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Abstract.-The present studies indicate that T4 infection induces an alteration in host ribosomes which restricts the translation of host and other T4-unrelated template RNAs but permits normal translation of T4 RNA. A heat-labile factor has been isolated from T4-infected cell ribosomes which, when combined with normal cell ribosomes, confers upon the latter the property of selective T4 template RNA translation.Recent reports from this laboratory1' 2 and others3 have provided evidence for the coding of transfer RNAs (tRNA) by the T4 genome. The relevance of phage-specific tRNAs to the viral replicative process is not known. Hattman and Hofschneider4 have shown that when Escherichia coli is infected with the RNA phage M12 and then superinfected with T4, progeny M12 phage is strongly inhibited or completely blocked. These investigators further reported that in mixed M12 and T4 infections, M12 protein synthesis is interfered with.5 It therefore seemed possible that T4 tRNAs might be involved in the "shut-off" of RNA phage protein synthesis and perhaps of host protein synthesis.In the course of work to determine the merits of this idea, it was observed that ribosomes from T4-infected cells were much less efficient for translation of MS2 and E. coli template RNAs than were ribosomes from uninfected E. coli, although translation of T4 RNA was similar for both types of ribosomes. This communication describes in vitro studies with normal and T4-infected cell ribosomes and compares their translational efficiency with different RNA templates.Materials and Methods.-Preparation of ribosomes and supernatant fraction: E. coli B cells, normal or 12 min infected with T4am61 (a lysozyme amber mutant, 10 PFU/cell), were ground with alumina (Alcoa 305, 2 gm/gm cells) in the cold, mixed with buffer A (10 mM Tris, pH 7.5, 30 mM KCl, and 10 mM magnesium acetate) plus 5 /g/ml of DNase (RNase-free), and then sedimented at 30,000 X g for 30 min. The S30 supernatant was adjusted to contain 6 mM 2-mercaptoethanol (MSH), centrifuged again as above, incubated at 370 for 1 hr after adding various reagents (ATP, PEP, PEP kinase, all 20 amino acids) as described by Nirenberg et al.,6 dialyzed overnight at 4°against two changes of buffer B (buffer A plus 2 mM MSH), and then centrifuged at 49,000 rpm for 2.5 hr in a Spinco no. 50 rotor. The top three fourths of the clear S165 supernatant was collected and used as the soluble supernatant fraction; the sedimented material was rinsed with buffer B, suspended in the same buffer, and centrifuged at 10,000 X g for 10 min. The nonsedimentable material was used as the source of unwashed ribosomes.

Abstract. The phenomenon of selective translation of T4 template RNA by ribosomes from T4-infected cells, or factors derived therefrom, has been extended to studies on the initiation of protein synthesis. A high-salt extract derived from T4-infected ribosomes inhibits the formation of initiation complexes of MS2 and Escherichia coli template RNA with uninfected ribosomes while efficiently supporting the formation of initiation complexes with T4 template RNA. T4 factors also permit T5 template RNA to bind to E. coli ribosomes, which indicates that the T4 selective effect is not exclusive for T4 templates. Other evidence indicates that T4 factors do not alter the process of polypeptide chain elongation.Various reports have now described specific changes in the translational machinery of E. coli when infected by T-even bacteriophage. These changes include alterations in host tRNAs'-3 and the enzymes which modify them,4'5 changes in aminoacyl-tRNA synthetases,6 and the synthesis of phage-coded tRNAs.7-9 Another induced alteration was found by Hsu and Weiss,'0 who observed that ribosomes from T4-infected cells efficiently translate T4 template RNA but are restricted in their translation of host and MS2 RNA's. This phenomenon is an early phage function and is not manifested when infection occurs in the presence of chloramphenicol'0 or with T4 ghosts (unpublished results of the authors).In the same report, Hsu and Weiss found that a high-salt extract of T4-infected ribosomes (T4 factors) imparts a similar property of "selective" T4 RNA translation when added to uninfected ribosomes. These findings were interpreted by the authors as a form of translational control exerted by the invading phage on its host. Subsequent reports by Schedl et al.,1' Dube and Rudland,"2 and Steitz et al.'3 confirm the above observations. Refs. 12 and 13 also show that T4 factors restrict the binding of host, f2, and R17 RNA to ribosomes but permit the binding of T4 RNA. In this communication, we provide independent evidence that T4 factors exert their restrictive effect at the level of the formation of initiation complexes, and that the apparent selective translation of T4 template RNA, in vitro, is not exclusive. In addition, evidence is presented which indicates that T4 factors exert no effect on the elongation of polypeptide chains.

A previous report from this laboratory has shown that certain derivatives of polycyclic aromatic hydrocarbons bind to 4X174 DNA and render it noninfectious. The present work describes the relationship between the extent of 4X174 DNA binding by (±) -anti-benzo [alpyrene-7,8-dihydrodiol-9,10- (1-3), it reacts covalently with nucleic acids (4, 5), and DNA conjugated with anti-BPdiolepoxide has been isolated from cultured cells exposed to BP (2, 6). In addition, several studies have shown that BP-diolepoxide is unusually active as a mutagenic agent for mammalian (1, 7) and bacterial cells (8,9), and a more potent carcinogen than the parent hydrocarbon in newborn mice (10).We have described a bacteriophage assay system for evaluation of the biological activity of polycyclic aromatic hydrocarbons (PAH) and related compounds (11)(12)(13) (14), except that the infected cell lysates obtained by lysozyme treatment were subjected to two successive centrifugations (15 min at 17,000 X g and 2 hr at 100,00 X g) for sedimentation of phage. The resuspended phages were then purified as reported (14). DNA was isolated from purified viruses by several extractions with phenol containing 1% sodium dodecyl sulfate, followed by dialysis against 10 mM Tris (pH 7.5)/1 mM EDTA. The concentration of OX DNA was determined on the basis that, in a 1-cm light path, 1 A260 unit is equivalent to 36 jsg/ml or 1.3 X 1013 molecules per ml (14).Radioactive 4X174am3 DNA was prepared by the same procedure described above, except that infection was carried out in the presence of [3H]thymidine (6 Ci/mmol) at a concentration of 20 nmol/ml. Isolation of DNA from the radioactive virus by phenol extraction gave a specific activity of 3 X 104 cpm/,ug of DNA.Infectivity Assay of gbX174am3 DNA. The infectivity of OX174am3 DNA was assayed by absorption with spheroplasts prepared from E. coli K12W1485 and plating with its sensitive host, E. coli HF4714, for plaque development, as reported (11, 13). Preparation of Radioactive Replicative Intermediates RF1 and RF2. 3H-Labeled 4X174am.3 phage, prepared as described above, was used to infect E. coli C in the presence of chloramphenicol (15 ug/ml). After 10 min of infection, DNA was extracted from the infected cells by the procedure of Brutlag et al. (15). The cell lysate was adjusted to contain 1 M NaCl and was extracted once with phenol; the nucleic acid was precipitated from the aqueous phase with ethanol. After resuspension in Tris/EDTA and sedimentation in a neutral sucrose gradient, the pellet showed two radioactive peaks, 21 S and 16 S, corresponding to the qX174 DNA replicative intermediates RF1 and RF2, respectively.

Several examples of viral-induced modifications of cellular ribonucleic acids have been reported. After infection of Escherichia coli with T-even bacteriophage, Sueoka and Kano-Sueokal 2 found quantitative as well as qualitative changes in leucyl-tRNA's when compared to uninfected cells by chromatography on methylated albumin-kieselguhr (.IMAE). ]More recently, Waters and Novelli,3 using reverse phase chromatography, were able to confirm the early changes in leucyltRNA after T2 infection of E. coli and, in addition, observed two new leucyl-tRNA peaks which appeared very late after infection. Wainfan, Srinivasan, and Borek4 have reported on the alteration in the relative' activities of the base-specific methylases after T2 infection.The presence of thiolated bases in sRNA and the demonstration that cell-free extracts catalyze the transfer of cysteine-sulfur to sRNA,5' 6 prompted an inquiry as to whether any changes occurred in sulfur-containing RNA's after viral infection. The present communication describes changes in the 1\IAK chromatographic profiles of S15-labeled sRNA after E. coli infection with T-even bacteriophages. Evidence is offered which indicates that these changes are viral-induced, and that they require protein synthesis to be manifest.Experimental.-Growth of cells and viral infection: E. coli B cells were grown in a medium that contained the following constituents per liter: 2 gm of NH4Cl, 6 gm of NaCl, 0.01 gm of MgCl2, 6 gm of Na2HPO4, 3 gm of KH2PO4, 0.026 gm of Na2SO4, 2 gm of glucose, and 10 mM Tris -HCl of pH 7.5. This medium was further supplemented with 0.04 vol of 3XD medium.7 The cell suspension (2%7 inoculum of an overnight culture) was vigorously shaken in a gyratory apparatus at 370 (generation time approximately 50 min) until a density of 7 X 108 cells/ml was reached, at which time L-tryptophan was added to a concentration of 100 ,gg/ml, and then T4 phage at a ratio of 13 plaque-forming units per cell. Under these conditions, cell death was found to be greater than 99% after 2.5 mim, and the number of infective centers was 9.5%/ or more of the bacterial cell count. The infected cell suspension was shaken at 370, and after a given time infection was stopped by the rapid addition of 0.5 Al NaN3 (2 ml per 100 ml of culture) and crushed ice, and rapid cooling in an ice-salt water bath to 4°. The cells were collected by centrifugation and either handled immediately for RNA extraction or stored frozen at -20°. Infection with the coliphages T2, T7, MS2, and OX174p-was carried out in a similar manner except that the infecting ratio of phage/cell was 10, and E. coli K12W1485 and E. coli C were used as the respective hosts for M\S2 and qX174p-phages.S35-labeling of cells: E. coli cells (either infected or uninfected) were labeled with S35 by the addition of radioactive Na2SO4 (New England Nuclear Corp.) to the culture medium. Cells were collected as described above except that nonradioactive 0.5 Al Na2SO4 (2 ml per 100 ml of culture) was added to the suspension at the same time as the azide. "P...