DNA from Bacteriophage Lambda: Molecular Length and Conformation

MacHattie, L.A.; Thomas, Charles A.

doi:10.1126/science.144.3622.1142

Cited by 159 publications

(37 citation statements)

References 9 publications

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“…This agrees with molecular length (33 million) (118) and sedimentation measurements (31 million) (28). As expected, M-13 could not be phosphorylated, presumably because it is a closed circle (119).…”

Section: End Labeling--assupporting

confidence: 87%

“…A further study of ~. circles with and without the protein film technique confirmed the requirements for circle formation and the apparent continuity of the united ends (118). Identification of circular DNA by electron microscopy is now routine (see 201,212), and under special conditions can even reveal ~X DNA single strands as closed rings (63).…”

Section: Lini~r or Circular Dnamentioning

confidence: 88%

“…(a) Electron microscopy: The lengths of ),DNA molecules deposited carbon grids (11) have been compared (118) with those visualized by protein film technique (106) (169) M23.0 (13) *~4.3 (165) .85 (105) . 78 (203) 28 (197) UrT (197) 45 (139) HMUrT (139) 43 (122) HMUrT ( (2) uo (1) no no (182) no (2) yes (2) yes (9) yes (2) yes (2) no (2) no (92) no (152)…”

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confidence: 99%

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The Anatomy of Viral DNA Molecules

Thomas

MacHattie²

1967

Annu. Rev. Biochem.

119

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Without known exception, purifi ed virus particles, the virions, contain a single type of nucleic acid-RNA or DNA-never both. This fact provides the most convenient definition of a virus particle (115). The distinction be tween RNA-containing and DNA-containing viruses appears to be a pro found one that reflects a fundamental difference in their life cycle and evo lutionary origin. This review will be concerned with the DNA-containing viruses.It is generally a greed that viruses are reproduced from their own nu cleic acid. This can take place in various host cells whose biochemical ma chinery always provides substantial assistance in the process and sometimes even modifies the virus or its nucleic acid in a nongenetic way. These facts do not alter the idea that the identity and biological stability of viruses de pend solely upon the information in their own nucleic acids. In this regard they are like other microorganisms. To the extent that these generaliza tions are correct (and some unusual exceptions are known), a perfect un derstanding of the anatomy of viral DNA molecules, including the se quence of nucleotides, would define all tha,t can be known about the func tional potential of the virus. The term virus properly embraces all phases of the viral life cycle (35).Viral DNA molecules spend only part of their existence in mature virus particles called virions. They are found in infected cells, often in multiple copies and, in the case of temperate viruses, the viral DNA molecule is in corporated as a provirus into the DNA of the host cell. In this location it almost (but not quite) evades physical and chemical study, although the presence and the structure of prophage have been genetically studied for many years. It is a matter of reasonable expectation, and some faith, that a viral DNA molecule does not gain or lose information (nucleotide se quences) during any stage of its life cycle. In those cases where this does occur, the resulting entity must be considered a different, though related, virus.Relying on the notion that viral DNA molecules maintain their infor mational integrity at every stage of their life cycle, one may expect to ob tain information of general significance through the study of DNA mole-

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Section: End Labeling--assupporting

confidence: 87%

Section: Lini~r or Circular Dnamentioning

confidence: 88%

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The Anatomy of Viral DNA Molecules

Thomas

MacHattie²

1967

Annu. Rev. Biochem.

119

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“…It is very unlikely that FV 3 DNA possesses cohesive ends as observed in lambdoid phages (29), because when the DNA was renatured either without prior exonuclease digestion or after complete denaturation, no circular molecules were observed.…”

Section: Materials and Methods Cells And Virus Fathead Minnow (Fhm) mentioning

confidence: 99%

The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant.

Goorha¹,

Murti²

1982

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

115

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We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques. The linear FV 3 DNA molecules (Mr _100 X 106) formed circles when partially degraded with bacteriophage A 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion. The results suggest that the DNA molecules contain direct terminal repeats. The repeated region composed about 4% of the genome. Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference. The tails presumably represent the terminal repeats. The formation ofduplex circles suggests that the FV 3 genome is circularly permuted. This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (4i) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with Hindm. From the above data, we conclude that the FV 3 genome is both circularly permuted and terminally redundant-unique features for an animal virus. their temporal appearance during the infection, size of the replicating molecules, and site of synthesis. During the first stage, the smaller replicating DNA molecules (equal to or less than twice the genome length) are synthesized exclusively in the nucleus early in infection (up to 3 hr after infection). During the second stage, extremely large replicating complexes are synthesized only in the cytoplasm and late in infection (after 3 hr after infection). The large replicative complex acts as the precursor for the production of mature viral DNA size molecules. * To complement these studies on DNA replication and to understand the structural organization of the genome, we have examined virion DNA by electron microscopy and restriction endonuclease analysis. In this paper, we show that the FV 3 genome is terminally redundant and circularly permuted-unique features for an animal virus. MATERIALS AND METHODS Cells and Virus. Fathead minnow (FHM) cell monolayerswere grown at 330C in plastic roller bottles with Eagle's minimal essential medium containing 5% fetal calfserum. A clonal isolate of FV 3 was used to infect cells at a multiplicity of 0.1 plaqueforming unit per cell. FV 3 virions were purified as described (12). To minimize nicks and gaps in the viral DNA, crude virus preparations were not sonicated, freeze-thawed, or pelleted.Extraction of Viral DNA. DNA from purified FV 3 preparations was extracted by the phenoVchloroform procedure (15).Digestion of DNA with A 5'-Exonuclease. The procedure of Grafstrom et al. (16) was used with some modifications. The reaction mixture (total volume 0.1 ml) containing DNA at 10 pg/ ml, enzyme (New England BioLabs) at 160 units/ml, bovine serum albumin at 50 pug/ml, 1 mM 2-mercaptoethanol...

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“…DISCUSSION We have established that there is a circular DNA form produced specifically after induction of Mu cts4 prophage and after infection with phage Mu cts4. Other temperate phages, e.g., lambda and P22, are known to produce circular DNA of a phage genome size in the course of viral development (16,17). Multiple-lengthed circles corresponding to dimers, trimers, and higher concatemers of 4X174 DNA have been described as well (18).…”

Section: Resultsmentioning

confidence: 99%

Isolation of Heterogeneous Circular DNA from Induced Lysogens of Bacteriophage Mu-1

Waggoner

González

Taylor

1974

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

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Covalently-closed circular DNA molecules are formed after induction of phage Mu cts4 and after infection with phage Mu ctsl. The circular molecules obtained after induction have a molecular length range from 36.5 to 156.7 kilobases as measured by electron microscopic techniques. These heterogeneous molecules have no consistent correlation to exact multiples of a Mu genome equivalent (37.3 i 1.2 kilobases). Direct evidence is given that these molecules contain phage Mu DNA that is covalently linked to other DNA sequences.Mu-1 is a temperate bacteriophage of Escherichia coli K12 that generates stable, random mutations in its host by linearly inserting its DNA into bacterial genes (1-3). The phage genome can be inserted in a clockwise or counter-clockwise orientation with respect to the bacterial chromosome (4), and the genetic and physical maps of prophage and vegetative phage are congruent (4, 5). Since the DNA isolated from mature virions is linear (2), it seemed reasonable to us that the ends of Mu DNA may orient prior to integration by forming a circular DNA molecule. Covalently-closed circular DNA molecules have been observed in Mu lysogens (5) and more specifically in lysogenic bacteria after induction of a heat-inducible mutant prophage (6). Here we further characterize these circular molecules by electron microscopic analysis. MATERIALS AND METHODSBacteria and Bacteriophage. Bacterial strains used in this study are described in Table 1. Mu cts4 is a heat-inducible mutant of phage Mu isolated in this laboratory by E. Raizen.Analysis of Intracellular Events. Cells were induced at 450 at a cell density of 1 to 2 X 108 cells per ml in T broth (2) supplemented with thymine (4 mg/ml). At 5 min after induction, [methyl-8H]thymidine (7 uCi/ml) was added, and 23 min later a 15-ml sample was removed and added to 15 ml of cold TES buffer (0.05 M NaCl, 0.05 M Tris, 0.005 M EDTA, pH 8.0), containing 0.01 M sodium azide. Incorporation of labeled thymidine was linear for 10 min under these conditions. The remainder of the culture was monitored for lysis by measuring decline in turbidity. The DNA was isolated by the Bazaral and Helinski procedure (8), collected, and counted as previously described (2). Fractions were dialyzed for 24 hr against a solution containing 0.8 M NaCl, 0.01 M EDTA, 0.05 M Tris (pH 8.5) and 24 hr against DNA buffer (0.25 M NaCl, 0.01 M EDTA, 0.05 M Tris, pH 8.5) and the absorbancy was determined at 260 nm.For the experiment in Fig. 1, a culture was grown and induced as above, but it was shifted to 37°at 35 min after induction. Samples were removed at various times and assayed appropriately on L agar (1) for colony forming units and on indicator strain C600 for chloroform-resistant and total phage Mitomycin Treatment. Cells were grown in T broth supplemented with thymidine (8 gg/ml) and then treated prior to induction with mitomycin C (10 ,g/ml) at 330 according to the procedure of Lindqvist and Sinsheimer (9). At the time of induction [methyl-3H]thymidine (1 0Ci/ml) was added and then at th...

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DNA from Bacteriophage Lambda: Molecular Length and Conformation

Cited by 159 publications

References 9 publications

The Anatomy of Viral DNA Molecules

The Anatomy of Viral DNA Molecules

The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant.

Isolation of Heterogeneous Circular DNA from Induced Lysogens of Bacteriophage Mu-1

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