Time and mode of fusion of human fibroblasts treated with polyethylene glycol (PEG)

Pontecorvo, G.; Riddle, P. R.; Hales, Anne

doi:10.1038/265257a0

Cited by 63 publications

(12 citation statements)

References 9 publications

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“…Pontecorvo et al (1977) have suggested that in human fibroblasts, PEG-mediated fusion occurs in very restricted areas at or near the tips of the filamentous processes of these cells.…”

Section: Discussionmentioning

confidence: 99%

Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Coetzee

Sirgel

Lecatsas

1979

Journal of General Microbiology

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Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 x 10-6 to 1.7 x 10-7 per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane. , 1978). There were three reasons for our choice. Firstly, in preliminary attempts to prepare spheroplasts by the combined method used (see Methods) the conversion frequency of Providence alcalifaciens strain P29 to spheroplasts was higher I N T R O D U C T I O NFodor

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“…Pontecorvo et al (1977) have suggested that in human fibroblasts, PEG-mediated fusion occurs in very restricted areas at or near the tips of the filamentous processes of these cells.…”

Section: Discussionmentioning

confidence: 99%

Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Coetzee

Sirgel

Lecatsas

1979

Journal of General Microbiology

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“…Their method, however, was modelled on a technique used for fusion of mammalian cells (Pontecorvo et a/., 1977) which employs a lower inolecular weight PEG (mol. wt 1540) for fusion, while the method described here is similar to the Bacillus system (Fodor & Alfoldi, 1976;Schaeffer et a/., 1976) which employs PEG 6000.…”

Section: Discussionmentioning

confidence: 99%

“…Polyethylene-glycol-induced protoplast (or cell) fusion has recently been demonstrated in several eukaryotic systems (Ferenczy, Kevei & Szegedi, 1975;Ann6 & Peberdy, 1976;Jones et al, 1976;Power et al, 1976;Pontecorvo, Riddle & Hales, 1977;Sipiczki & Ferenczy, 1977) and in the prokaryotic genus, Bacillus (Fodor & Alfoldi, 1976;Schaeffer, Cami & Hotchkiss, 1976). This technique appears to have potential broad utility to effect genetic recombination in prokaryotic micro-organisms, particularly in those lacking classical recombination systems.…”

Section: Introductionmentioning

confidence: 99%

Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

Baltz

1978

Journal of General Microbiology

145

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Conditions for highly efficient genetic recombination in Streptomyces by protoplast fusion are described. Protoplasts of S. fradiae and S. griseofuscus were formed by a modification of the glycine-lysozyme-lytic enzyme method (Okanishi, Suzuki & Umezawa, 1974). Regeneration of cells from protoplasts was monitored throughout the growth cycle and was most efficient when cells of either S. fradiae or S. griseofuscus were taken from the transition phase between the exponential and stationary growth phases. Fusion of protoplasts carrying different auxotrophic or chromosomal drug-resistance markers was achieved by treatment with polyethylene glycol, and high frequencies of stable genetic recombinants were obtained. Strain development and genetic analysis of several other economically important Streptomyces species, however, has been hindered by the apparent lack of natural fertility in these strains, and the lack of transduction and transformation systems (Hopwood et al., 1973). A more general means to effect genetic recombination in Streptomyces would therefore be quite useful.Polyethylene-glycol-induced protoplast (or cell) fusion has recently been demonstrated in several eukaryotic systems (Ferenczy, Kevei & Szegedi, 1975; Ann6 & Peberdy, 1976;Jones et al., 1976;Power et al., 1976;Pontecorvo, Riddle & Hales, 1977;Sipiczki & Ferenczy, 1977) and in the prokaryotic genus, Bacillus (Fodor & Alfoldi, 1976;Schaeffer, Cami & Hotchkiss, 1976). This technique appears to have potential broad utility to effect genetic recombination in prokaryotic micro-organisms, particularly in those lacking classical recombination systems. A key factor in the utility of this technique is the capability of fused bacterial protoplasts to regenerate cell walls and thus viable cells. In this communication, I describe a highly efficient genetic recombination system for Streptomyces using protoplast fusion and efficient cell regeneration.Downloaded from www.microbiologyresearch.org by

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“…C42 and A9 cells were plated together in 60 mm tissue culture dishes 24 h before hybridization in a ratio of 70% mouse cells: 30% human cells (Davidson and Ephrussi, 1970) at a density such that the monolayer was subconfluent at the time of hybridization (Pontecorvo et al, 1977). Hybridization was induced by treatment of cells with a 45% (w/v) solution of polyethylene glycol (M, 6000.…”

Section: Methodsmentioning

confidence: 99%

Identification of cell surface polypeptides encoded by human chromosomes 2 and 5 in human-mouse somatic cell hybrids

Carlin¹

1983

Cytogenet Genome Res

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Iodinated cell surface polypeptides of human-mouse somatic cell hybrids were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and high resolution two-dimensional gel electrophoresis in an effort to identify human gene products. Expression of two cell surface polypeptides was positively correlated with retention of specific human chromosomes: a polypeptide of molecular weight 250,000 and isoelectric point 8.3 with the human 5, and a polypeptide of molecular weight 250,000 and isoelectric point 7.0 with the human 2.

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Time and mode of fusion of human fibroblasts treated with polyethylene glycol (PEG)

Cited by 63 publications

References 9 publications

Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

Identification of cell surface polypeptides encoded by human chromosomes 2 and 5 in human-mouse somatic cell hybrids

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