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PubMed1991

Physical methods for the transformation of plant cells.

Transfer and expression of foreign genes in adult plants and their progeny has been achieved by acceleration of DNA-coated particles or microinjection techniques. Cultured cells or embryoids served as targets for the introduction of marker genes that were stably expressed in the nucleus or the chloroplast. Cloned genes from the maize anthocyanin pathway were regulated in a tissue-specific manner when transferred into maize by particle acceleration. In spite of these successes, stable transformation efficiency was low due to uneven particle distribution and cell death after bombardment. Transferred genes did not always segregate in a Mendelian fashion in the succeeding generation, and additional efforts of embryo rescue or shoot grafts were needed to obtain viable progeny from original transformants. New technical advances such as the helium-driven particle gun may improve transformation rates in the future, but some problems of cell manipulation remain.

Journal Article↗
PubMed1998

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination.

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.

Alleles↗
PubMed2001

Activation of c-Abl kinase activity and transformation by a chemical inducer of dimerization.

c-Abl is a non-receptor tyrosine kinase that is activated in human leukemias by the fusion of Bcr or Tel sequences to the Abl NH(2) terminus. Although Bcr and Tel have little in common, both contain oligomerization domains. To determine whether oligomerization alone is sufficient to activate c-Abl, we have generated and characterized an Abl protein that can be activated selectively with the chemical inducer of dimerization, AP1510. Mutant Abl proteins with one (c4F1) or two (c4F2) copies of the AP1510 binding motif (FKBP) transformed NIH 3T3 cells in a ligand-dependent manner with the c4F2 protein 60-fold more potent than c4F1. Both chimeric proteins exhibited ligand-dependent dimerization in vivo, suggesting that the increased transformation efficiency of the c4F2 mutant reflects more effective dimerization rather than formation of higher order oligomers. In the absence of ligand, c4F2-expresssing fibroblasts morphologically reverted and arrested in G(1). In Ba/F3 cells, the c4F2 chimera exhibited ligand-dependent kinase activation, transformation to interleukin 3-independent growth, and relocalization of the fusion protein from nucleus to cytoplasm. These results demonstrate that dimerization alone is sufficient to activate the Abl kinase and provide a method to regulate conditionally c-Abl activity that will be useful for studying the normal physiological role of c-Abl and the mechanism of transformation and leukemogenesis.

3T3 Cells↗
PubMed2004

Agrobacterium-mediated transformation of Bangladeshi Indica rices.

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.

Agrobacterium tumefaciens↗
PubMed1992

The HIV tat gene transforms human keratinocytes.

Skin disorders are frequently seen in patients with the acquired immune deficiency syndrome (AIDS). Since many of these cutaneous manifestations are accompanied by an early onset of epidermal hyperplasia, the keratinocyte is a candidate for infection by the human immunodeficiency virus (HIV). We now report that the HIV tat gene, under the control of the viral long terminal repeat (LTR), can efficiently transform human keratinocytes in culture. Our finding suggests that this activity of the tat gene may be responsible for the epidermal hyperplasia that accompanies psoriasis and precedes the development of squamous cell and basal cell carcinomas in AIDS patients.

Animals↗
PubMed1994

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Animals↗
PubMed2005

In vitro plant regeneration and genetic transformation of Dichanthium annulatum.

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.

Acetophenones↗
PubMed1999

Development of a system for genetic manipulation of Bartonella bacilliformis.

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.

Bacteriological Techniques↗
PubMed1981

Addition, deletion, and substitution of long nonhomologous deoxyribonucleic acid segments by genetic transformation of Haemophilus influenzae.

A complete EcoRI digest of Haemophilus influenzae phage HP1 deoxyribonucleic acid (DNA) was mixed with incomplete digests of various H. influenzae R plasmids, sealed with T4 ligase, and transformed into an HP1 lysogen. Most of the chloramphenicol- and tetracycline-resistant transformants did not produce phage although they possessed all the phage genes examined. They also did not transfer antibiotic resistance by conjugation. DNA lysates from them transformed other lysogens to resistance and to loss of phage production at different but quite high frequencies (addition of long DNA segments). They themselves could be transformed efficiently to strains with a wild prophage (deletion of long DNA segments). It was concluded that lysogenic cultures had been constructed with various DNA inserts in their prophages carrying antibiotic resistance genes from the R plasmids. The site of insertion was determined by genetic crosses. DNAs with inserts that transferred with lower efficiency were more sensitive to ultraviolet radiation. This supports the view that insert transfer efficiencies reflect the sizes of the insert.

Anti-Bacterial Agents↗
PubMed2006

Chrysanthemum (Dendranthema x grandiflora).

This chapter provides reproducible methods for the transformation of Dendranthema x grandiflora 'Shuhou-no-chikara' (standard) and 'Lineker' (spray) stem internode thin cell layers (TCLs) and conventional stem internode explants using Agrobacterium strains carrying a binary vector with beta-glucoronidase (GUS) reporter and nptII selector genes. Transformation efficiencies are reported at the kanamycin selection (callus formation and plantlet rooting), GUS assay, polymerase chain reaction (PCR), and Southern analysis levels. Notes on regeneration improvement through the use of TCLs, as well as the effective use of sonication for both regeneration and generation of transformants, stimulation of Agro-infection and elimination of Agrobacterium, are included.

Agrobacterium tumefaciens↗
PubMed2005

Bleomycin stimulates targeted gene repair directed by single-stranded oligodeoxynucleotides in BHK-21 cells.

Synthetic single-stranded oligodeoxynucleotides (ssODNs) designed for single nucleotide mismatch can induce targeted gene repair (TGR) in chromosomal DNA. TGR directed by ssODNs is an ideal method for gene manipulation because it precisely exchanges single nucleotides in targeted DNA without additional modification to the genome. However, TGR efficiency is still insufficient for practical use. Here, we report that bleomycin (BLM), which breaks double-stranded DNAs, stimulates TGR directed by ssODNs in BHK-21 cells. To evaluate TGR efficiency, transformants of BHK-21 cells permanently expressing mutant EGFP genes were established. Microscopy and restriction fragment length polymorphism (RFLP) analysis clearly showed that the mutant EGFP gene was restored to a normal EGFP gene with an ssODN comple-mentary to a transcribed strand. TGR efficiency was measured by counting the number of GFP-fluorescent cells with a flow cytometer. TGR efficiency of the ssODN complementary to a transcribed strand was significantly higher than that of ssODN complementary to a non-transcribed strand, indicating that a strand bias existed. Exposure of cells to BLM stimulated TGR directed by ssODN in a dose-dependent manner, and high concentrations of BLM (750 ng/ml) abolished strand bias on TGR efficiency. These results suggest that BLM stimulates TGR directed by ssODNs in BHK-21 cells, probably due to activation of the DNA repair system.

Animals↗
PubMed1994

[Actinomycete plasmid and integrative vectors based on DNA of the temperate Phi C31 actinophage, determining limitation of lytic development of phage Phi C31, not dependent on repressor].

Bireplicon plasmids were constructed. The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome. Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance. The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S. lividans. The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S. lividans. No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA. The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants. The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids. As a result actinomycete monoreplicon plasmids were formed. The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.

Bacteriolysis↗
PubMed1992

A novel, highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis.

We have developed a gene-cloning system in Micromonospora olivasterospora, a fortimicin A (astromicin) producer. Plasmids of Micromonospora from two strains of M. olivasterospora were used for construction of the vectors. Two antibiotic-resistance genes, nmrA and nmrB, cloned from a neomycin-producing Micromonospora, were introduced into these plasmids for the selection of transformants. In a new protoplasting protocol for lysozyme-resistant bacteria, protoplasts of M. olivasterospora were found in short-time incubation with lysozyme and transformed efficiently, indicating that the method was suitable to shotgun cloning. Using this system, seven biosynthetic genes for fortimicin A were cloned. Their physical maps revealed that at least four of these genes were clustered. Analysis of a cosmid library of M. olivasterospora showed that eleven biosynthetic genes and a self-defense gene existed in a region of approx. 25 kb of DNA.

Aminoglycosides↗
PubMed2005

Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells.

Introduction of foreign genes into bacterial cells (transformation) is used for supplementing defective genes or providing additional biological functions. Transformation can be achieved using either chemical or physical methods, e.g., electroporation. Bulk electroporation offers several advantages over chemical methods, including high transformation efficiency, but its application is limited due to the high numbers of cells and plasmids needed as a result of the high death rate of cells during this process, and the difficulty in electroporating single cells. Synthetic inorganic gene nanocarriers have received limited attention in the transformation of bacterial cells. Here we present a plasmid delivery system based on water dispersible multi-walled carbon nanotubes (CNTs) that can simultaneously target the bacterial surface and deliver the plasmids into the cells via temporary nanochannels across the cell envelope. Transformation experiments performed on E. coli provide evidence for the high potential of CNTs for nanoscale cell electroporation.

Escherichia coli↗
PubMed1975

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.

Culture Media↗
PubMed1983

Long terminal repeat enhancement of v-mos transforming activity: identification of essential regions.

The transforming efficiency of recombinant DNA clones containing the Moloney sarcoma virus v-mos sequence was enhanced by introducing the Moloney sarcoma virus long terminal repeat (LTR) in either the 5' or 3' position relative to v-mos. We analyzed the polyadenylated RNA expressed in cells transformed by these recombinant DNA clones and examined the structural integrity of integrated copies of the DNA. In each case, we demonstrated the presence of v-mos containing RNA transcripts in the polyadenylated RNA and showed that these RNA transcripts are consistent with the structure of the transfected DNA. The analysis of DNA from these transformed cells showed that the relative positions of the v-mos and LTR sequences within the transfected DNA were conserved in the integrated DNA copies. These results demonstrate that a single LTR can successfully enhance the transforming activity of v-mos from either a 5' or a 3' relative position. The results from the transfection analysis of recombinant clones containing only portions of the LTR introduced 3' to v-mos demonstrate that the essential region of the LTR responsible for the enhancement of transformation is a region within the unique 3' sequences of the LTR containing the 73-base-pair tandem repeat sequence.

Animals↗
PubMed1990

Analysis of the phenotype and the restriction enzyme mapping level of mutations induced by the new mutagen glycidyl methacrylate.

Glycidyl methacrylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMA-bound pBR322 were used to transform Escherichia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, ApRTCS and ApSTcR, in the transformants and a deductive mutant ApsTcs in the nontransformants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant ApRTcS, four of seven maps were changed, some sites were shifted to other resistant gene regions, for example, sites of Bg/I, EcoRI, HindIII, HincII, etc., and there was a new recognition site for HincII (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation.

Ampicillin Resistance↗
PubMed1997

Transformation of rice mediated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

Agrobacterium tumefaciens↗