Uptake of Single-Stranded Bacteriophage DNA by Isolated Tobacco Protoplasts

Suzuki, Masahiko; Takebe, Itaru

doi:10.1016/s0044-328x(76)80089-x

Cited by 38 publications

(14 citation statements)

References 22 publications

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“…DNA uptake by plant protoplasts is known to be promoted by addition of poly-L-lysine (11,13,15). The beneficial effects of poly-L-lysine are due to its protective action on the nuclear membrane-bound DNA against DNase.…”

Section: Discussionmentioning

confidence: 99%

“…High mol wt poly-cations are known to increase DNA uptake by plant protoplasts (11,13,15) as well as to stimulate infection of viral RNA (1). As shown in Table II at a concentration of 1 ,ug/ml, poly-L-lysine (mol wt 150,000) considerably enhanced DNA binding and uptake.…”

Section: Methodsmentioning

confidence: 98%

“…Because plant protoplasts lack a cell wall they may have distinct advantages over normal plant cells for exogenous DNA uptake (5,11,13,15). Incorporated DNA may be degraded and reutilized for de novo DNA synthesis prior to genetic expression.…”

mentioning

confidence: 99%

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DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Ohyama

Pelcher²,

Horn³

1977

Plant Physiol.

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DNA binding and uptake by ucdei isolated from soybean (Glycine max L. Meff.) protopbsb were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drsicay with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occumd at pH 6 and the procea was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dTJ-[dA-dTJ) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-L-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.Genetic modification of higher plants by feeding exogenous DNA has been reported (4,8,14). Because plant protoplasts lack a cell wall they may have distinct advantages over normal plant cells for exogenous DNA uptake (5,11,13,15). Incorporated DNA may be degraded and reutilized for de novo DNA synthesis prior to genetic expression. Exogenous DNA must be stabilized as well as replicated before the genetic information can be expressed in the cell. A possible mechanism for stabilization is the integration of DNA into host genome. Exogenous DNA must therefore penetrate through the cytoplasm, and must be adsorbed to nuclear membrane without being degraded.Some investigators have observed that DNA taken up by cells was bound to the nuclei using radioautographic techniques (6, 7) and by analyzing nuclei isolated from protoplasts fed labeled DNA (15). This paper describes some of the factors affecting in vitro DNA binding and DNA uptake by nuclei isolated from soybean protoplasts. MATERIALS AND METHODSCell Culture and Preparation of Protoplasts. Soybean cells (Glycine max L. Merr.) (SB-1) grown in 1-B5 medium (3) were used throughout this work. Protoplasts were prepared by enzymic removal of cell wall as described previously (10).Isolation of Nuclei from Protoplasts. Protoplasts washed with 0-B5 medium (sucrose-free) containing 0.275 M sorbitol were suspended to 0-B5 (sucrose-free) medium containing 10 mm MgCl2, 1 mm 2-mercaptoethanol, and 0.5% Triton X-100 (medium A) plus 0.275 M sorbitol. The protoplasts were disrupted by a Dounce homogenizer with 10 gentle strokes. The homoge-NRCC No. 15963. 98 nate was passed once through a layer of Miracloth and twice through a triple layer of Miracloth. Filtrate was layered on 4 ml of medium A containing 0.4 M sorbitol and centrifuged at 400g for 5 min in an International model HN centrifuge. The pellet was suspended in 2 ml of medium A containing 0.275 M sorbitol. This step was repeated twice to remove debris, cytoplasmic organelles, and starch granules. The nuclei suspension was layered on stepwise gradients of 5 ml of 0.5 M sorbitol in medium A and 2 ml of 1 M sorbitol i...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 98%

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DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Ohyama

Pelcher²,

Horn³

1977

Plant Physiol.

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“…In animal cells, DNA coprecipitated with calcium phosphate is adsorbed onto the cells and taken up into the cells in the continued presence of excess CaCl2 (12). On the other hand, protoplasting is essential for plant cells to take up DNA (26,31,35).…”

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confidence: 99%

Transformation of protoplasted yeast cells is directly associated with cell fusion.

Harashima¹,

Takagi²,

Oshima³

1984

Mol. Cell. Biol.

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“…Several reagents have been reported which protect plasmid DNA from degradation, including poly-L-ornithin (8) and Zn2+ (12,19). Thus, the encapsulation technique using REV liposomes can be substituted for these reagents.…”

Section: Resultsmentioning

confidence: 99%

Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts

Uchimiya¹,

Harada²

1981

Plant Physiol.

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Reverse-phase evaporation lipid vesices (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 13 molar ratio, encapsulated approximately 30% of exogenously supple recombinant DNA vector, pBR322. The DNA sequestered in REV lposomes was highly tolerant to DNase.A two-step procedure was developed, which involves encapsulation of DNA with liposomes using one-tenth phosphate-buffered saine-05 molar mannitol, followed by incubation of liposome-DNA with protoplasts in phosphate buffer-O.5 molar mannitol.About 11% of liposome-encapsulated DNA was transferred into protoplasts, whereas 6% uptake was observed in the control. Although some degradation of Incorporated DNA occurred inside protoplasts, 50% of the total radioactivity resolved by 0.8% agarose gel was associated with Artificial lipid vesicles (liposomes) have been used as vehicles for transferring biologically active materials such as proteins, mRNA, and chromosomes into mammalian cells in culture (5,14,16,18). Some instances have been presented demonstrating liposome-mediated transfer of materials into plant protoplasts. These include a fluorescent marker, chloroplasts via multilamellar lipid vesicles (3, 6, 21), and bacterial RNA entrapped in large unilamellar lipid vesicles (13).Special attention has been paid to the transfer of genetic materials, particularly recombinant vehicles (plasmid DNA), into plant protoplasts with the aid of liposomes. Also, several attempts have been made to insert bacterial plasmids into plant protoplasts (7,12). In most cases, the major problem encountered in the transfer of naked DNA into protoplasts seems to be the degradation of DNA molecules before their transfer to the protoplasts (4,10,15 MATERIALS AND METHODS Protoplasts. Protoplasts were prepared from suspension cultured cells of Daucus carota by a modified method previously described (22). Cultured cells, S to 6 days old, were incubated in a solution containing 1% Cellulase Onozuka (Kinki Yakult MFG, Nishinomiya, Japan), 0.2% Macerozyme (Kinki Yakult), 0.01% Pectolyase (Seishin Pharmaceutical, Nagareyama, Japan), and 0.5 M mannitol. After incubation for 2 to 3 h at 25 C with gentle shaking, protoplasts were passed through two layers of Miracloth (Calbiochem) and centrifuged at 150g for 1.5 min. Pelleted protoplasts were suspended in 0.5 M mannitol and recentrifuged. Protoplasts were thus washed at least twice and maintained in an appropriate buffer solution containing 0.5 M mannitol.Preparation of I'HIpBR322. [3H]pBR322 was prepared by a method reported elsewhere (1). Specific radioactivity obtained was 62,000 cpm/,ug DNA.REV Liposomes. REV liposomes were prepared according to a modified method developed by Szoka and Papahadjopoulos (20).Hydrogenated phosphatidyl choline (egg lecithin) (3.2 ,tmol; 2.5 mg) and stearylamine (0.95 ,umol; 0.265 mg) (both from P. L. Biochemical, Milwaukee, WI) were mixed in chloroform contained in a 10-ml round-bottom flask. Chloroform was evaporated under N2 gas with a gentle rotation. When a film on the inside wall o...

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Uptake of Single-Stranded Bacteriophage DNA by Isolated Tobacco Protoplasts

Cited by 38 publications

References 22 publications

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Transformation of protoplasted yeast cells is directly associated with cell fusion.

Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts

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