Increase In Transformation Frequency Research Articles

Absence of a dose-rate effect in the transformation of C3H 10T1/2 cells by alpha-particles.

The findings of Hill et al. (1984) on the greatly enhanced transformation frequencies at very low dose rates of fission neutrons induced us to perform an analogous study with alpha-particles at comparable dose rates. Transformation frequencies were determined with gamma-rays at high dose rate (0.5 Gy/min), and with alpha-particles at high (0.2 Gy/min) and at low dose rates (0.83-2.5 mGy/min) in the C3H 10T1/2 cell system. alpha-particles were substantially more effective than gamma-rays, both for cell inactivation and for neoplastic transformation at high and low dose rates. The relative biological effectiveness (RBE) for cell inactivation and for neoplastic transformation was of similar magnitude, and ranged from about 3 at an alpha-particle dose of 2 Gy to values of the order of 10 at 0.25 Gy. In contrast to the experiments of Hill et al. (1984) with fission neutrons, no increased transformation frequencies were observed when the alpha-particle dose was protracted over several hours.

Pichia pastoris as a host system for transformations.

We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.

Pichia pastoris as a host system for transformations.

We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.

Integrative transformation of the yeast Yarrowia lipolytica

We have derived a DNA-mediated transformation system for the yeast Yarrowia lipolytica based on the lithium acetate method Ito et al. (1983) developed for Saccharomyces cerevisiae. The first plasmid used, pLD25, contains the Y. lipolytica LEU2 gene (coding for the enzyme beta-isopropylmalate dehydrogenase, EC 1.1.1.85) on a 6.6 kb piece of DNA inserted into pBR322. The recipient strain ATCC 20688 contains the rarely reverting mutation leu2-35. The Y. lipolytica LEU2 gene complements leuB mutants in Escherichia coli and leu2 mutants in S. cerevisiae and it also hybridizes weakly to the S. cerevisiae LEU2 gene. Y. lipolytica transformation frequencies of up to 104 Leu+ cells per microgram of DNA, per 108 viable cells have been obtained from plasmds linearized with restriction enzymes. The more than 100-fold increase in transformation frequency obtained by using linearized DNA instead of intact plasmid resembles the situation seen in S. cerevisiae for site-directed integrative transformation (Orr-Weaver et al. 1981). The transformants were stable when grown in non-selective medium. We found that pLD25 integrated at the leu2 region when either linear or intact plasmid was used to transform Y. lipolytica.

Genetic transformation of molybdenum-starved Azotobacter vinelandii: increased transformation frequency and recipient range

Azotobacter vinelandii Nif− mutants UW3, UW45, and UW91 that did not become competent for genetic transformation after growth in Fe-limited competence induction medium did become competent when Mo starvation also was imposed. Mo starvation of other Fe-limited competent strains increased their frequency of transformation. Mo starvation also increased the frequency of transformation of strain UW1 with heterologous DNA from Azotobacter, Rhizobium, and Beijerinckia. Mo starvation did not increase the competence of the encapsulated A. vinelandii strain ATCC 12837. The substitution of mannitol or sorbitol for glucose in the Fe-limited competence induction medium, however, increased strain 12837 transformation frequency over 200-fold.

Neoplastic Transformation Is Enhanced by Multiple Low Doses of Fission-Spectrum Neutrons

The neoplastic transformation of C3H mouse 10T1/2 cells was measured induced by fission-spectrum neutrons delivered at a high dose rate in five fractions over 4 days. The transformation frequency was significantly enhanced over that due to single equivalent total doses. These new data, in the low dose region, demonstrate an increased transformation frequency by fractionated versus single exposures of high-dose-rate fission-spectrum neutrons; an increase equal to that observed with low-dose-rate fission-spectrum neutrons (i.e., 0.086 rad/min). Estimates of the dose modifying factor (DMF), based upon the ratio of the initial linear portions of the induction curves for high and for low dose rates, suggest the same DMF (approximately 7.8) for both five daily fractions of high-dose-rate neutrons and for low-dose-rate neutrons. However, when these results are compared to those following high-dose-rate 60Co gamma rays (100 rad/min), the relative biological effectiveness (RBE) for low-dose-rate fission-spectrum neutrons based upon slope ratios is 19.6; similarly, the RBE relative to five daily fractions of 60Co gamma rays is 78.8.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

Transformation of Neurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA.

We have conducted a detailed study of 108 qa-2+ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which the qa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass the qa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-BglII qa-2+ DNA fragment might possess an ars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying the qa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal resident qa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.

Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus.

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.

A re-appraisal of the BHK 21 clone-13 cell transformation system with respect to the assay conditions and the mutational origin of the transformed phenotype.

Two BHK 21/13 cell lines (BHK-ICI and BHK-A3) were used for studies on cell transformation. The BHK-ICI cells formed micro-colonies in agar while the BHK-A3 cells did not. The size distribution of colonies in agar derived from BHK-ICI cells was homogeneous and did not permit a distinction between untransformed micro-colonies and transformed macro-colonies. Moreover, the transformation frequency in BHK-ICI cells was influenced substantially by cell density, as densities greater than 500 cells/ml reduced the observed transformation frequency, whereas no reduction in transformation frequency was observed for BHK-A3 cells for densities up to 10(4) cells/ml. Therefore, we recommend the use of a BHK cell line that does not form micro-colonies for the transformation assay. In contrast with published data, obtained at high cell density with BHK cells that form micro-colonies, induction of cell transformation in BHK-A3 cells by treatment with 4NQO did not behave as a mutational trait. (i) Continuous treatment did not lead to a steady increase in transformation frequency but to a reduction in transformation frequency after the second day of exposure. (ii) A continuous treatment up to 24 h proved to be about five times more effective in inducing cell transformation than a one hour acute treatment. The results support the suitability of the BHK cells as an assay for cell transformation if the adverse effects of cell density are met and the formation of micro-colonies is prevented. The use of BHK-A3 cells at a density of 10(4)/ml fulfills these requirements.

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.

Cloning of antibiotic resistance and nutritional genes in streptomycetes.

Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66 were adjusted to generate reproducibly a population of cloned genes likely to represent the entire genome. Factors which influence the recovery of viable transformants include: growth phase of the mycelium, ionic and osmotic characteristics of the medium during protoplast formation and transformation, and moisture content and protoplast density during regeneration. A modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes, using one of two plasmid vectors. Viomycin resistance genes on BamHI or PstI fragments were cloned from S. vinaceus genomic DNA into S. lividans, using the SLP1.2 vector. At least three different S. vinaceus BamHI fragments (1.9, 5.8, or 8.5 kilobases) confer viomycin resistance; only one PstI fragment (4.3 kilobases) was found. Recombinant plasmids were all able to produce lethal zygosis and to be transferred by conjugation within S. lividans. SCP2 was used to clone S. coelicolor A3(2) genes that "complemented" the auxotrophic mutation hisD3, argA1, or guaA1. Recombinant DNA technology can now be applied to economically and academically interesting problems unique to streptomycete molecular biology.

Effect of growth rate and nutrient limitation on the transformability of Escherichia coli with plasmid deoxyribonucleic acid.

The observed transformation frequency by plasmid deoxyribonucleic acid of Escherichia coli grown in continuous culture was found to depend on both the steady-state growth rate and the type of nutrient used to limit growth. With carbon, nitrogen, or phosphorus limitation, the faster the growth rate, the higher the transformation frequency. The increase in transformation frequency associated with higher rates was shown to be due to more transformable cells in the population rather than an increased efficiency of deoxyribonucleic acid uptake. Growth rate had relatively little effect on the transformability of cells from sulfate- and Mg2+-limited chemostats, indicating that some factor other than the growth rate must influence the frequency of transformation. Regardless of the nutrient limitation or the growth rate, no transformants were obtained in the absence of CaCl2.

Effect of dimethylsulfoxide on transformation ofBacillus subtilis in vitro

Transformation ofBacillus subtilis was carried out in the presence of dimethyl sulfoxide and polyethylene glycol. The frequency of transformation ofB. subtilis was increased by 0.1% dimethyl sulfoxide but was not appreciably changed by polyethylene glycol. It is suggested that the increase in frequency of transformation was due to the effect of dimethyl sulfoxide on permeability of the cell membrane or to changes in membrane deoxyribonuclease activity.

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The transformation efficiency of Escherichia coli prepared by the calcium-heat shock procedure has been increased sixfold by growth of cells in 0.5 M sucrose and the addition of 1 μg/ml lysozyme along with DNA. A mutant of E. coli, hyt , has been selected which has a tenfold increased efficiency of transformation.

A method for detecting carcinogenic organic chemicals using mammalian cells in culture.

A method for testing organic chemicals for their carcinogenic potential is described. Baby hamster kidney cells (BHK-21/C1 13) were exposed to different doses of test compound in liquid tissue culture medium containing rat liver post-mitochondrial supernatant and cofactors (S-9 mix) to aid metabolism, but without serum. Survival of cells following exposure to the compound was assessed by cloning in liquid growth medium. Transformation was assessed by colony growth in semi-solid agar. The dose-response curve for survival was used to determine the LC50 of the compound. A dose-response curve for transformation was constructed and a 5-fold increase in transformation frequency at the LC50 was regarded as a positive test result. The method may also be used for testing gaseous compounds. Cells grown in monolayers and overlaid with serum-free medium and S-9 mix were exposed to vinyl chloride gas mixed with air. After exposure, the treated cells were trypsinized, resuspended in growth medium, and survival and transformation assays performed. The methods described are illustrated by examples taken from an evaluation study using 120 compounds and found to be more than 90% accurate in distinguishing between carcinogens and non-carcinogens.

Enhancement of simian virus 40 infection in simian and human cells by permissive cell extracts.

SummaryThe sensitivity of semipermis-sive cell cultures to SV40 infection can be enhanced by a component of uninfected, simian cells (CV-1) that are permissive for SV40 infection. Enhancement is demonstrated by 10- to 40-fold higher plating efficiency in simian cells, 10- to 30-fold increase in virus production by human cells and 3-fold increase in frequency of transformation in a line of human diploid fibroblasts after inoculation of mixtures containing CV-1 extracts and virus. The enhancer is recovered from cultured cells by a mild extraction procedure and is not sedimented by centrifugation at 20,000g for 1 hr. The active component of the supernate appears to be protein. Incubation of virus with enhancer preparations for 20 min at 23° results in the formation of a complex that is not dissociated by dilution. Although these properties of the SV40 enhancer are similar to those described previously for an enhancer of an enterovirus, the SV40 enhancer differs from the enterovirus enhancer in its host and v...

Role of deoxyribonuclease in genetic transformation. II. RNA as an inhibitor of doexyribonuclease activity in pneumococcal strains.

It was found that an inhibition of the relatively low deoxyribonuclease activity in the highly transformable and stable Pneumococcus R6bd strain results in a decrease of transformation frequency. The opposite is true about the Pneumococcus PMSII57 strain where an inhibition of its high deoxyribonuclease activity results in an increase of transformation frequency. It was observed that ribonucleic acid and especially t-RNA inhibits deoxyribonuclease activity in pneumococcus strains. Hence the kinetics of the effect of t-RNA as a function of time was examined with respect to the frequency of transformants in recipient cells of R6bd strain. It was found that t-RNA inhibits transformation. Maximum inhibition occurs if t-RNA is added to the DNA-bacterial complex at the time of penetration of transforming DNA into recipient cells. The results indicate that in a highly transformable and stable Pneumococcus strain the penetration of DNA is associated with the regulation of optimal deoxyribonuclease activity.