Translation of avian sarcoma virus RNA in Xenopus laevis oocytes.

Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and $\text{p12}\text{15}$, upon injection of avian sarcoma virus 70S or 35S RNA into $\text{Xenopus}$ oocytes has been presented. Further, we show that tRNA^trp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.     

RNA polymerase activity in double-stranded ribonucleic acid virus particles from Aspergillus foetidus.

RNA polymerase activities have been detected in purified particles of $\text{Aspergillus foetidus}$ viruses S and F. Incorporation of [³H]-UTP into acid insoluble RNA was dependent on ATP, GTP, CTP and magnesium ions. No pretreatment of the particles was required and the rate of reaction was proportional to the amount of virus added. In the conditions used RNA synthesis by $\text{A. foetidus}$ virus S was complete in 4 h. The reaction could be stimulated by Triton X-100, but was unaffected by heat shock, dithiothreitol, potassium chloride or ammonium chloride; it was inhibited by ethidium bromide but not by actinomycin D. The major reaction product was single-stranded RNA, as indicated by its sensitivity to degradation by ribonuclease A. This is the first report of synthesis of single-stranded RNA by a double-stranded RNA mycovirus.     

Methylation of late adenovirus 2 nuclear and messenger RNA.

Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [³H-methyl]-methionine and [¹⁴C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ${}^3\text{H}{}^{14}\text{C}$ ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T₂ and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m⁷ GpppN^mpNp and m⁷ GpppN^mpN^mpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA $\text{72}$, 2012–2016).     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Template specificities of the RNA dependent DNA polymerase of different murine C-type virus particles.

The crude RNA dependent DNA polymerase of seven different C-type viruses (AMV, Kirsten-MSV produced by NRK or $\text{NIH}\text{3T3}$ cells, Moloney-MuLV, Kirsten-MuLV, the murine myeloma associated virus (MuMAV) from FLOPC-1 and MOPC-21) was analyzed for their ability to utilize four different synthetic $\text{RNA}\text{DNA}$ hybrids or three different $\text{DNA}\text{DNA}$ duplexes as templates. The polymerases from AMV and murine sarcoma or leukemia viruses were distinctly different in their template stimulated activities and the two MuMAV polymerases were different from all of the other enzymes. MuMAV RDDPs were not stimulated by any of the synthetic $\text{RNA}\text{DNA}$ hybrid templates to the same level as the enzymes of the other C-type viruses and their ability to distinguish between templates was also different.     

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.     

A rapid procedure to detect in situ DNA/RNA hybrids

We developed a new method for detecting DNA/RNA hybrids formed $\text{in}\text{situ}$ using $\text{anti-}\text{DNA}\text{RNA}$ antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesised $\text{in}\text{vitro}$ from cloned $\text{Drosophila}$ histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.     

The broad spectrum antiviral agent ribavirin inhibits capping of mRNA.

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common target for a chemotherapeutic agent in both DNA and RNA viruses is the “capping” reaction of mRNAs which $\text{inter}\text{alia}$ involves the formation of a guanine pyrophosphate structure at the 5′ terminus by mRNA guanylyl transferase. We have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped.     

Synthesis of diphtheria toxin in E. coli cell-free lysate

An $\text{E. coli}$ cell-free lysate was used to translate $\text{C. diphtheriae}$ RNA from nontoxinogenic C₇(?), C₇ infected with β tox⁺ corynebacteriophage, and $\text{C. diphtheriae}$ strain PW8. De novo synthesis of toxin was detected by immune precipitation with antitoxin, ADP-ribosylation of mammalian elongation factor 2 and rabbit skin test. The results indicated that toxin is produced in the $\text{E. coli}$ protein synthesizing system primed with RNA from cells infected with tox⁺ bacteriophage and is absent in systems primed with RNA from C₇(?) cells.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, $\text{dP1}\text{dt}\text{= M·f·n·}\text{A}\text{t}$, where $\text{dP1}\text{dt}$ = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and $\text{A}\text{t}$ = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ($\text{dP1}\text{dt}$). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo^?1 hr^?1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo^?1 hr^?1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (f^zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f^zygotic and $\text{dP1}\text{dt}$, the rate of elongation of nascent histone polypeptide chains ($\text{A}\text{t}$) during the first 6 hr of development was estimated; $\text{A}\text{t}$ remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.     

Carbon monoxide and hydroxymercuribenzoate sensitivity of a fatty acid (omega-2) hydroxylase from Bacillus megaterium.

DNA-free minicells of $\text{Escherichia coli}$ will not allow growth of phage T-7, nor is RNA synthesis stimulated by phage infection. Thus, these miniature cells seem not to contain $\text{in vivo}$ RNA polymerase activity. However, DNA-dependent RNA polymerase activity can be unmasked in extracts with poly(dA-T) and Mn²⁺. This activity may represent a cytoplasmic pool of inactive RNA polymerase in normal cells.     

A template independent, rifampicin sensitive poly (A).poly (U) synthesizing activity present in Bacillus subtilis.

A template independent poly (A)·poly (U) synthesizing activity has been isolated from $\text{Bacillus subtilis}$. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn⁺⁺ (Mg⁺⁺ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to $\text{Escherichia coli}$ holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).     

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ($\text{E.}\text{coli}$ endotoxin) and an attenuated bacteria ($\text{B.}\text{pertussis}$ vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.     

RNA synthesis in Escherichia coli during K + -depletion

During K⁺ depletion of a mutant of $\text{Escherichia}$$\text{coli}$ which cannot concentrate this cation, protein synthesis is inhibited but RNA formation continues. The RNA produced during K⁺ depletion was analyzed by gel electrophoresis. It was found that 4S, 5S and 23S RNA were synthesized by K⁺-depleted cells whether uninfected or infected with phage T4. In addition, an RNA species moving close to 16S (presumably 17S) and material of about 6–10S were made during K⁺ depletion. These species of RNA were not evident in growing cells. Methylation of RNA is severely inhibited during K⁺ depletion.     

Studies on the mode of action of calciferol. VI. Effect of 1,25-dihydroxy-vitamin D3 on RNA synthesis in the intestinal mucosa

Vitamin D (calciferol) has been postulated [A.W. Norman, $\text{Science 149}$, 184 1965] to mediate its physiological effects via an inductive process which produces new RNA and proteins. This was tested directly by studying the effect of the biologically active form of calciferol, 1,25-dihydroxycholecalciferol to stimulate $\text{in vivo}$ the pulse labeling of intestinal mucosa by ³H uridine. The maximum stimulation of RNA labeling occurs 6 hours after the intracardial administration of 325 pmoles of 1,25-dihydroxy-cholecalciferol: the first effects were apparent within 3–4 hours. Also, actinomycin D was shown to block both 1,25-dihydroxy-cholecalciferol stimulated Ca²⁺ transport and RNA synthesis. The chronology of events occurring after administration of 1,25-dihydroxy-cholecalciferol suggest that the primary biochemical response of intestinal mucosa to 1,25-dihydroxy-cholecalciferol is probably the initiation of RNA and protein synthesis.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.