Intra- and Interspecies Virus Transfer in Aspergilli via Protoplast Fusion

Intra- and interspecies transfer of dsRNA viruses between blackAspergilliandAspergillus nidulansstrains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of blackAspergillusandA. nidulansstrains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found amongA. nidulansstrains, transfer among blackAspergilliwas frequently unsuccessful. The lack of virus transfer between blackAspergillusisolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible blackAspergilli. The inefficient transfer of dsRNA viruses between blackAspergilliis not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of blackAspergilliare discussed.     

L-谷氨酸温度敏感突变株的选育

采用黄色短杆菌TJ1为出发菌株,根据代谢控制发酵原理,利用紫外线、硫酸二乙酯进行诱变,定向选育出具有寡霉素抗性、谷氨酸氧肟酸盐抗性的温度敏感突变株TMGO106。然后,以温度敏感突变株TMGO106和产酸率高（10.5%以上）的天津短杆菌TG961为新株,通过原生质体融合技术,成功地选育出了产酸率高的融合子CN1021（13．6g/dl,糖酸转化率达60％）,在6m^3发酵罐上中试其L－谷氨酸产量达14．6％,糖酸转化率达62．8％,并且该菌株系温度敏感型菌株,可用于谷氨酸强度发酵。     

二元融合子与荧光假单胞菌耐药性遗传标记的筛选

应用定性和定量药物敏感性试验与诱变相结合,筛选出荧光假单胞菌56-12-10菌株的遗传标记为链霉素抗性,二元融合子AM-1菌株的遗传标记为环丙沙星抗性,确定了在这两种细菌原生质体融合试验中,选择培养基中链霉素的应用浓度为300Iu/ml,环丙沙星应用浓度为100Iu/ml。     

Effect of protoplasting on antibiotic activity and resistance in the strain Streptomyces hygroscopicus 155]

The influence of protoplasting and protoplast regeneration in the presence of polyethylene glycol on antibiotic activity, components of antibiotic complexes and antibiotic resistance in Streptomyces hygroscopicus 155 was studied. It was shown that the protoplasting and protoplast regeneration influenced the antibiotic activity. The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity. The processes also affected the components of the antibiotic complexes but had no effect on the strain resistance to some antibiotics.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

用原生质体融合技术选育2—酮基—L—古龙酸高产菌株的研究

对革兰氏阳性的地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）Ｈ１９和革兰氏阴性的２－酮基－Ｌ－古龙酸产生菌Ｓ１９的原生质体的制备条件进行了研究,并采用聚乙二醇作诱导剂进行了两菌株的原生质体融合,用链霉素作为抗性标记对融合子进行了选择。从１７株产生２－酮基－Ｌ－古龙酸的融合子中选出了一株连续传代八次产酸高且产量稳定的融合子１５号。融合子１５号具有两个亲本菌株所具有的一些特性。     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f ). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data. Received: 16 July 1996 / Accepted: 2 December 1996     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f?). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data.     

Interspecific recombinants of Bacillus thuringiensis x Bacillus cereus

The possibility of interspecies recombination was shown by using protoplast fusion method. The Bacillus thuringiensis var. galleriae strain 48S Thi Nic Gua Rifr Strr and 56R Gua Rifr, and also Bac. cereus carrying the plasmid pBC16 responsible for resistance to tetracycline (150 mcg/ml) were used. Recombinants were selected on the medium containing rifampicin and tetracycline. They were shown to combine the properties of both parents. The majority of recombinants were resistant to phages Tg4 and Td15 and represented the mean level of sensitivity to phages Tg12, Tg13 and Td14. Examination of the plasmid profiles of recombinants revealed that their resistance to tetracycline was due to the plasmid with mobility analogous to pBC16. It was concluded that the protoplast fusion method can be used to obtain recombinants between relatively remote species of microorganisms.     

Protoplast Fusion of Lactobacillus casei

Lactobacillus casei ATCC 7469 was successfully converted to protoplasts by treatment with endo-7V-acetyl muramidase in sucrose phosphate buffer. For full hydrolysis of cell walls, a high concentration of sucrose and a cold shock were necessary. Mg²⁺ ions enhanced the stability of protoplasting cells. The cell wall regeneration of protoplasts was more effective on gelatin-induced regeneration medium than with the soft overlay method. The optimal concentration of gelatin was 2.5%. The frequency of regeneration was found to be about 6% for the protoplast prepared by enzyme treatment for 20 min. The mutants having streptomycin resistance and rifampicin resistance, as selection markers for the detection of fusion, were isolated by UV irradiation and NTG treatment. These mutants were stable for at least several transfers. Protoplast fusion was carried out using PEG (50% solution of polyethyleneglycol, M.W. 6,000). The frequency of protoplast fusion was found to be about 10^-5.     

Some peculiarities concerning the contraction of the gametes in Spirogyra maxima (Hass. Wittrock)

Conclusions We are of opinion that CZURDA and LLOYD did only see the last partof those stadia that characterize the examined process of fusion.One can easily imagine that CZURDA and LLOYD continued theirobservations only long enough to see a fusion follow those stadiawhere the male protoplast was already returned before the mouth ofthe conjugation canal. They mention indeed that the male protoplastnever loses its contact with some part of the conjugation process.In those cases one never sees a female protoplast contracted withoutthe male one being contracted at the same time. One can howeversee a male protoplast contracted without the female one being soalready. Hence their conclusions that the male protoplast shouldcontract first.If any importance is to be attached to the fact that the timesbetween the contraction of the male protoplast and the fusion arerather constant, compared with those between the contraction of themale protoplast and the fusion, it could be that the male protoplastplays a leading part in the process of fusion.     

酿酒酵母和产朊假丝酵母原生质体的形成、再生及属间融合

使用由亚硝基胍诱变所得到的营养缺陷型作为单倍体融合亲株的核基因标记,同时也采用线粒体球红霉素抗性突变株的小菌落形式作为融合亲株的线粒体基因标记。酿酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)两亲株原生质体的制备是用对数生长早期的细胞在蜗牛酶和0.7M KCl及β-巯基乙醇或二巯基苏糖醇的作用下完成的。二者的原生质体的形成率在30—60分钟内达到90—99％。原生质体再生率,酿酒酵母最高为29—35％,产朊假丝酵母为7.5％。两亲株的原生质体在35％PEG(M.W.6,000),10mM CaCl_2条件下被诱导融合。在基础培养基上,长出以营养互补为标记的融合菌株。融合频率为10~(-5)—10~(-6)。试验表明,这些融合菌株具有杂种的性质。其中一株杂合子在同化D-木糖、纤维二糖等的能力上比亲株明显增强。     

豌豆根瘤菌与新疆中华银瘤菌原生质体的属间隔合研究

以青霉素和氯霉素分别作为Rhizobium leguminosoum USDA2370和Sinorhizobium xinjiangnesis CCBAU110)的抗药性标记。利用原生质体融合技术,成功地获得了USDA2370和CCBAU110的属间隔合菌株。该融合菌株可分别在双亲寄主植物上结瘤。融合菌株在细菌形态、大小、菌落特征及蛋白质电泳图谱上与亲本菌株均有所不同。融合菌株与USDA23703的DNA同源性为56．6％,而与CCBAU110的DNA同源性为10．2％。     

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.     

原生质体培养在芸薹属蔬菜作物中的研究进展

原生质体是遗传转化的优良受体。在原生质体培养过程中会产生很多无性系变异,还可以产生体细胞杂种,这些都为蔬菜育种提供了新的途径。介绍了原生质体培养及原生质体融合方法的研究进展,综述了原生质体培养在芸薹属蔬菜育种中所取得的研究成果,并对今后研究的方向进行了展望。     

Transformation of Saccharomyces cerevisiae with yeast mitochondrial DNA linked to two micron circular yeast plasmid

Mitochondrial DNA from a petite mutant of yeast carrying an oligomycin resistance determinant has been ligated in vitro to 2 μm yeast plasmid DNA. The recombinant DNA so produced has been used to transform an oligomycin sensitive strain of Saccharomyces cerevisiae to oligomycin resistance at a frequency approaching 50 times the spontaneous mutation rate to oligomycin resistance. The majority of transformants showed genetic properties suggesting that recombination between the transforming DNA and the resident mtDNA has occurred. The properties of a subclass of oligomycin resistance transformants suggested that in these cells the transforming DNA has not become stably integrated into the mitochondrial genome of the recipient cell.     

Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.     

Transfer of functional adenovirus E1A transcription activator proteins into mammalian cells by protoplast fusion

Human adenovirus 2/5 E1A proteins were used to evaluate protoplast fusion as a method of transferring functional proteins into mammalian cells. Both the E1A 13 and 12 S mRNA products expressed in Escherichia coli are shown to activate in trans adenovirus gene expression following transfer into monkey kidney cells by protoplast fusion. Approximately 20% of the recipient mammalian cells exhibited positive nuclear E1A-specific immunofluorescence following fusion with protoplasts containing E1A protein. E. coli-expressed E1A protein was modified post-translationally in Vero cells following protoplast fusion, as evidenced by its shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. These results establish protoplast fusion as a simple rapid method for examining the functional activity, intracellular distribution, and post-translational modification of E. coli-expressed proteins in intact mammalian cells.