Physical and Chemical Properties of Rna From the Bacterial Virus R17

Mitra, Sankar; Enger, Mette; Kaesberg, Paul

doi:10.1073/pnas.50.1.68

Cited by 63 publications

(15 citation statements)

References 12 publications

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“…1 for RI. The OD (260 m,u) was 0.832; the patterns were photographed at 8, 16, and 24 min after reaching speed (52,640 rev/min); the temperature was 20.0 C; and r/2 was 0.06. degradation products (22). In contrast, RI was a completely heterogeneous population of molecules, as might be expected from the pattern of RI obtained by sucrose density gradient centrifugation (11).…”

Section: Resultsmentioning

confidence: 77%

“…Indeed, the value of / which yields a molecular weight closest to Mw (theoretical) = 2.9 X 106 is again 2.5 x 106. Mitra et al (22) pointed out that the value of /3 should not be greater than 2.5 X 106 for R17 RNA and Eigner and Doty (5) showed that (i) / = 2.5 X 106 for native DNA, and (ii) / = 2.3 X 101 for denatured DNA (although the values varied between 1.6 X 106 and 2.6 x 106 in this case). Furthermore, /3 has been shown to have the value of 2.5 X 106 for many synthetic polymers (8).…”

Section: Discussionmentioning

confidence: 80%

“…Since AX and XO (distance of meniscus from the rotor center) were constant, then The latter equation was programmed on the LOCI-2 computer (Wang Laboratories, Inc., Tewsbury, Mass.). The absorbancy index of R17 RNA at 260 m,u was determined, by the method of Mitra, Enger, and Kaesberg (22), to be 23.9 cm2/mg, compared with their value of 23.3 cm2/mg. Since this value depends on the initial optical density at 260 mru of the RNA and therefore can vary considerably, these values are in very close agreement.…”

Section: Methodsmentioning

confidence: 99%

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Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Franklin¹

1967

J Virol

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Replicative intermediate (RI) is considered to be the double-stranded ribonucleic acid (RNA) template for synthesis of viral RNA, with bound nascent single-stranded viral RNA. A theoretical description of RI is based on the analysis of a steady state of biopolymerization on a template which determines not only nucleotide sequence, but also chain length. The hydrodynamic properties of RI isolated from Escherichia coli infected with bacteriophage R17 are compared with those of RNA isolated from R17 (single-stranded RNA) and of replicative form (RF) isolated from E. coli infected with R17. RF is double-stranded RNA template without any single-stranded component. Whereas S for R17 RNA is a function of the ionic strength (F/2) of the solvent, S is almost invariant with r/2 for RF. By contrast S for RI lies between the sedimentation constants for R17 RNA and RF and S varies with r/2 as does R17 RNA. The weight distribution of S for RI demonstrates the heterogeneity of this material, and the variation in the weight distribution with ionic strength demonstrates the duality of structure in RI. Using S and [X], the Mw for RI is estimated to be 2.6 x 106 daltons, as compared with the theoretical value of 2.9 X 106 daltons. 65 on July 11, 2020 by guest

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Section: Resultsmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

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Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Franklin¹

1967

J Virol

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“…For the small RNA-containing bacteriophages, whose RNA has a molecular weight of 1.1 X 106, both hydrodynamic and spectroscopic data show that 60-80% of the RNA exists in the form of short base-paired loops (1)(2)(3)(4)(5). Recently, sequence analysis has also shown that the bacteriophage RNA contains many internal regions which are self-complementary and which could produce hairpin loops by base pairing (6,7).…”

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confidence: 99%

Studies on secondary structure of single-stranded RNA from bacteriophage MS2 by electron microscopy.

Jacobson¹

1976

Proc. Natl. Acad. Sci. U.S.A.

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A method allowing the demonstration and study by electron microscopy of secondary structure of viral RNA has been developed. Single-stranded RNA from the bacteriophage MS2 has been analyzed in the electron micro-scope in the presence of various concentrations of MgCI2.Depending on the salt concentration, the molecules display one to three large open loops which range in size from 10 to 20% of the total RNA lengh, and smaller closed loops which are approximately 3-5% of the total RNA length. Within one spreading, the conformation of the molecules is variable.However, the average complexity of the molecules increases with increasing salt, and individual loops which are infrequent at low salt increase in frequency with increasing salt. By analyzing the manner in which the individual loops appeared, it was possible to show that all molecules could be described by one basic pattern of secondary structure formation.Secondary structure, that is, the presence of short, helical, base-paired hairpin loops, has been demonstrated in singlestranded RNA by a variety of physical techniques. For the small RNA-containing bacteriophages, whose RNA has a molecular weight of 1.1 X 106, both hydrodynamic and spectroscopic data show that 60-80% of the RNA exists in the form of short base-paired loops (1-5). Recently, sequence analysis has also shown that the bacteriophage RNA contains many internal regions which are self-complementary and which could produce hairpin loops by base pairing (6, 7). Calculations based on the known free energies of the individual base-pairs suggest that the most stable molecules are those in which the individual loops are relatively short, generally under 50 nucleotides in length. Although it is not known whether more than one stable arrangement of loops is possible, the specificity of the fragments obtained after nuclease digestion suggests that one arrangement is preferred (8). In addition to the physical data, a variety of genetic and biochemical experiments suggest that specific secondary structure occurs in phage RNA molecules, and is probably responsible for the regulation of both transcription and translation during viral replication (9).Recent studies have shown that specific patterns of loops can be visualized on ribosomal RNAs in the electron microscope (10, 11). These studies show that secondary structure mapping can be used, in ways similar to denaturation mapping of double-stranded DNAs (12), to obtain information about the biological properties of RNA molecules. To date, studies of this sort have generally been unsuccessful with RNAs of lower (G+C) content, (13), and it has therefore been suggested that the hairpin loops which occur on nonribosomal RNAs may be too small to be visualized in the electron microscope. The one exception is a recent report on the possible occurrence of two specific loops on the RNA of .RNAs from the small RNA-containing bacteriophages have a moderate (G+C) content and messenger function. Due to the large amount of information available about their secondar...

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“…I n order to make either interpretation possible, MS2 RNA must remain, a t least partially, double-stranded during the infection. Since 700/, of the MS2 RNA is doublestranded [16], it seems unlikely that a break in one of the double-strand regions of the RNA makes the RNA fall apart. The possibility that the RNA is repaired cannot be excluded, especially in the light of the recent report of an E. coli RNA ligase [17].…”

Section: 1~0~ [S]mentioning

confidence: 99%

Killing of ³²P‐Labelled Single‐Stranded‐RNA Bacteriophage MS2

Lupker

Glickman²,

Rörsch³

et al. 1973

European Journal of Biochemistry

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The killing of highly 32P-labelled bacteriophage MS2 by transmutation was studied. The efficiency of killing found was 0. 60 & 0.08. This value is significantly lower than the value expected for a single-stranded bacteriophage. These results can be interpreted by assuming either that the MS2 RNA is replicated with the breaks caused by radiophosphorus decay, or that the breaks are repaired before replication. Preparation of 32P-Labelled Phage MS2 was prepared in the following way. The host strain was grown to a Concentration of 108 cells/ml in Tris-Cas medium, after which the culture was centrifuged for 10 min a t 4000 rev./min. The bacteria were resuspended in Tris-Cas medium to which carrier-free 3,P (The Radiochemical Centre, Amersham, England; specific activity 40-90 Ci/mg) was added to the specilk activities given in the text.After various prelabelling times (indicated in the text) the bacteria were infected by adding MS2 to a concentration of 108 p. f.u./ml. The infected bacteria were incubated 2.5 to 3 h at 37 "C after which CHC1, was added. The lysates were centrifuged for 20 min a t 4000rev.lmin. MS2 titers of lysates so obtained were not lower than 2 x 1O1O p.f.u./ml and generally in the range of 1 to 2 x loll p.f.u./ml.The clarified lysates were diluted 1 in 100 in freezing medium. Aliquots were frozen in a solid C0,-ethanol bath and stored in a Revco low-temperature Freezer a t -80 "C. The controls were treated the same way, except that the 32P was added after the bacteria were lysed. Storage of unlabelled MS2 in the way mentioned above did not result in a loss of viability of the phage during the course of the experiments (4 to 6 weeks).Phage lambda was grown as described by Clickman et al. [5]. Titrations MS2 was titrated using strain KA 52 as indicator. The bacteria were grown to a concentration of 3 x 108 cells/ml in Luria Broth containing CaCl, to a concentration of 0.01 M. The usual soft-agar overlay technique was used. Lambda was titrated as described elsewhere [5].

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Physical and Chemical Properties of Rna From the Bacterial Virus R17

Cited by 63 publications

References 12 publications

Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Studies on secondary structure of single-stranded RNA from bacteriophage MS2 by electron microscopy.

Killing of ³²P‐Labelled Single‐Stranded‐RNA Bacteriophage MS2

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Physical and Chemical Properties of Rna From the Bacterial Virus R17

Cited by 63 publications

References 12 publications

Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid

Studies on secondary structure of single-stranded RNA from bacteriophage MS2 by electron microscopy.

Killing of 32P‐Labelled Single‐Stranded‐RNA Bacteriophage MS2

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Killing of ³²P‐Labelled Single‐Stranded‐RNA Bacteriophage MS2