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At least 523 records · Page 29Linked to original sources
PubMed2006

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics.

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

Blotting, Southern↗
PubMed1998

Properties of tumour suppressor p53 in murine hepatocyte lines transformed by hepatitis B virus X protein.

Persistent infection by hepatitis B virus (HBV) correlates with the prevalence of hepatocellular carcinoma. It has recently been demonstrated that the complete viral genome very efficiently transforms the immortalized murine hepatocyte line FMH202 in vitro. Here it is shown that the viral transactivating protein X (HBx) is sufficient to transform FMH202 cells, albeit with lower efficiency. Clonal cell lines expressing HBx mRNA in moderate or high amounts grew in soft agar and formed tumours in nude mice. Growth efficiency in soft agar of HBx transformed cell lines was much lower than that of cell lines transformed with the complete genome, and latency of tumour induction in nude mice was significantly longer after inoculation of HBx than of HBV transformed FMH202 cell lines. A marker of complete transformation, p53, was found to be phosphorylated more strongly in HBx transfected cell lines than in controls, and a cellular kinase was found to be associated with p53 complexes from HBx transformed cell lines. p53 was of wild-type conformation and was located in the nucleus of transformed cells.

Animals↗
PubMed1978

Transforming activities of trichloroethylene and proposed industrial alternatives.

Three chlorinated hydrocarbons, proposed or already in use as industrial substitutes for the hydrocarbon trichloroethylene, were tested for in vitro transforming potential in a Fischer rat embryo cell system (F1706), which previously has been shown to be sensitive to transformation by chemical carcinogens. Trichloroethylene and the three substitutes (1, 1, 1 trichloroethane, tetrachloroethylene and methylene chloride) all were found to induce transformation, the three substitutes being equal or more efficient transforming agents.

Animals↗
PubMed1988

A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells.

We have constructed a new SV40 mutant, T147, that makes a large T antigen that is only 147 amino acids long. We show that the T147 T antigen is a phosphoprotein that is as stable as wild-type T antigen and that the SV40 viral origin binding activity of the T147 T antigen is reduced at least 100-fold relative to that of wild-type T antigen. Most importantly, we demonstrate that cloned T147 DNA transforms rat F111 cells to anchorage independence as efficiently as cloned wild-type SV40 DNA and that cloned T147 DNA also efficiently transforms C3H10T1/2 mouse cells in a focus assay.

Animals↗
PubMedDate not supplied

Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus.

Polyoma virus is a papovavirus that productively infects mouse cells. In cells of other species, such as rat cells, polyoma virus is virtually unable to replicate, and a small proportion of infected cells become stably transformed. The ability of polyoma virus to transform infected cells is determined by genes that encode the large, middle and small T antigens and which are found in the early region of the virus genome. We have inserted the transforming region of polyoma virus into a murine leukaemia virus (MLV) vector, to generate a replication-defective transforming retrovirus which for the first time allows efficient transformation of mouse cells by the polyoma virus middle T gene. During the life cycle of this recombinant virus the intervening sequence present in the original polyoma virus middle T gene was removed. The recombinant virus that we have constructed is analogous to other acutely transforming retroviruses, and demonstrates that the polyoma middle T gene is a dominant transforming oncogene.

Animals↗
PubMed2000

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Blotting, Southern↗
PubMed1994

Transformation of Bartonella bacilliformis by electroporation.

Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae. The bacterium is an intracellular parasite of human erythrocytes. To date, members of the family Bartonellaceae have not been transformed by standard chemical methods. We report the first successful transformation of a member of the Bartonellaceae family, B. bacilliformis, by the method of electroporation. The optimal conditions for electroporation of B. bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 x 10(5) was obtained. Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 micrograms/mL. Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA. Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B. bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization. Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.

Bartonella↗
PubMed1998

Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression.

We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.

Clone Cells↗
PubMed1990

C127 cells resistant to transformation by tyrosine protein kinase oncogenes.

C127 is a nontumorigenic mouse cell line widely used in in vitro transformation assays due to its normal morphological appearance and its very low levels of spontaneous transformation. We now report that C127 cells are resistant to transformation by tyrosine protein kinase oncogenes derived from growth factor receptors such as the retroviral v-fms and the human trk transforming genes. In contrast, these cells could be efficiently transformed by members of the ras oncogene family and by serine/threonine kinase oncogenes such as v-mos and v-raf. C127 cells were also found to be resistant to transformation by v-src, the prototype of a large family of tyrosine protein kinase oncogenes whose products are associated with the inner side of the plasma membrane. However, morphologically normal C127 cells expressing pp60v-src acquired a transformed phenotype upon continuous passage in vitro. Somatic cell hybrids (neoR, hygroR) obtained by fusion of G418-resistant C127 cells expressing p70trk (neoR) and hygromycin-resistant NIH3T3 cells (hygroR) exhibited transformed properties as determined by their ability to grow in semisolid agar. In contrast, no such growth was observed when these neoR p70trk-containing C127 cells were fused to control hygroR C127 cells. These results indicate that C127 cells may either lack or express insufficient levels of certain critical substrate(s) necessary for the onset of transformation by tyrosine protein kinase oncogenes.

Animals↗
PubMed1989

The oncogenicity of jun.

The oncogenic transforming potential of the jun oncogene was investigated with viral constructs that contain various terminal deletions of v-jun, c-jun and recombinants between the viral and cellular genes. Cellular jun can induce transformation of chicken embryo fibroblasts with a low efficiency. High efficiency transformation is dependent on alterations in the major transactivator domain. A jun deletion that lacks the transactivator domain is non-transforming. These observations are compatible with the conclusion that transcriptional regulation plays an important part in jun oncogenesis. In cells transformed by viral jun and expressing high levels of viral jun expression of the cellular jun is not constitutively upregulated. Cellular jun may therefore not contribute to transformation in these cells.

Animals↗
PubMed1993

H-ras and raf-1 cooperate in transformation of NIH3T3 fibroblasts.

We examined the effect of overexpression of growth factor-regulated second messenger enzymes, alone and in combination, on transformation of NIH3T3 cells. Signal transducers included phospholipase C-gamma (PLC-gamma), protein kinase C-gamma (PKC-gamma), and two proto-oncogenes, c-H-ras and c-raf-1. Three of these proteins, PLC-gamma, PKC-gamma and Raf-1, did not transform NIH3T3 cells alone or in combination. c-H-ras, which under its own promoter control has low transforming activity, also did not cooperate with PLC-gamma or PKC-gamma. In contrast, the combination of normal or oncogenic p21 H-Ras with the Raf-1 kinase dramatically increased transformation efficiency. The level of Ras protein required for transformation was reduced in Raf-1 co-transfectants, implying that, at low levels of p21 Ras, p74 Raf-1 is rate limiting. As transformation by Ras depends on jun-mediated transcriptional events, we also examined H-ras and c-raf-1 cooperation in transcriptional transactivation of TPA-responsive element (TRE)-dependent reporters. Like the H-ras/c-raf-1 cooperation in transformation, we observed this synergistic stimulation of TRE-dependent transcription. This pathway for transformation and transcriptional activation by increased levels of normal Ras and Raf may be important in tumors that show overexpression but lack mutationally activated forms of these two proto-oncogenes.

3T3 Cells↗
PubMed2003

Dictyostelium discoideum transformation by oscillating electric field electroporation.

Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High-efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes, or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for D. discoideum, its efficiency is relatively low and its reproducibility is weak. Here we optimized the oscillating electroporation technique for D. discoideum transformation and compared it to the exponential decay electroporation. A 20-fold increase in the efficiency was resproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in D. discoideum.

Adaptation, Physiological↗
PubMed2003

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants.

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Bacillus thuringiensis Toxins↗
PubMed1987

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.

Agaricales↗
PubMed2005

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N.

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.

Agaricales↗
PubMed1989

Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers.

An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed. Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers. Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans. The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene. Co-transformation frequencies using unselected DNA varied between 50 and 65%. The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr. This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state.

Aspergillus nidulans↗
PubMed1986

Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene.

Approximately 2 kilobases (kb) of additional DNA can be packaged into wild-type virions of human adenovirus type 5 (Ad5). To extend this limit, a helper independent Ad5 cloning vector was constructed by deleting most of early region 3 (E3) from map coordinates 78.5 to 84.7 and essentially all of early region 1 (E1) from coordinates 1.0 to 10.6. E3 is nonessential for adenovirus replication in cultured cells, and E1 is nonessential when the virus is propagated in 293 cells which constitutively express the E1 gene products. The resulting new virus, dlE1,3 is about 5.5 kb shorter than wild-type Ad5 and therefore should be able to accept up to 7.5 kb in foreign DNA. To test the usefulness of this vector, the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) along with its regulatory sequences was inserted into the unique XbaI site of dlE1,3 (at map position 78.5/84.7). The resulting recombinant virus, Adtk, expressed the HSV tk at a low level (as compared with HSV-1) in infected cells; however, tk expression was markedly enhanced when Adtk-infected cells were superinfected with a tk- mutant of HSV. Furthermore, the Adtk virus efficiently transformed tk- mouse cells (line LTA) to the tk+ phenotype. At a low efficiency, it was also possible to transform tk- human cells (line 143), and tk+ transformants of both mouse and human origin have been established as permanent lines.

Adenoviruses, Human↗
PubMed1994

High-efficiency electrotransformation of the yeast Schwanniomyces occidentalis.

A method has been developed for introducing heterologous DNA rapidly and efficiently by electropermeabilization into the yeast Schwanniomyces occidentalis. A transformation efficiency as high as 2 x 10(5) transformants/microgram of plasmid DNA was obtained with a square-wave electric pulse of 2.17 kV/cm during 18 ms. Small quantities of DNA (5 ng) can be used to transform 3 x 10(8) cells. The main parameters which have been optimized are: presence of adenine in the culture medium, pretreatment of the cells with dithiothreitol during the exponential growth phase of the cells, amount of cells treated, and pulse-field strength and duration. Competent cells can be stored to allow electrotransformation whenever needed.

Adenine↗