Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions.

Solutions containing polyethylene glycol MW 6000 (PEG) induced fusion of protoplasts of Penicillium chrysogenum. Balanced heterokaryons were formed by fusion of nutritionally complementing protoplasts. Heterokaryotic fusion products were obtained up to a frequency of 4% of the number of protoplasts, surviving the fusion treatment. Investigation of the conditions, necessary to achieve this high fusion frequency, showed that supplementing the PEG solution with Ca++ and adjustment to high pH gave the best results. Mechanisms of fusion of fungal protoplasts by PEG, calcium and alkaline pH are discussed in view of the obtained results.     

Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions

Solutions containing polyethylene glycol MW 6000 (PEG) induced fusion of protoplasts of Penicillium chrysogenum. Balanced heterokaryons were formed by fusion of nutritionally complementing protoplasts. Heterokaryotic fusion products were obtained up to a frequency of 4% of the number of protoplasts, surviving the fusion treatment. Investigation of the conditions, necessary to achieve this high fusion frequency, showed that supplementing the PEG solution with Ca⁺⁺ and adjustment to high pH gave the best results. Mechanisms of fusion of fungal protoplasts by PEG, calcium and alkaline pH are discussed in view of the obtained results.     

Fusion of protoplasts in Streptomyces antibioticus

The analysis of Streptomyces antibioticus recombinants isolated under non-selective conditions for stability of inheritance of parental properties demonstrated the existence of non-stable diploids and stable haploid recombinants. Also, genetic analysis of heteroclones isolated on selective medium after plating spores of regenerants was conducted. The results of analysis of haploid recombinant and heteroclones pointed to linkage of genetic loci and the possibility to use the technique of protoplast fusion for establishment of genetic map of S. antibioticus.     

Biosynthesis of oleandomycin by cultures of Streptomyces antibioticus obtained after regeneration and fusion of protoplasts

The ability of Streptomyces antibioticus strains to synthesize oleandomycin is studied under the effect of regeneration and fusion of protoplasts. The production of strains-regenerants with an increased (by 30-50%) synthesis of oleandomycin is possible. Regenerants of mutants resistant to the proper antibiotic retain a high level of the oleandomycin synthesis more stably. Variations in the antibiotic-production ability are considered in regenerant populations of various generations.     

Studies of the biosynthesis of actinomycin in protoplasts from Streptomyces antibioticus.

Protoplasts actively synthesizing actinomycins have been prepared from Streptomyces, antibioticus. They showed an absolute requirement for the presence of oxygen, galactose, and alkaline earth ions. Sucrose was most efficient as an osmotic stabilizer. However, in air-saturated buffer the protoplasts seemed to be slightly inhibited in their metabolism. This is expressed by the appearance of 4-methyl-3-hydroxyanthranilic acid and the inability to utilize [1?¹⁴C]sarcosine for actinomycin synthesis. Evidence has been obtained that sarcosine and N-methyl-l-valine are not free precursors of the peptide-bound N-methyl-amino acids. It is further demonstrated that synthesis of actinomycin IV and actinomycin V differ from each other with respect to their different susceptibilities against the changings in the physiological environment of the protoplasts. Actinomycin synthesis is severely reduced when protoplasts are incubated in the presence of 10^?3, m methionine.     

Structural changes induced by glycine on Streptomyces antibioticus

Abstract Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus .     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

Kinetics and ultrastructure of sheep fibroblast fusion induced by polyethylene glycol. Comparison with endogenous cell fusion induced by Visna virus]

The authors compare the fusion of sheep fibroblasts induced by low multiplicities of infection using visna virus and by high concentrations of polyethylene-glycol. In the case of Visna virus cell fusion is of the endogenous type, while fusion induced by polyethylene-glycol is of the exogenous type. The ultrastructural features are discussed for each type of cell fusion. The main differences between the two systems involve the intracellular microfilaments and Golgi apparatus.     

Glutamate-induced uptake of proline by Streptomyces antibioticus.

Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized.     

Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose

To determine whether polyethylene glycol (PEG) causes growth of liposomes by affecting them directly or indirectly, vesicles composed of phosphatidylcholine were exposed to increasing concentrations of Mr 15 000-20 000 PEG or Mr 40 000 dextran either by direct mixing or across a dialysis membrane. After incubation at room temperature and dilution below at least 5% (w/w) polymer, the vesicles were monitored for fluorescence energy transfer and for absorbance at 400 nm. PEG induced the same levels of dequenching or lipid mixing and increased turbidity, regardless of whether the vesicles had been mixed directly with or dialyzed against PEG. These changes occurred within 5-15 min of polymer application. It is concluded that the increased lipid mixing and/or increased turbidity, indicating vesicle growth, resulted from an indirect effect of PEG on the vesicles--most likely dehydration. Dextran, in contrast to PEG, induced less dequenching and/or less turbidity increase when vesicles were directly mixed with, as opposed to dialyzed against, dextran. Although dextran not in contact with vesicles and with osmotic activity comparable to PEG was able to cause a degree of membrane fusion similar to that of PEG, therefore, the dehydrating effect of dextran could be mitigated if it were allowed to interact with vesicles. In further support of membrane dehydration as a precursor to membrane fusion, lipid mixing among sonicated and sonicated, frozen-thawed vesicles dialyzed against sucrose increased as a function of sucrose concentration. Vesicle morphology generally determined the maximal degree of membrane fusion inducible by the polymers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction of mRNA to plant protoplasts using polyethylene glycol

A method is described to deliver mRNA to plant protoplasts using polyethylene glycol for transient expression. In order to optimize delivery and subsequent expression, we have examined a number of parameters that affect expression levels including the requirement for Mg²⁺ and Ca²⁺ ions, carrier nucleic acid, and the linearity of the dose-response. Delivery of mRNA using PEG results in levels of expression that are comparable to those obtained with electroporation. This method is particularly useful for those species in which electroporation does not work well. Moreover, the use of PEG to deliver mRNA is an inexpensive alternative to electroporation in that it does not require an electroporator.     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

New antibiotic-producing Streptomyces TT-strain,generated by electrical fusion of protoplasts

Interspecific electrofusion between protoplasts of multhiomycin-producing Streptomyces antibioticus aAL Ala⁻Leu⁻ and neomycin-producing S. fradiae fHL His⁻Leu⁻ was done. When the concentration of both protoplasts increased to 1 × 10⁹ protoplasts/ml, the frequency of fusion attained was 18.6%. The addition of multithiomycin and neomycin to the regeneration medium was very effective for screening for fusants able to produce new antibiotics. The ability to produce new antibiotics was very unstable in the fusants. After several subcultures, fusants selected as new antibiotic producers (10 strains) lost this ability with one exception (TT-strain). The antibiotic produced by the stable TT-strain clone was purified and characterized to some extent. It was active on a range of Gram-positive bacteria and distinguished from multhiomycin and neomycin by bioautographic analysis.     

The fusion of erythrocytes by treatment with proteolytic enzymes and polyethylene glycol.

Polyethylene glycol (PEG) has been utilized to induce homokaryocyte formation in avian and mammalian erythrocytes previously treated with proteolytic enzymes. PEG of molecular weight 6,000-7,5000 was found superior to 1,500 and 20,000 MW PEG. Cells exposed to protease alone, prior to PEG treatment, fused to a high degree (60-95% multinucleated cells), whereas trypsin or pepsin treatment alone allowed very little fusion (2.5%). Trypsin lowered the effectiveness of protease when used in combination. Cells which were not treated with proteolytic enzymes agglutinated in the presence of PEG but did not fuse to a significant extent (0.01%). Fusion was also markedly dependent upon the rate at which PEG was eluted during the fusion process. Electron microscopy indicated that fusion began during the elution of PEG from the agglutinated cells.     

Polyethylene-glycol induced fusion of bacterial protoplasts

Summary Direct selection for recombinants by supplemented minimal media from polyethylene-glycol (PEG)-induced fusion of protoplasts of polyauxotrophic strains of B. megaterium revealed striking physiological influences on the yield of recombinants. Cytoplasmic state of the protoplasts to be fused, rather than genetic events, determined the number of colonies obtained on the selection media. It is suggested that the physiological effects primarily influenced the ability of the fused protoplasts to revert to bacillary form.     

Use of Streptomyces sp. 26-115 protoplasts inactivated by heating in fusion experiments

When heated at 55 degrees C for 30 or 60 minutes protoplasts of auxotrophic mutants of Streptomyces sp. 26-115 producer of actinomycin C (active and inactive variants) lost their capacity for regeneration. The protoplasts heated at at 55 degrees C for 30 minutes and not for 60 minutes maintained some ability to yield recombinants on fusion under the effect of PEG 6000. Unlike the parent active strain, the colonies formed by the spores of the prototrophs yielding on fusion of the intact protoplasts showed wide ranges of antibiotic activity against M. flavus while a significant part of the colonies was inactive. The use of the inactive variant protoplasts heated at 55 degrees C for 30 minutes in the fusion procedure increased the proportion of the inactive variants.