated with membrane bound polysomes since the latter have been shown to synthesize mainly the virion glycoprotein in an $\text{in vitro}$ translation system.     

Adenyl cyclase of oxyntic cells its association with different cellular membranes

The adenyl cyclase of the oxyntic, or acid-secreting, cells of bullfrog gastric mucosa has been found to be a membrane-bound enzyme. A method has been developed to isolate the adenyl cyclase rich membrane fractions in a hypotonic medium containing dithiothreitol, which has been found to protect the hormonal resposivenes of the adenyl cyclase.Highest specific activity of adenyl cyclase was localized in a light membrane fraction which also had abundant K⁺-stimulated ATPase and K⁺-stimulated $\text{p-}\text{nitrophenyl}$ phophatase and very low cytochrome $\text{c}$ oxidase activty. The three gastric secretagogues tested, namely histamine, pentagastrin and methylcholine, significantly stimulated the adenyl cyclase activity of the light membrane fraction.After treatment with 10 mM Mg⁺ further subfractionation of the light membrane fraction on a sucrose density gradient yielded light membrane subfraction 1, light membrane subfraction 2 and light membrane subfraction 3 in order of increasing densities. The three subfractions had different enzymatic and chemical properties. Adenyl cyclase activity has been found to be distributed in all three subfractions. However, the hormonal responsiveness of the three fractions was quite different. Light membrane subfraction 2 could be stimulated by all three secretagogues, light membrane subfraction 1 by histamine and methylcholine, while light membrane subfraction 3 was refractory to all three secretagogues. On the basis of the cholesterol to phospholipid molar ratio, RNA content, glycoprotein content and the enzymatic data it is suggested that light membrane subfraction 1 and light membrane subfraction 2 are of general plasma-membrane type, while light membrane subfraction 3 is largely of cytoplasmic origin.     

The role of triiodothyronine in the regulation of synthesis rate and translatable mRNA level of fatty acid synthetase in a preadipocyte cell line

Triiodothyronine (T₃) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob₁₇ preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6–10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 40–42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T₃ included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T₃ effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T₃ would mainly affect fatty acid synthetase as a pretranslational level.     

Half-lives and relative amounts of stored and polysomal ribosomes and poly(A)+ RNA in mouse oocytes

Growing mouse oocytes were labeled in vitro with [³H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.     

Interferon inhibition of hexose monophosphate shunt activity as the mechanism of blocking differentiation of a preadipose cell line

Mouse L cell interferon (IFN $\text{α}\text{β}$) inhibited the differentiation of mouse 3T3-Li fibroblasts into adipocytes. IFN $\text{α}\text{β}$ also inhibited hexose monophosphate shunt (HMP) activity in these cells. HMP activity is required for the reducing power necessary for the conversion of 3T3-Li fibroblasts to adipocytes. Both IFN blockage of differentiation and HMP activity were reversed by the reducing agent 2-mercaptoethanol, probably through interaction with membrane receptors and not through direct inactivation of IFN. Several non-antiviral effects of IFN $\text{α}\text{β}$ on cellular function, including differentiation and immunoregulation, may be mediated at the biochemical level through blockage of HMP activity.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.     

Messenger RNA of mouse immune interferon (MuIFN-gamma)

A lysate of $\text{Staphylococcus aureus}$ ($\text{S. aureus}$) induced high-titered (10^3.6 ～ 10^4.6 units/ml) mouse immune interferon (MuIFN-γ) in spleen cell cultures. mRNA was extracted from such cells, purified by oligo(dT) cellulose chromatography and sucrose gradient centrifugation, and injected in $\text{Xenopus laevis}$ oocytes. Fractions containing RNA of 15 to 16 S gave rise to IFN activity, that was characterized as IFN-γ by virtue of its acid lability, neutralization by antiserum to partially purified MuIFN-γ, and lack of neutralization by antiserum to NDV-induced L-cell IFN.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.