Infection of Acholeplasma laidlawii by MVL51 virus

Liss, Alan; Maniloff, Jack

doi:10.1016/s0042-6822(73)81013-x

Cited by 63 publications

(36 citation statements)

References 19 publications

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“…Acholeplasma was used for virus propagation and as indica MVL51, a group LI mycoplasmavirus, was used (6,7). JAI cells were assayed as colony forming ur tryptose agar plates, and viruses were assayed as ph units (PFU) on (8).…”

Section: Methodsmentioning

confidence: 99%

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Replication of mycoplasmavirus MVL51: Attachment of MVL51 parental DNA to host cell membrane

Das

Maniloff

1976

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

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The results presented here demonstrate that, de the multiplicity of infection, a certain percents tracellular parental viral DNA sediments with membrane and that this DNA exists mostly in E forms. These data also suggest that RF replicatic at membrane sites and that the maximum nui available per cell is two to three. There is no transf label to progeny viruses at the multiplicities of ih in this study. MATERIALS AND METHODSOrganisms, Media, and Buffer. Acholeplasma was used for virus propagation and as indica MVL51, a group LI mycoplasmavirus, was used (6, 7). JAI cells were assayed as colony forming ur tryptose agar plates, and viruses were assayed as ph units (PFU) on JAI lawns. PFU/cpm), and after 5 min unadsorbed viruses were removed un) containing by centrifugation. The washed infected cell pellet was resus-;ht 2 X 106(3).pended in warmed tryptose broth, and at specified intervals the n shown (4) to infected culture was pulse labeled for varying periods with )uble-stranded[3H]thymidine. Labeling was terminated by adding an equal vo forms: RFI volume of ice-cold Tris-EDTA-NaCl-NaCN buffer and cenLicked circles).trifuging at 12,000 rpm for 10 min at 5°. The cell pellet was roduced from washed twice with Tris-EDTA-NaCl buffer and finally reins to form a suspended in the same buffer. The cell suspension was gently is a short-lived lysed by freezing in liquid nitrogen and thawing. Three cycles viral infection of freeze-thawing were found to lyse the cell suspension combasal medium, pletely, as judged by the loss of turbidity. r tryptose meSucrose Gradient Centrifugation. A subcellular, fast sedionly a 10 min menting complex was isolated as follows: a 4 ml CsCl shelf, e resolved by containing 1.2 g/ml of CsCl in 40% sucrose, was placed at the and extrusion bottom of the centrifuge tube, over which 34 ml of 5-20% or killing the (wt/vol) linear sucrose gradient buffered by Tris-EDTA-1 M NaCl was formed. One milliliter of infected cell lysate was pending upon layered on top of the gradient and centrifuged for 3 hr at 24,000 age of the inrpm in a Beckman L3-50 centrifuge in an SW27 swinging the host cell bucket rotor. IFI and RFII Deproteinized fast and free sedimenting complexes were rn takes place analyzed by sedimentation through a 38 ml 5-20% (wt/vol) mber of sites linear high salt sucrose gradient as described (4). One milliliter .er of parental of sample was layered on top of the gradient and centrifuged nfection used for 16 hr at 5°at 24,000 rpm in an SW27 rotor. For all these gradient analyses, 1.2-ml fractions were collected from the top of the gradient using an ISCO density gradient fractionator.

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Section: Methodsmentioning

confidence: 99%

“…In richer dium used in the studies reported here, there is 4 latent period and the SSII component cannot b sucrose gradient analysis (4). Virus assembly a through the cell membrane occur without lysing cells (6).…”

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confidence: 99%

Replication of mycoplasmavirus MVL51: Attachment of MVL51 parental DNA to host cell membrane

Das

Maniloff

1976

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

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“…A. laidlawii JAl and K2 and M. capricolum Kid have been described previously (7,15,30). A. laidlawii was grown in tryptose medium and assayed as CFU on tryptose agar plates (7), and M. capricolum was grown in mycoplasma broth base medium and assayed as * Corresponding author.…”

Section: Methodsmentioning

confidence: 99%

Heat shock response in mycoplasmas, genome-limited organisms

1990

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We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JAl and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 The heat shock response has been found in every organism which has been studied (see references cited in references 2, 13, 14, and 21). It is characterized by synthesis of a group of specific proteins in response to a sudden temperature increase in the environment of the organism. The major heat shock proteins have been highly conserved during the evolution of procaryotes and eucaryotes (see references cited in reference 32). Other types of environmental stress also induce synthesis of some or all of the heat shock proteins (e.g., see reference 35).Mycoplasmas are the smallest free-living cells (18). They arose by degenerate evolution from a branch of the eubacterial phylogenetic tree containing gram-positive eubacteria with DNA having low G+C contents (16,29,37,39). Therefore, mycoplasma evolution from eubacteria must have involved a significant loss of genetic information (16,29). Mycoplasma genome sizes fall into two ranges (for a review, see reference 28): 1,500 to 1,700 kilobase pairs (kb) (about one-third of the genetic capacity of Escherichia coli) and 700 to 800 kb (about one-sixth of the genetic capacity of E. coli). The latter genome sizes approach the theoretical limit for the minimal amount of genetic information in a free-living organism (19).We have investigated whether mycoplasmas have heat shock proteins, i.e., whether the apparently universal heat shock response has been conserved during the genome reductions accompanying mycoplasma evolution. In this report, we describe studies of the heat shock response in Acholeplasma laidlawii K2 and JAl, with genome sizes of 1,646 to 1,719 kb (26,27), and Mycoplasma capricolum Kid, with a genome size of 724 kb (27). MATERIALS AND METHODSCells, virus, and media. A. laidlawii JAl and K2 and M. capricolum Kid have been described previously (7,15,30). A. laidlawii was grown in tryptose medium and assayed as CFU on tryptose agar plates (7), and M. capricolum was grown in mycoplasma broth base medium and assayed as * Corresponding author.CFU on mycoplasma broth base plates (6). Acholeplasma virus L2 was grown and assayed as PFU on A. laidlawii host cell lawns (23). E. coli JM101 (40) was grown in LuriaBertani medium (31).Temperature shift. Although the mycoplasmas used in these studies have optimum growth temperatures of 37°C (4), to have at least a 10°C increment between incubation temperatures before and after shift up, cells for heat shock studies were grown at 32°C to an A610 of 0.1, corresponding to about 108 CFU/ml (33). The doubling time at 32°C of the mycoplasmas used in these studies was about 140 min. Samples (1 ml) were then shifted to a higher temperature to induce a heat shock response or kept at 32°C as a control.For studies using E. coli, an overnight cultu...

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“…Such a region in L51 might be useful for insertion of foreign DNA for cloning in mycoplasmas. In addition, since L51 phage particles longer than unit length have been observed in virion preparations (3,15), there may not be strong constraints on the amount of DNA that L51 can package. Hence, the L51 deletion mutants described here may allow development of a mycoplasma cloning vector.…”

Section: Methodsmentioning

confidence: 99%

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine

Sladek¹,

Nowak²,

Maniloff³

1986

J Bacteriol

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Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JAl and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JAl cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JAl. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JAI restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In constrast, JAl cells (which contain no methylated DNA bases) have a newly discovered type of restrictionmodification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JAl cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JAl, which retains the JAl modification phenotype, was isolated, indicating that JAl cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.Mycoplasmas are procaryotes that have no cell walls: each cell is bounded only by a lipid-protein cell membrane (12). They are the smallest known free-living cells and have genomes 20 to 40%o the size of those of typical eubacteria (12, 16). These organisms arose by degenerative evolution as a branch of the gram-positive eubacteria (16,30).Some strains of the mycoplasma Acholeplasma laidlawii are hosts for four different bacteriophages (4, 18). Mycoplasma phage L51 is a bullet-shaped virion containing circular, single-stranded (SS) DNA of 4.5 kilobases (kb). Mycoplasma phage L3 is a T7-like virion containing linear, doublestranded (DS) DNA of 39 kb. Mycoplasma phage L2 is an enveloped virion containing circular, superhelical, DS DNA of 11.8 kb. Mycoplasma phage L172 is an enveloped virion containing circular, SS DNA of 14 kb.Bacteria have restriction-modification systems, which may have evolved to protect cells against infection by foreign DNA (for a review, see reference 27). Restriction involves endonucleases that recognize specific nucleotide sequences (.4 bases in length [11,24]) and cleave the DNA molecule at or near the sequence. A cell protects its DNA from endonuclease cleavage with a methylase that recognizes the same DNA sequence as the endonuclease and methylates a specific nucleotide within that sequence (2, 10). Specifically modified (methylated) cellular DNA ...

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Infection of Acholeplasma laidlawii by MVL51 virus

Cited by 63 publications

References 19 publications

Replication of mycoplasmavirus MVL51: Attachment of MVL51 parental DNA to host cell membrane

Replication of mycoplasmavirus MVL51: Attachment of MVL51 parental DNA to host cell membrane

Heat shock response in mycoplasmas, genome-limited organisms

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine

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