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At least 19 recordsLinked to original sources
PubMed1975

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.

Azo Compounds↗
PubMed1975

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.

Coliphages↗
PubMed1978

Transfection with replicating DNA from the temperate Bacillus bacteriophage phi 105 and with T4-ligase treated phi105 DNA: the importance in transfection of being longer than genome-length.

Replicating phage DNA extracted from Bacillus subtilis infected with phage phi 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature phi 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role intransfection.

Bacillus subtilis↗
PubMed1979

The effect of factors causing induction of DNA breaks on transfection of chicken cells by the XC DNA, and the kinetics of appearance of virus-producing cells after transfection.

The effect of BUdR and 4NQO treatment of the recipient chicken fibroblast cultures on the efficiency of transfection by the XC DNA was investigated. The efficiency of transfection was 2-fold higher when the recipient cultures were incubated in the presence of 10 micrograms BUdR/ml in medium 199 (48%) than when cultures were not treated (25%). The efficiency was not further increased by exposure of BUdR-treated cultures to visible light for 20 min (31%). Growth of BUdR-treated cultures decreased after light irradiation which indicated that BUdR was incorporated into the host cell DNA. Treatment of the recipient cultures with 4NGO at a concentration of 0.4 micrograms/ml for 2 h before transfection exerted only unfavourable effects on transfection efficiency. In cultures positive in transfection, transformation was first detected 3 days after exposure to DNA by the infectious centre assay, with a frequency of 4.76 +/- 6.46 transformed cells per 10(6) cells.

4-Nitroquinoline-1-oxide↗
PubMed1975

A quantitative theory of transfection in B. subtilis.

A theory of transfection is developed which describes three different types of experiments in transfection: The concentration dependence of transfection, transfection with marker rescue, and the mapping function in transfection crosses. The theory is applicable to transfection systems exhibiting quadratic or higher order dose response (APP1, AP50, SP82). It pictures the essential process in transfection as follows: transfecting DNA molecules, having suffered inactivating events during uptake or intracellularly, have to recombine prior to replication under elimination of these lesions. The probability for recombination, successful in this sense, is calculated as a function of the number of DNA molecules within the competent cell, the mean number of inactivating events per DNA molecule, and the crossover probability per nucleotide. Under the assumption of random distribution of inactivating events over the population of DNA molecules and homogeneous crossover probabilities the theory explains on a quantitative basis a number of experimental observations in transfection, as for instance the relative efficiencies of different helper phage in transfection with marker rescue, the third order concentration dependence in SP50 transfection, and the high recombination frequencies observed in transfection crosses.

Bacillus subtilis↗
PubMed1978

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

Bacteriophages↗
PubMed2026

Impact of transfection optimization on adeno-associated virus purification performance and vector quality.

Recombinant adeno-associated virus (rAAV) has shown great promise as a viral vector for gene therapy. However, efficient manufacture of high-quality rAAV to meet clinical demands remains challenging. Here, we optimized rAAV production via a design of experiments (DoE) approach to evaluate the effects of five transfection parameters on vector genome titer, full particle ratio (FE ratio), and viral protein (VP) stoichiometry. The DoE model showed that no single set of transfection conditions could maximize all three responses simultaneously. Subsequently, the materials from DoE-optimized transfections were purified by affinity chromatography followed by anion exchange chromatography (AEX). We observed that AEX recovery declined when capsid loading was high, a situation caused by the combination of high titer with low FE ratio, reducing some of the gains in titer achieved by transfection optimization. Furthermore, AEX had limited capacity to enrich full particles from materials with a very low initial FE ratio. Finally, materials from DoE-optimized transfections showed improved transduction efficiency, which was associated with the ratios of VP1 and VP2 to total VP in the capsid. Overall, this study demonstrates that transfection-derived quality attributes affect purification outcomes and overall vector quality. Our findings highlight the necessity of strategically balancing multiple quality attributes during transfection optimization and provide insights for integrated upstream transfection and downstream purification development.

Adeno-associated virus↗
PubMed1977

DNA transfection of ecotropic murine leukemia viruses in mouse cell cultures.

DNA's were isolated from cells chronically infected with N-, B-, or NB-tropic murine leukemia viruses and tested for infectious activity in various mouse cell cultures. Early detection of the DNA transfection is facilitated by growing the DNA-recipient cells in medium containing 10(-6) M hydrocortisone. Appropriate shearing of the DNA preparations may increase the efficiency of the transfection. With these procedures virus production of the transfected cells can be detected by XC plaque assay as early as 4 days after DNA inoculation in NIH 3T3 cells. Susceptibility of the mouse cell cultures to DNA transfection does not parallel their susceptibility to virion infection. Progeny viruses derived from the transfection show the same N- or B-tropic host range property as do the parent viruses.

Animals↗
PubMed1978

Transfection and transformation of Agrobacterium tumefaciens.

The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10(-7) transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10(-7) transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10(-8). These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce "opine" synthesis in Crown-gall plant cells.

Bacteriological Techniques↗
PubMed1978

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.

Bacteria↗
PubMed1978

Transfection of Streptococcus pneumoniae with bacteriophage DNA.

It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966).

Bacteriophages↗
PubMed1978

Transmission of B77 virus double mutant LA334 markers by transfection.

Successful transfection of Brown Leghorn chicken fibroblasts was carried out with DNA isolated from duck cells transformed by the LA334 mutant of avian sarcoma virus B77. Transfection of duck cells was negative. The four viruses recovered after transfection were all temperature-sensitive for transformation. Two were fully temperature-sensitive for replication, as shown by analysis of virus replication, by characterization of virus particles produced at the nonpermissive temperature using density gradient centrifugation, and by electron microscopic examination. The other two viruses were only partially temperature-sensitive for replication. The results suggest that both the src and gag regions of the avian sarcoma virus genome are transferred simultaneously during transfection, probably by a single integral provirus copy.

Avian Sarcoma Viruses↗
PubMed1977

Application of the transfection technique for segregation of avian tumor viruses.

The possibility of clong avian tumor virus (ATV) by the transfection technique was studied. The attempts required efficient facilitator for the uptake of exogenous cellular DNA by chick embryo cells (CEC) to increase transfection efficiency. The calcium method was more efficient than the DEAE-dextran method for the uptake of tritium-labeled DNA, and the calcium method was used in further segregation studies of ATV by transfection of cellular DNA from ATV-infected cells. The test viruses included the BAI-A strain of avian myeloblastosis virus (a mixture of subgroup A and B viruses) and the B77 strain of avian sarcoma virus (a mixture of transforming and transformation-defective viruses). Analysis of the recovered progeny viruses after transfection revealed that they were indeed segregates of the parent viruses.

Animals↗
PubMed1975

Transfection with heteroduplex SPP1 DNA: a pyrimidine dimer induced influence on the conversion pattern.

Bacillus subtilis competent cells were transfected with SPP1 heteroduplices having pyrimidine dimers in one of the strands. The data obtained reveal that excision repair of the pyrimidine dimers influences the ratio of wild type versus mutant progeny observed in "normal" heteroduplex transfection. With increased exposure of one strand to UV dose the percentage of infective centers having the unirradiated strand genotype shows an increase. A comparison of the transfection data in her+ and her- host excludes asymmetric replication as the cause of the observed changes in the conversion pattern. The data can be explained on the basis of a dimer induced co-excision of the mismatched region. In addition transfection data from wild type/deletion mutant heteroduplices where the strand of mutant origin was irradiated exclude the possibility of the wild type loop being excised during uptake.

Bacillus subtilis↗
PubMed1976

Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of Lambda DNA.

The transfecting activity of linear lambda DNA is 100 times higher in calcium treated E. coli K12 (lambda i434) than in non-lysogenic strains: the levels of transfection are 1-2.10(7) and 1-2.10(5) infective centers per 1 mug of lambda DNA respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated "helper" phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms lambda DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.

Calcium↗
PubMed1975

[Transfection of Escherichia coli using bacteriophage MS2 RNA].

The authors present materials for comparison of three methods of E. coli infection by the RNA of the MS2 bacteriophage. The cells accepted the RNA at the stage of spheroplasts (obtained with the aid of a lysozyme and EDTA) with the efficacy of up to 1.5 X 10(5) IC/mug of the RNA. Diethylpyrocarbonate was used as a transfection stimulant. The product of transfection was identical to the MS2 bacteriophage. The efficacy of transfection in the calcinated system and on the frozen-defrosted bacteria was lower than on the spheroplasts (10(2) IC/mug of the RNA) which was largely associated with the formation of "latent infectious centres". Formation of latent negative colonies was characteristic of bacteriophage MS2, but formation of these colonies was intensified when calcinated cells were used for transfection.

Coliphages↗
PubMed1979

A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection.

The longest DNA molecules synthesized by endogenous reverse transcription in detergent-permeabilized Moloney murine sarcoma virus (Mo-MSV) virions (clone G8-124) are double-stranded DNA molecules of 5,8 kilobase pairs (kbp). This DNA species has been purified by sedimentation of total in vitro synthesized Mo-MSV DNA through neutral sucrose gradients. A physical map of the positions of the cleavage sites for a series of restriction endonucleases has been derived for this 5.8 kbp DNA. Mo-MSV DNA synthesized in vitro was found to induce morphological transformation of NIH-3T3 mouse fibroblasts upon transfection. The foci had a morphology indistinguishable from that of Mo-MSV-induced foci, and the induced transformed phenotype was stable. The 5.8 kbp double-stranded DNA (dsDNA) purified by agarose gel electrophoresis also induced focal transformation. Furthermore, gel-purified, restriction endonuclease-generated fragments of 5.8 kbp dsDNA containing the region from 2.8--4.9 kbp on the physical map of Mo-MSV DNA were able to induce foci. In contrast, endonuclease-generated DNA fragments lacking this region on the map were unable to transform cells upon transfection. When transformants derived by transfection with 5.8 kbp dsDNA were infected with Moloney murine leukemia virus (Mo-MLV) helper virus, Mo-MSV was rescued from a small portion of these cells, suggesting the establishment of the complete viral genome in these cells. One Mo-MSV DNA fragment, spanning 2.8--4.9 kbp on the physical map, was generated by cleavage of 5.8 kbp DNA with endonucleases Hind III + Sal I and currently represents our maximum estimate for the size of the transforming region of the Mo-MSV genome. This fragment includes the Mo-MSV sequences which are found in the DNA of uninfected mouse cells.

Base Sequence↗