Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transformation efficiency”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources
PubMed2026

Development of a highly efficient prime editing platform for cucurbits enables breeding of multi-disease-resistant cucumber.

The prime editing (PE) system is a precise genome editing technology that works efficiently in monocots; however, its application is limited by low editing efficiency in dicots, particularly Cucurbitaceae and Solanaceae plants. Here, we first significantly improved the transformation efficiency by introducing spectinomycin in cucurbits, then used the tomato elongation factor 1-alpha (SlEF1α) promoter to enhance PE protein expression, and incorporated the Csy4 ribonuclease to process pegRNAs, collectively addressing multiple constraints limiting PE efficiency in cucurbits. The optimized PE systems, particularly Csy4-PE6d, achieved an average desired editing frequency of 80.83% at targeted loci in cucumber via stable genetic transformation, with frequencies reaching up to 100% at certain sites. Moreover, Csy4-PE6d generated homozygous edits in 36.43% of transgenic lines and demonstrated robust editing activity in melon, pumpkin, and potato. Using the Csy4-PE6d tool, we generated heritable edited cucumber lines with dual resistance to bacterial angular leaf spot and downy mildew by targeting the CsSGR gene. Collectively, this optimized system substantially enhances PE efficiency in Cucurbit crops, providing an effective solution to common challenges such as low editing efficiency and limited heritability in these species.

Disease Resistance↗
PubMed1998

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

Adenosine Triphosphatases↗
PubMed1983

Development of DNA-mediated transformation systems for plants.

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.

Journal Article↗
PubMed1998

Cloning of a trans-spliced glyceraldehyde-3-phosphate-dehydrogenase gene from the potato cyst nematode Globodera rostochiensis and expression of its putative promoter region in Caenorhabditis elegans.

Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.

Amino Acid Sequence↗
PubMed1996

Electrotransformation of Clostridium thermosaccharolyticum.

Transformation of the thermophile Clostridium thermosaccharolyticum ATCC 31960 was achieved using plasmid pCTC1 and electroporation. Evidence supporting transformation was provided by Southern blots, detection of the plasmid in 10 out of 10 erythromycin-resistant clones, retransformation of E. coli and C. thermosaccharolyticum with plasmid DNA isolated from C. thermosaccharolyticum, and a proportional relationship between the number of transformants and the amount of DNA added. Transformation efficiencies were very low for plasmid DNA prepared from E. coli (0.6 transformants mg-1 DNA), although somewhat higher for plasmid DNA prepared from C. thermosaccharolyticum (52 transformants mg-1 DNA). Transformation-dependent erythromycin resistance indicates that an adenosine methylase gene originating from Enterococcus faecalis, a mesophile, is expressed in C. thermosaccharolyticum. The plasmid pCTC1 appears to be replicated independently of the chromosome, as indicated by visualization of recovered plasmid on gels, and retransformation using recovered plasmid. pCTC1 is maintained in C. thermosaccharolyticum at both 45 and 60 degrees C. Restriction analysis showed little or no rearrangement occurred upon passage through the thermophile.

Blotting, Southern↗
PubMed1992

Direct DNA hybridization screening of primary yeast transformants in the construction of targeted yeast artificial chromosome (YAC) libraries.

A simple and inexpensive method for direct, large-scale DNA hybridization screening of primary yeast colonies following introduction of foreign DNA into Saccharomyces cerevisiae by spheroplast transformation is presented. An optimally thin layer of standard regeneration agar containing the transformed spheroplasts is spread on selective plates using carefully controlled temperature conditions; the colonies regenerate initially within the thin layer of hardened regeneration agar, but as they grow, greater than 90% of them break the surface and become accessible to direct lifting and screening methods. The technique avoids the manual or mechanized picking of transformants from within the regeneration top agar, does not require specialized (and often expensive) regeneration matrices such as alginate or low-melting-point agarose, and yields transformation efficiencies and screening results identical to those obtained using the standard spheroplast transformation protocol. We demonstrate the utility of this method for the construction of a targeted human-YAC library from a hamster hybrid cell line that contains chromosome 13 as its only human DNA.

Blotting, Southern↗
PubMed2003

The spirochetal chpK-chromosomal toxin-antitoxin locus induces growth inhibition of yeast and mycobacteria.

Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin-antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the the spirochetal chpK gene into a mycobacterial expressing vector led to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and Mycobacterium bovis BCG. However, few mycobacterial transformants were obtained. This result could be due to plasmid structural modifications leading to disruption of chpK expression, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.

Bacterial Toxins↗
PubMed2004

Microbial transformations of chalcones: hydroxylation, O-demethylation, and cyclization to flavanones.

Microorganisms were examined for their potential to catalyze biotransformation reactions that mimic plant biosynthetic processes. Specifically, microorganisms were screened for their abilities to transform selected chalcones to flavonoid and other products. Aspergillus alliaceus UI 315 efficiently transformed 3-(2' ',3' '-dimethoxyphenyl)-1-(2'-hydroxyphenyl)propenone (2'-hydroxy-2,3-dimethoxychalcone) (1) to several products, all of which were characterized by UV, NMR, and mass spectral analyses. A. alliaceus cyclized 1 to three flavanones and to O-demethylated and hydroxylated chalcones, some of which functioned as intermediates in the cyclization process. Inhibition studies using SKF525A, metyrapone, and phenylthiocarbamide with whole cell reactions showed that as many as three cytochrome P450 enzymes may be involved in these reactions. One enzyme catalyzed chalcone cyclization; another, O-demethylation; and a third, hydroxylation of chalcones. Flavonoid products are racemic, unlike the same products that are stereoselectively cyclized in plants. This work shows that microorganisms are capable of cyclizing chalcones to form flavonoid products, thus affording a mimic of plant biosynthetic processes.

Aspergillus↗
PubMed1994

A rapid and simple method for Bacillus subtilis transformation on solid media.

Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample containing 1 microgram DNA and 10(7) exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10(-5), as compared to 10(-4) routinely obtained by the method of transformation in liquid medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell density, medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiological conditions.

Bacillus subtilis↗
PubMed2005

Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis.

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (DeltapyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (DeltapyrF DeltatrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (DeltapyrF DeltatrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (DeltapyrF DeltatrpE DeltahisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

Archaeal Proteins↗
PubMed1970

Growth and development of competence in the group H streptococci.

The growth and development of competence by group H streptococci, strain Challis, were compared in synthetic, semisynthetic, and complex media with respect to the cultural conditions required, time of onset and persistence of competence and transformation efficiency. Provided that cultural conditions were strictly controlled in the synthetic system, transformation frequencies of 1% or above were routinely observed. The initial pH must be between 7.3 and 7.6, and the addition of freshly prepared bicarbonate ion was required. Furthermore, competence was sensitive to the degree of initial agitation of the culture. There was no evidence that "step-down" or "unbalanced" growth conditions were required. Competence could be provoked in the incompetent strain Wicky, growing in complex or semisynthetic media, by the addition of heat-killed or filtered cultures of strain Challis prepared during the competent period of growth in synthetic medium.

Albumins↗
PubMed2001

Natural genetic transformation of Streptococcus mutans growing in biofilms.

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.

Amino Acid Sequence↗
PubMed1984

Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

Animals↗
PubMed2000

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms.

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.

Acinetobacter↗
PubMed2004

A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation.

Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes.

Chromosomes, Artificial, Bacterial↗
PubMed1976

A quantitative analysis of the susceptibility of human leukocytes to transformation by Epstein-Barr virus.

Susceptibility of lymphocyte-enriched cell fractions isolated from human umbilical cord blood and adult peripheral blood to transformation by the B95-8 strain of Epstein-Barr virus (EBV) was investigated quantitatively. Minimum multiplicity of input of virus (50% transforming dose) per cell (MOI) necessary to induce maximum level transformation of cord cells ranged from 0.02 to 0.2. The frequency of initially transformed cells (fraction of transformable cells) in the cord cell samples from two different individuals was estimated to be 2.6 to 6.2%. In this system, the appearance of cells positive for EBV-associated nuclear antigen (EBNA) paralleled the growth curve of transformed cells. About 70% of the latter were EBNA-positive. In adult cell preparations from two individuals, 1.8 and 0.03%, respectively, of the cells were transformable indicating larger individual variations in sensitivity to EBV than in cord cells. The EBV susceptibility was also determined by the transforming efficiency (TE) expressed as the negative log of the virus dilution which induces transformation in 50% of cell cultures infected at an MOI of 0.2. From the TE value, a minimum MOI which induces transfromation could be calculated. Also by this test it was shown that the EBV susceptibility of adult cells was not only lower but also much more variable between individuals than that of cord cells. There was no correlation between the susceptibility of cells and the titer of anti-EBV antibody in donors' sera. In cultures of mixed cord cells and adult cells known to have low EBV susceptibility, the minimum MOI increased in proportion to the amount of adult cells.

Adult↗
PubMed1979

Lethal effect of protamine and histone on competent Bacillus subtilis cells. Inhibition of genetic transformation by protamine in sublethal concentration.

Under experimental conditions of genetic transformation, protamine and total histone were bactericidal for Bacillus subtilis cells. The abilities to cause lethality were very similar for both, either protamine or histone, with no antagonistic effects amongst these natural polycations. With both basic proteins acting simultaneously the enhancement was higher than a summation of the separate lethal effects. Sublethal concentration of protamine added at the beginning of transformation time, produced a strong inhibition of transforming efficiency. The same concentration added later than 10 min from the start of transformation had no inhibitory effect. These facts together with the absence of inhibition by simple pretreatment of DNA alone as well as the cell protection by protamine against lytic activity of lysozyme, suggest a protamine-cell surface interaction which impedes DNA uptake events.

Bacillus subtilis↗
PubMed1985

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae.

The survival of plasmid YRp12 treated in vitro with ultraviolet- or gamma-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrimidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After gamma-irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability. More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.

DNA Repair↗