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.     

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.     

Transfection: enhancement by assembly protein of bacteriophage R17.

Assembly protein was isolated by DEAE cellulose chromatography from disrupted R17 bacteriophage and reconstituted with purified R17 phage RNA. Following reconstitution, ¹²⁵I labeled assembly protein co-sediments with 27S R17 phage RNA in a sucrose gradient. SDS-polyacrylamide gel analysis of the 27S ¹²⁵I labeled protein-RNA complex confirmed that assembly protein was the only phage protein associated with the RNA. The specific infectivity (PFU/μg RNA) of the R17 phage RNA-assembly protein complex was 35-fold greater than that of R17 phage RNA when assayed on $\text{Escherichia coli}$ spheroplasts. Infectivity of both preparations was destroyed by treatment with pancreatic ribonuclease A. Furthermore, the assembly protein-RNA complex was infectious for intact cells whereas phage RNA was not infectious. Infectivity of this 27S complex for intact cells was totally eliminated by pretreatment with ribonuclease.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

Complete amino acid sequence of the coat protein of the Pseudomonas, aeruginosa RNA bacteriophage PP7

As part of a project intending to assess the evolutionary kinship between the RNA coliphages and RNA bacteriophages of other bacterial genera, we have sequenced the coat protein of $\text{Pseudomonas}\text{,}\text{aeruginosa}$ RNA phage PP7. Like the coat proteins of coliphages MS2 and Qβ and of the broad host range RNA phage PRR1, PP7 coat protein (127 residues) is highly hydrophobic, and contains a cluster of basic residues between positions 40 to 60. Minimal mutation distance values were calculated for comparison of PP7 coat protein with each MS2, Qβ and PRR1 coat proteins. Application of the Moore-Goodman criterion to those values, shows that these four RNA bacteriophage coat proteins very likely descent from a common ancestor.     

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.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

A role for ribonuclease III in synthesis of bacteriophage T4 transfer RNAs.

Synthesis of T4 tRNA^Gln depends on normal levels of $\text{Escherichia}\text{coli}$ ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA^Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA^Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA^Gln.     

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.     

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.     

Intracellular location of human fibroblast interferon messenger RNA

Human diploid fibroblast (FS-4) cells were induced to produce interferon mRNA by exposure to poly(rI)·poly(rC) plus cycloheximide. The intracellular location of interferon mRNA was investigated by differential centrifugation of the cytoplasm into a membrane (pellet) and a free (supernatant) fraction, followed by injection of mRNA isolated from either fraction into $\text{X}\text{.}\text{laevis}$ oocytes. When translation in FS-4 cells was prevented, most (85–90%) of the interferon mRNA activity was found in the free fraction. However, when translation was permitted, most (80–95%) of the interferon mRNA activity was found in the membrane fraction. These results are consistent with the predictions of the “signal hypothesis” (Blobel and Dobberstein, J. Cell Biol. 1975, $\text{67}$:835) for secretory proteins.     

Transfection of Vibrio cholerae by bacteriophage phi 149 DNA

DNA isolated from Choleraphage ø149 of Group IV was infectious when mixed with competent $\text{V.}\text{cholerae}$ cells. The cells were competent during mid-log phase of growth. The infectivity of phage DNA was destroyed by deoxyribonuclease but not by ribonuclease or pronase. About 5 min is required for the establishment of the DNase resistant state. The dose response curve for transfection suggested that 2 to 3 molecules of DNA are required to produce one infections center. An infectivity of 5 × 10⁴ infectious center per μg of DNA was obtained.     

Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.