Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Release of the inhibition of messenger RNA translation in extracts of interferon-treated Ehrlich ascites tumor cells by added transfer RNA

In an extract of Ehrlich ascites tumor (EAT) cells which had been “preincubated” for 45 min to lower endogenous protein synthesis (S30_C) the translation of exogenous encephalomyocarditis (EMC) viral mRNA proceeds at a constant rate for over 90 min. In a similarly treated extract of interferon-treated EAT cells (S30_INT) the translation proceeds at a lower rate than in the S30_C for about 30 min and then stops. The impairment of the translation in the S30_INT is mediated by one or more inhibitors. After the cessation of translation the viral mRNA in the S30_INT is in large polysomes. The size of these changes little (if any) during a further 15 min incubation. The addition of mouse tRNA (but not ribosomal RNA or $\text{E. coli}$ tRNA) to the S30_INT after the cessation of viral mRNA translation results in the restart of translation at a rate close to that in the S30_C. This effect of tRNA is diminished by pactamycin, which inhibits peptide chain initiation but not elongation. These results indicate that addition of tRNA allows the elongation of incomplete peptide chains and the initiation of new chains. The need for added tRNA may be due to the fact that in S30_INT the amino acid acceptance of some of the endogenous tRNA species (but not of added tRNAs) is impaired. This impairment is pronounced for leucine and very slight, if any, for five other amino acids tested (i.e. isoleucine, methionine, phenylalanine, threonine, and valine).     

Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon.

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.     

Inhibition of protein synthesis directed by added viral and cellular messenger RNAs in extracts of interferon-treated Ehrlich ascites tumor cells. Location and dominance of the inhibitor(s)

Protein synthesis directed by exogenous (viral or cellular) messengers is impaired, but endogenous protein synthesis is not affected in an extract of interferon-treated Ehrlich ascites tumor cells (INT-extract). Protein synthesis directed by exogenous messengers is also impaired in a mixture of an INT-extract with an extract from control cells. This reveals that the impairment is due to one or more inhibitors in the INT-extract. The nondialyzability of the inhibitor(s) is probably an indication of large molecular size. In a not incubated INT-extract much of the inhibitory activity is in the high speed sediment fraction i.e., is presumably bound directly or indirectly to ribosomes. During incubation of the extract most of the inhibitory activity is released into the high speed supernatant fraction. The dose-response curve shows that in our conditions the translation of cellular messengers (from mouse L cells) is as sensitive to impairment by the inhibitor(s) as that of viral messengers (from reovirus or from encephalomyocarditis virus).     

Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.     

Relationship between changes in the translational apparatus and actinomycin production in Streptomyces antibioticus.

As previously reported (G. H. Jones, 1975), transfer ribonucleic acids (tRNA's) and ribosomes from actinomycin-producing cultures of Streptomyces antibioticus show a decreased ability to function in aminoacylation and translation as compared with the corresponding components from younger cells. Further, specific changes in the isoacceptor patterns are revealed when tRNA's from actinomycin-producing cells are compared with those of younger cells by reverse- phase column chromatography. A specific glycyl-tRNA species is eliminated from the reverse-phase profile of tRNA's from actinomycin-producing S. antibioticus cells as compared with younger cells. Changes in isoacceptor patterns were also observed for the amino acids methionine, valine, phenylalanine, and leucine. Actinomycin synthesis was inhibited by growing S. antibioticus cells in the presence of alpha-methyl-DL-tryptophan. Inhibition of actinomycin synthesis reversed the changes in tRNA observed in normally grown control cultures, although it had no demonstrable effect on the growth of the cells. Thus, tRNA from 48-h-old, alpha-methyl-tryptophan-grown cells had amino acid acceptor activity that was equal to or greater than that of tRNA from 12-h-old, normally grown cells. Similarly, the reverse-phase chromatographic pattern for glycyl-tRNA's from 48-h-old, alpha-methyl-tryptophan-grown cells was identical to that of the glycyl-tRNA's from 12-h-old, normally grown cells. In contrast, the ability of ribosomes from 48-h-old, alpha-methyl-tryptophan-grown cells to function in polypeptide synthesis in vitro was essentially identical to that of 48-h-old, normally grown cells. Ribosomes from 12-h-old, normally grown cells were severalfold more active in in vitro polypeptide synthesis.     

Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells.

In vitro systems for the aminoacylation of transfer ribonucleic acid (tRNA) and for polypeptide synthesis have been constructed from young (12-h cultures, not producing actinomycin) and old (48-h cultures, producing actinomycin) cells of Streptomyces antibioticus. When Escherichia coli aminoacyl-tRNA synthetases were used to acylate S. antibioticus tRNA's, it was observed that, per absorbance unit of tRNA, the tRNA's from 48-h cells had a lower ability to accept the amino acids, leucine, serine, pheynlalanine, methionine, and valine than did the tRNA's from 12-h cells. Individual differences were observed between aminoacyl-tRNA synthetases from 12-h cells and those from 48-h cells with respect to the rate and extent of aminoacylation of E. coli tRNA with the five amino acids listed above. In vitro systems for the synthesis of polyphenylalanine have been constructed from 12- and 48-h cells. Ribsomes and soluble enzymes from 12-h cells are more efficient than those from 48-h cells in supporting polyphenylalanine synthesis, and, although the activity of both systems can be stimulated by the addition of E. coli tRNA, the higher level of incorporation observed in the unstimulated 12-h system (ribosomes and soluble enzymes) is maintained. Indeed, the difference in capacity for polyphenylalanine synthesis between in vitro systems from 12- and 48-h cells is greater when the systems are maximally stimulated by E. coli tRNA. Cross-mixing experiments reveal that enzymes from 48-h cells support a slightly higher level of polyphenylalanine synthesis than enzymes from 12-h cells with ribosomes from either cell type, and that the ribosomes are the primary agents responsible for the decreased efficiency of the in vito system from 48-h cells are compared with that from 12-h cells. To determine whether ribosome-associated factors were responsible for the relative inefficiency of the ribosomes from 48-h cells in translation, salt-washed ribosomes from 12- and 48-h cells were examined for their abilities to catalyze polyphenylalanine synthesis. Even after salt washing, ribosomes from 12-h cells were about five times higher in specific activity (counts per minute of polyphenylalanine synthesized per absorbance at 260 nm of ribosomes) than equivalent amounts of ribosomes from 48-h cells. Analysis of the proteins of salt-washed ribosomes of the two cell types by acrylamide gel electrophoresis suggests that the relative amounts of individual proteins present on ribosomes from 12-h cells are different from the amounts present on ribosomes from 48-h cells. These results are discussed in terms of the regulation of translation in S. antibioticus.     

Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.     

Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney

The tRNA from Ehrlich ascites tumor cells is deficient in the modified nucleoside Q (queuosine). Continuous infusion of Q base (queuine) to tumor-bearing mice reverses the deficiency of Q in Ehrlich ascites tRNA, and coincidently, causes an inhibition of tumor growth.     

Infidelity of translation of encephalomyocarditis viral RNA with tRNA from human malignant trophoblastic cells.

We have investigated tRNA from the human malignant trophoblastic cells (BeWo cell) and human chorionic tissue for the translation of specific mRNAs, in a tRNA-dependent protein synthesizing system from Ehrlich ascites cells. BeWo cell tRNA and chorionic tRNA supported oviduct mRNA or encephalo-myocarditis (EMC) viral RNA directed amino acid incorporation into polypeptides equally effectively. Polypeptides synthesized with oviduct mRNA and tRNA from both sources were identical upon sodium dodecylsulfate polyacrylamide gel electrophoresis. But the EMC RNA directed polypeptides synthesized with BeWo cell tRNA were different from those synthesized with chorionic tRNA. A polypeptide (molecular weight 58,000) was apparently not synthesized and the synthesis of a faster moving component (molecular weight, 14,000) was enhanced when BeWo cell tRNA was used. These results imply a functional difference in tRNA from human malignant cells compared to their normal counterpart.     

Isolation and characterization of a guanine insertion enzyme, a specific tRNA transglycosylase, from Escherichia coli.

A guanine insertion enzyme (tRNA transglycosylase) was purified to a homogeneous state from Escherichia coli B by ammonium sulfate fractionation and DEAE-cellulose, DEAE-Sephadex A-50, phosphocellulose, and Sephadex G-200 column chromatographies. The molecular weight of the enzyme, which appeared to be a single polypeptide, was 4.6 X 10(4) by sodium dodecyl sulfate gel electrophoresis. The enzyme catalyzes exchange of guanine with guanine located in the first position of the anticodon of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp, but unlike the enzymes isolated from rabbit reticulocytes and Ehrlich ascites tumor cells it does not catalyze the exchange of guanine with queuine (7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) present in these tRNAs. The pH optimum of the reaction was 7.0, and the pH1 value was 4.6 to 4.8. The reaction required Mg2+ ion. 7-Methylguanine inhibited guanine insertion, but the other purine analogues tested were not inhibitory and could not replace guanine.20     

Limitation of reticulocyte transfer RNA in the translation of heterologous messenger RNAs.

The effect of various tRNAs on protein synthesis was investigated using a tRNA-dependent cell-free system from Ehrlich ascites cells. Ascites cell tRNA and rabbit liver tRNA were found to promote efficient translation of globin mRNA, oviduct mRNA, and encephalomycarditis (EMC) viral RNA. In contrast, reticulocyte tRNA participated efficiently only in the translation of globin mRNA; the translation of oviduct mRNA AND EMC viral RNA in the presence of reticulocyte tRNA resulted in the synthesis of relatively few large mature proteins and the accumulation of discrete, smaller polypeptides. These results suggest that isoaccepting tRNA species required for the synthesis of ovalbumin and EMC viral protein (but not hemoglobin) are probably functionally absent in reticulocyte tRNA, causing a premature, nonrandom termination of synthesis of these proteins. This provides preliminary evidence that variations in tRNA populations, frequently observed between different cell types, are large enough to define and perhaps regulate the proteins that the cell is capable of synthesizing.     

Uridylation of U6 RNA in a nuclear extract in Ehrlich ascites tumor cells

The uridylation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells was examined. This reaction required ATP or GTP, although these nucleotides were not incorporated into U6 RNA itself. ATP and GTP could be replaced by their nonhydrolyzable analogues ATP gamma S and GTP gamma S. Therefore, hydrolysis of ATP or GTP is not necessary for the uridylation of U6 RNA, indicating that these nucleotides are effectors of this reaction. By chromatographies of a nuclear extract of Ehrlich ascites tumor cells on phosphocellulose and DEAE-cellulose, U6 RNA could be separated from an enzyme adding a uridine residue(s) to this RNA.     

Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu.

Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P). Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's. The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion. Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another. It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species. On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine.     

Transfer RNA and aminoacyl tRNA synthetases in hormone dependent and independent mammary tumors of GR mice: II. Isoacceptor tRNA's in hormone dependent and independent mammary tumors of GR mice.

The chromatographic elution profiles of 15 aminoacyl tRNA's from dependent and independent mammary tumors of GR mice have been studied using the reversed phase chromatography (RPC 5). The seryl tRNA from the dependent tumor displayed three isoacceptor peaks while only two isoacceptor peaks were observed in the case of the independent tumor when the tRNA's were charged in the presence of the GR mice liver enzyme. Charging of the tRNA's with radioactive leucine by homologous and heterologous enzyme revealed major differences in the leucyl isoacceptor species. The homologous dependent tumor system charges five leucyl tRNA species while the independent system only charges four. The leucyl tRNA from the dependent tumor has a new peak which is only recognized by its own enzyme, but this peak is either suppressed or completely absent in the independent tumor.     

IMP-cyclohydrolase/transformylase from ehrlich-ascites carcinoma (author's transl)]

The IMP-cyclohydrolase/transformylase enzyme was purified from Ehrlich ascites tumor cells by ammonium sulfate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex. The electrophoretically pure enzyme has a molecular weight of about 350000, estimated by a gel filtration, and an isoelectric point of 6.2. It is composed of 8 subunits with a molecular weight of 46000. Every subunit is composed of two different proteins with a molecular weight of 18000 and 28500. Some further characteristics of the enzyme are reported.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Partial derepression of the isoleucine-valine enzymes during methionine starvation is Salmonella typhimurium.

Methionine starvation of methionine auxotrophs in the presence of excess branched-chain amino acids results in a partial derepression of the isoleucine and valine enzymes. Reversed-phase chromatography indicated that isoleucine, valine and leucine tRNA were altered during methionine starvation. In addition, the total tRNA isolated from cells under these conditions were undermethylated. The observed derepression may be caused by the inability of methyl-deficient tRNA's to participate adequately in normal regulatory functions.     

Interaction of Escherichia coli glutaminyl-tRNA synthesis with noncognate tRNA''s.

Several noncognate tRNA's from Escherichia coli were mischarged with glutamine by E. coli glutaminyl-tRNA synthetase if dimethylsulfoxide was present in the reaction mixture. Kinetic analysis of the mischarging revealed that dimethyl sulfoxide stimulated the misacylation by affecting the maximum velocity. Several noncognate tRNA's were shown to interact with glutaminyl-tRNA synthetase as measured by their ability to protect the enzyme against thermal inactivation or to replace cognate tRNA in stimulating glutamine-dependent ATP-PPi exchange reaction. These tRNA's, however, did not coincide with those which were mischargeable with glutamine.