Neutral Sucrose Gradients Research Articles

Carcinogenesis and aging. II. Modifying effect of aging on metabolism of methyl(acetoxymethyl)nitrosamine and its interaction with DNA of various tissues in rats.

Young (3 month-old) and old (14 month-old) female outbred rats received a single i.p. dose (13 mg/kg) of N-[14C]methyl-Nacetoxymethylnitrosamine [( 14C]DMN-OAc), which, under these conditions, selectively induces intestinal tumours. Complete DMN-OAc breakdown occurred within 30 min in both young and old rats but exhalation of 14CO2 continued for over 1 h in young rats and over 3 h in old rats. The highest level of methylation in both young and old rats was found in the DNA of epithelial cells of the colon and in other adjacent abdominal organs (liver and uterus). The initial capacity for excision of the O6-methylguanine from liver DNA was greater in young animals, but further this DNA adduct was repaired more efficiently by the liver of old rats. In the DNA of ileal and colonic enterocytes, O6-methylguanine excision was higher in young than in old animals. The DNA tertiary structure, measured by the sedimentation pattern of nucleoids in neutral sucrose gradient, was damaged in old rats, and, to a lesser extent, in young rats. The non-uniform pattern of DNA damage in young and old animals may be associated with differing carcinogenic effects of DMN-OAc in rats of different ages, a hypothesis which is currently under test.

Escherichia coli host factor required specifically for the phi X174 stage III reaction: in vitro identification and partial purification.

A cell-free extract prepared from phi X174-infected Escherichia coli cells sustained in vitro synthesis of viral DNA (stage III reaction) when supplemented with fraction II from uninfected cells. The reaction was dependent upon deoxyribonucleoside triphosphate, ATP, added phi X174 replicative form I DNA template, and the fraction II from uninfected cells. This reaction differed from the stage II reaction (semiconservative replication of duplex replicative form DNA) by the production of stable viral protein-DNA complexes sensitive to anti-phi X174 antiserum. Three types of protein-DNA complexes were identified, 50S, 92S, and a 114S complex that cobanded in CsCl and cosedimented in neutral sucrose gradients with a phi X174 phage marker. The sensitivity of these complexes to anti-phi X174 antiserum and Staphylococcus aureus provided a relatively rapid biochemical assay for direct measurement of the amount of DNA synthesized by the stage III reaction. With this assay, an E. coli factor (SIII) required specifically for the synthesis of viral protein-DNA complexes was identified and purified 200-fold from uninfected E. coli cells. The partially purified SIII factor was required for the synthesis of DNA and viral protein-DNA complexes in the phi X174-infected cell extracts and could not be replaced by rep protein, single-strand binding protein, or DNA polymerase III holoenzyme.

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.

Adenovirus DNA replication in vivo. Properties of short DNA molecules extracted from infected cells

Adenovirus type 5 (Ad5) DNA replicating in intact HeLa cells was pulse-labeled with [ 3H]thymidine extracted by the procedure of Hirt and analyzed on neutral sucrose gradients. In addition to viral replicative forms (over 33 kb), a slowly sedimenting species of DNA (0.05–3 kb) was observed. Hybridization analysis showed that this DNA contained about 30% Ad5 DNA sequences. Analysis of the sensitivity of this DNA to the 5′ OH-specific spleen exonuclease after alkali or RNAase treatment revealed that about 80% of these molecules contained ribonucleotides. DNA in this fraction was labeled at the 5′ end with 32P and hybridized together with control 3H-labeled Ad5 DNA to HpaI restriction fragments immobilized on nitrocellulose paper. Many of these small molecules were located near the termini. Alkali treatment prior to hybridization decreased the 32 P 3 H ratio throughout the genome. This suggests that some of these Ad5 molecules possess ribonuncleotides and therefore may be intermediates in discontinuous DNA replication. Longer molecules (over 0.5 kb) were found sedimenting with viral replicative forms and mature DNA. The Ad5 molecules in this fraction showed no evidence of alkali-labile termini.

In vivo quantitation of N-3 and N-7 alkylpurines induced by alkylnitrosoureas

Alkylation at the N-3 and N-7 sites of purines in rat mammary gland DNA induced in vivo by N-ethyl- N-nitrosourea (ENU) and N-methyl- N-nitrosourea (MNU) was quantitated using a newly developed, quantitative, and relatively rapid assay that does not require the use of radiolabeled DNA or radiolabeled alkylators. The assay was based on the selective depurination of the alkylated N-3 and N-7 purine sites in nonradiolabeled DNA followed by hydrolysis at neutral pH to form single-strand breaks in the DNA at each alkylated site. The number of single-strand breaks and thus alkyl- N-3 and alkyl- N-7-purines was determined from the number-average molecular weight of the nonradiolabeled DNA denatured by formamide and sedimented through 5 to 20% neutral sucrose gradients. It was found that MNU alkylates more effectively at the N-3 and N-7 positions of purines than ENU, as expected. These sites appeared to be lost from the rat mammary gland DNA within 24 h. These data are in agreement with those obtained using other approaches.

Frog virus 3 DNA replication occurs in two stages.

Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus.

Characterization of in vitro DNA breakage and crosslinking induced by bis(isopropylamine)-trans-dihydroxy-cis-dichloroplatinum(IV)

Bis(isopropylamine)-trans-dihydroxy-cis-dichloroplatinum(IV) (CHIP or JM-9), a derivative of Cisplatin, was found to have DNA breakage and interstrand cross-linking activities in vitro. DNA breakage was detected by alkaline and neutral sucrose gradient analysis, agarose gel electrophoresis, and alkaline ethidium bromide fluorescence assay employing covalently closed circular PM2 DNA. DNA cross-linking activity was detected by alkaline sucrose gradient analysis and by the "snap-back" assay employing PM2 DNAs. Non-sulfhydryl-containing reducing agents, e.g., NaBH4 and NADPH, stimulated both cross-linking and breakage activities. Alkaline buffers, cyanide, or sulfhydryl group containing agents inhibited both types of activities. The hydroxyl free radical scavenger sodium benzoate (100 mM) was found to inhibit 99% and 25% of DNA breakage and cross-linking activities, respectively, suggesting DNA breakage and cross-linking may be independently mediated.

Increased disulfide-mediated condensation of the nuclear DNA-protein complex in lymphocytes during postnatal development and aging

Previous studies with liver cells suggested that disulfide (SS) bonds may play a role in maintaining the compact structure of the condensed chromatin fraction in the interphase nucleus and in the formation of a more condensed nuclear DNA-protein complex with advancing age. In liver cells (generally postmitotic) the molecular species participating in these SS bonds appeared to be located in the relatively micrococcal nuclease resistant fraction of the nuclei. This fraction has been noted to contain a nuclear protein skeletal structure with which DNA associates. In the present study we investigated the SS mediated condensation of the nuclear DNA-protein complex in a non-postmitotic cell population (mouse spleen cells) as a function of postnatal development and aging, and the mechanism of the SS mediated condensation. The experiments involved lysis of live spleen cells on top of neutral sucrose gradients in the presence of varying concentrations of NaC1, or NaC1 plus varyingconcentrations of a positive supercoiler of DNA followed by sedimentation of nuclear DNA-protein complexes arising from these lyses. The results suggest that the SS mediated condensation of the nuclear DNA-protein complex previously found in liver cells may also occur in non-postmitotic (spleen) cells, and that it begins early in life and continues to increase during later aging. The molecular species participating in the SS bonds involved in the condensation appeared to be undissociable from DNA by 2.0 M NaC1, a concentration known to dissociate histones and most of the nonhistones from DNA, leaving behind a subgroup of nonhistone proteins. Studies by other workers have shown that such tightly DNA associated nonhistone proteins are largely homologous with the proteins of a nuclear protein skeletal structure obtained by extensive nucleolytic digestion of nuclei. Our present data are consistent with a role for SS bonds in the association of regions of nuclear DNA with the elements ofa nuclear protein skeletal structure. In addition we present evidence that a partial

Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.

We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.

Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β

Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m 2) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.

Alkali-sensitive sites in DNA from human cells treated with ultraviolet light, 1'-acetoxysafrole or 1'-acetoxyestragole.

The formation and repair of alkali-labile sites in the DNA of human cells treated with 254 nm u.v. light, 1'-acetoxyestragole (1'-AcO-E) or 1'-acetoxysafrole (1'-AcO-S) have been studied. DNA was analysed by sedimentation in alkaline sucrose gradients after the cells had been layered on the gradients in lysis solution for 15 h (long lysis) or for only 0.75 h (short lysis). With the long lysis technique, a dose of 20 J/m2 resulted in 0.2-0.4 strand breaks/10(8) daltons while treatment of cells with 0.5 mM 1'-AcO-E or 1'-AcO-S caused 0.1-0.3 strand breaks/10(8) daltons. In excision repair proficient T98G cells, one third to two thirds of these strand breaks disappeared upon 4 h incubation after exposure to each of the three agents. In excision repair deficient xeroderma pigmentosum fibroblasts (XPA), the alkali-labile sites produced by 1'-AcO-E or 1'-AcO-S were still repaired, although those resulting from u.v.-irradiation were not. Similar characteristics were observed after the short lysis period. The sedimentation velocities of nucleoids, prepared from treated XPA cells, in neutral sucrose gradients containing ethidium bromide, did not reveal the presence of overt strand breaks in the DNA, suggesting that the lesions were of a type in which the sugar-phosphate backbone was intact but sensitive to hydrolysis by alkali. The contribution of this type of damage to the total DNA damage produced by the agents was estimated to be less than 1% for u.v., and less than 2.5% for 1'-AcO-E and 1'-AcO-S.

DNA double strand breaks in Ehrlich ascites tumour cells at low doses of x-rays. I. Determination of induced breaks by centrifugation at reduced speed.

DNA double strand breaks (dsb) were determined in Ehrlich ascites tumour cells at doses down to 5 Gy. The method is based on the separation of DNA from other components by heating in a solution of pronase and detergents held in wide-mouth syringes, which were also used to facilitate the application of the released high molecular weight DNA to sucrose gradients. Purified DNA was sedimented in neutral sucrose gradients at low speed to reduce speed artifacts. The sedimentation profiles were analysed using a computer program and the number of dsb was determined by simulation of random breaks in the mass distribution of the control sample and by comparison of this simulated profile with that of the irradiated one. The number of dsb formed was proportional to X-ray dose in the range of 5 to 2000 Gy. The induction per dose was found to be nmr-1 D-1 = (11.7 +/- 2) x 10(-12) Gy-1.

Effects of Hyperthermia on the Sedimentation of Nucleoids from HeLa Cells in Sucrose Gradients

ROTI ROTI, J. L., AND PAINTER, R. B. Effects of Hyperthermia on the Sedimentation of Nucleoids from HeLa Cells in Sucrose Gradients. Radiat. Res. 89, 166-175 (1982). The changes in sedimentation distance of nucleoids in neutral sucrose gradients in the presence of various concentrations of the intercalating dye ethidium bromide are believed to reflect changes in the amount of DNA supercoiling within the nucleoid. When HeLa cells were heated to 450C for 30 min before cell lysis, sedimentation of their nucleoids was markedly affected; at all concentrations of ethidium bromide in the gradient, nucleoids from heated cells sedimented farther than those from unheated cells. After cellular protein was labeled with [3H]leucine for 15 to 17 hr, approximately 4% of the total 3H radioactivity cosedimented with nucleoids from unheated cells. This percentage increased with heating time (10-70 min) and temperature (44-460C); e.g., after 20 min at 450C the fraction of 3H cosedimenting with nucleoids was 7%. The heat-induced change in nucleoid sedimentation distance was a linear function of the percentage of 3H cosedimenting with nucleoids. These results show that hyperthermia causes an increase in the protein content of nucleoids. The additional protein affects sedimentation by increasing the mass of the nucleoids and by decreasing the amount of DNA available for supercoiling and thereby the efficiency of DNA rewinding.

DNA double strand breaks in Ehrlich ascites tumour cells at low doses of x-rays. II. Can cell death be attributed to double strand breaks?

The induction and repair of DNA double strand breaks (dsb) in early stationary Ehrlich ascites tumour cells by X-rays was determined using an improved sedimentation technique in neutral sucrose gradients. The disappearance of dsb was followed during post-irradiation incubation of the cells and was interpreted as dsb repair. Kinetics were approximated by exponential functions with time constants of t37 = 3.0 +/- 0.7 hours ('conditioned' medium) and t37 = 2.0 +/- 0.5 hours (growth medium). Maximal repair was reached after 24 hours and the relationship of the remaining breaks with dose was interpreted on the basis of a recombination repair model. Using these dsb data and on the assumption of one dsb being a lethal event, cell survival curves were calculated for different repair times and compared with experimental curves. It was shown that cell survival curves can be interpreted on the basis of one unrepaired dsb being a lethal event, when dsb repair continues for about 11 hours after plating the cells on nutrient agar.

A modified neutral sucrose centrifugation method for rapid detection of dna damaged by γ-radiation and repair in human lymphocytes

1. 1. The influence of 60Co γ-radiation on the sedimentation behaviour of human lymphocyte nucleoids in neutral sucrose gradients was studied with the aid of a modified fluorescent monitor system consisting of the DNA-binding dye, Hoechst 33258 and a vertical centrifuge rotor. 2. 2. A combined method employing the sedimentation procedure and the incorporation of tritiated nucleosides was utilized to evaluate the repair of γ-radiated DNA.

Depurination of DNA as a possible mutagenic pathway for cells.

The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly [d(A-T)] or poly [d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases. The ability of Escherichia coli DNA polymerase I to copy over apurinic sites was demonstrated using single-stranded circular DNA of bacteriophage 0X174 as a template and starting DNA synthesis at a fixed point. Analysis of the newly synthesized 0X174 restriction fragments on neutral and alkaline sucrose gradients shows that synthesis proceeded past apurinic sites. When using depurinated 0X174 DNA containing the am3 amber mutation as a template for copying by E. coli DNA polymerase I, an increased reversion to wild type is observed after transfection into E. coli spheroplasts. The enhancement in reversion frequency is proportional to the extent of depurination, suggesting that depurination is also mutagenic during copying natural DNA in vitro. When noncopied depurinated 0X174 am3 DNA is transfected in E. coli spheroplasts, no increase in reversion frequency is observed above background level. However, when the spheroplasts are derived from bacteria in which the SOS response had been induced by UV irradiation, a substantial increase is observed for depurinated molecules, whereas no increase is observed for nondepurinated templates, suggesting in vivo mutagenesis at depurinated sites. In each of the different assay systems investigated, the increase in misincorporation or reversion frequency is a linear function of the number of sites and is abolished by treatment of the depurinated templates with alkali, which rapidly induces strand breakage at apurinic sites.

Variables that may influence the alkaline sedimentation pattern of herpes simplex virus DNA.

Herpes simplex virus (HSV) DNA sediments homogeneously on a neutral sucrose gradient but heterogeneously on an alkaline sucrose gradient. Several factors that may influence the alkaline sedimentation pattern of HSV DNA were examined: e.g., the host cell, cell density at time of infection, multiplicity of infection, and the starting material for HSV DNA purification (whether HSV-infected cells or cell-free virus). Based on alkaline sedimentation analysis, these factors appear to play little or no role in the amount of intact single-stranded HSV DNA observed.

Identification of a packaged cellular mRNA in virions of rous sarcoma virus.

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.

Extremely underwound chromosomal DNA in nucleoids of mouse sarcoma cells

The superhelical properties of chromosomal DNA from cells of a mouse sarcoma were investigated in neutral sucrose gradients containing ethidium bromide. Removal of negative supercoiling from the DNA of the sarcoma cells required a substantially higher dye concentration than was necessary in the case of DNA from cultured mouse fibroblasts. The calculated value of the mean superhelical density in malignant cells (σ = −0.14) appears abnormally high compared with the value (σ = −0.09) obtained for DNA of mouse fibroblasts. Chromosomal DNA from mouse sarcoma cells is therefore concluded to be highly deficient in helical turns.

Inhibition of protein synthesis by vaccinia virus: III. The effect of ultraviolet-irradiated virus on the inhibition of protein synthesis

The relationship between limited transcription and the ability of vaccinia virus to cause inhibition of protein synthesis (shutoff) was studied in HeLa cells infected with ultraviolet (uv)-irradiated vaccinia virus. Protein synthesis is shutoff, at 4 hr after infection, in cells infected with virus uv irradiated up to 9600 ergs/mm 2, however, shutoff does not occur in cells infected with virus uv irradiated at 28,800 ergs/mm 2. The extent of shutoff in cells infected with uv-irradiated virus is related to cytoplasmic virus-induced RNA. Polyacrylamide gel electrophoretic analysis (PAGE) of the polypeptides in cells infected with uv-irradiated virus shows that no viral proteins are detected even when cells are infected with virus irradiated at the lowest dose (1920 ergs/mm 2). In order to determine if this loss of ability to cause shutoff by uv-irradiated virus is related to transcription or to damage to virus structural components, several morphological and biochemical properties of the virus were examined. Even with the highest dose of irradiation used (28,800 ergs/mm 2): (1) Virus morphology is not visibly altered. (2) Enzyme activities associated with the virus particle, such as terminal riboadenylate transferase and deoxyribonuclease (neutral and acidic) are not affected. (3) The state of the viral DNA as determined by velocity sedimentation in alkaline and neutral sucrose gradients shows that a low degree of single-stranded DNA nicks and proteins crosslinked to the viral genome are found. (4) Adsorption of uv-irradiated virus to cells is unaltered. Studies on penetration and uncoating show a conversion of subviral particle 1 to subviral particle 2 (first stage uncoating) in cells infected with virus uv irradiated up to 9600 ergs/mm 2 but not in cells infected with virus irradiated at 17,280 or 28,800 ergs/mm 2. These data suggest that inhibition of protein synthesis is related to virus-induced RNA synthesis.