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PubMed2001

A convenient and rapid method for genetic transformation of E. coli with plasmids.

A convenient and rapid method for the genetic transformation of Escherichia coli with plasmids is proposed. By mixing the recipient cells and plasmid DNA and spreading them directly on selective medium plates containing Ca2+, the so-called 'plate transformation' could achieve almost the same transformation efficiency as the classical transformation method with calcium. The whole protocol takes only about 2 min, its simplicity compared favorably, not only to the usual protocol, but also to all other documented modifications.

Calcium Chloride↗
PubMed2000

Envelope-dependent transactivation by the retroviral oncoprotein v-Rel is required for efficient malignant transformation of chicken spleen cells.

The retroviral oncoprotein v-Rel is a chimeric protein that has 11 helper virus-derived Envelope (Env) amino acids (aa) at its N terminus. Within these N-terminal Env aa of v-Rel there are three aa substitutions compared to the Rev-A helper virus Env. These aa substitutions have previously been shown to impart a number of unique properties onto v-Rel, including increased transforming and transactivating ability. In this study, we have analysed the sequence requirements for the Env aa to influence several properties of v-Rel. Phe residues at aa 3 and 9 are critical for an N-terminal transactivation function of v-Rel, and the analysis of several Env mutants demonstrates that transactivation ability parallels the transforming ability of v-Rel. Substitutions of conservative aa, such as leucine and tyrosine, for Phe 3 and 9 are tolerated for transactivation in chicken embryo fibroblasts and for transformation of chicken spleen cells. In contrast, the substitution of 10 Phe residues at the N terminus of v-Rel does not enable transactivation, indicating that a distinct structure surrounding Phe-3 and Phe-9 is essential for v-Rel function. We also show that the addition of the v-Rel Env aa to the N terminus of human c-Rel can enable it to activate transcription. Taken together, these results indicate that Phe residues at positions 3 and 9 have been selected for their ability to enhance the oncogenicity of v-Rel by increasing its ability to activate transcription.

Amino Acid Substitution↗
PubMed1985

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.

Adenoviruses, Human↗
PubMed2000

High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus.

In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacterium-sour orange (Citrus aurantium L.) interactions, such as culture medium, explant source and culture conditions, were studied to assess competence for transformation in such callus and to improve transformation frequency. Cell divisions and redifferentiation from the transgenic cells leading to transformation events were stimulated more by a combination of benzylaminopurine (BAP) + naphthalene-acetic acid (NAA) in the regeneration-selection medium than by BAP alone. Both age and source of the sour orange plant material affected transformation frequency. Explants from 4-month-old seedlings grown in the greenhouse showed higher transformation frequency than younger and older plant materials, indicating that they had a more suitable balance between dedifferentiated cells competent for transformation and Agrobacterium virulence. Enhancement of transformation frequency enabled us to incorporate the coat protein gene of citrus tristeza virus (CTV) in a sufficient number of sour orange plants to be able to evaluate this strategy for producing CTV-resistant plants.

Journal Article↗
PubMed1988

Highly efficient genetic transformation of Bacillus subtilis attached to sand grains.

Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 degrees C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml-1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 microgram ml-1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.

Adsorption↗
PubMed1989

Genetic transformation in encapsulated clinical isolates of Haemophilus influenzae type b.

Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x 10(-4) to 1 x 10(-6). The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.

Cell Membrane↗
PubMed2003

Automatic particle detection through efficient Hough transforms.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when a very large number of images are required to achieve three-dimensional reconstructions at near atomic resolution. Investigation of fast, accurate approaches for automatic particle detection has become one of the current challenges in the cryoEM community. At the same time, the investigation is hampered by the fact that few benchmark particles or image datasets exist in the community. The unavailability of such data makes it difficult to evaluate newly developed algorithms and to leverage expertise from other disciplines. The paper presents our recent contribution to this effort. It also describes our newly developed computational framework for particle detection, through the application of edge detection and a sequence of ordered Hough transforms. Experimental results using keyhole limpet hemocyanin (KLH) as a model particle are very promising. In addition, it introduces a newly established web site, designed to support the investigation of automatic particle detection by providing an annotated image dataset of KLH available to the general scientific community.

Algorithms↗
PubMed1999

Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus.

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.

Aspartate Carbamoyltransferase↗
PubMed2000

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria.

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10(3) transformants microg of DNA(-1)) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.

Animals↗
PubMed2002

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.

Bacteriophages↗
PubMed1996

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1.

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3 By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.

Amino Acid Sequence↗
PubMed2003

All-fiber spot-size transformer for efficient free-space optical interconnecting devices.

A new concatenated configuration of optical fibers is proposed to improve misalignment tolerance and working distance in a free-space optical interconnection. The structure is composed of three segments: a thermally expanded core fiber, a coreless silica fiber, and a claddingless graded-index fiber. A numerical analysis for the design of each fiber segment is performed completely, and the device characteristics are experimentally evaluated in terms of the tolerances for a 1-dB coupling-loss increment against longitudinal, transverse, and angular misalignments.

Journal Article↗
PubMed1989

Resistance to neoplastic transformation induced by nonterminal differentiation.

Cell clones derived from undifferentiated 3T3 T mesenchymal stem cells show a low rate of spontaneous transformation but can be efficiently transformed by a variety of carcinogens. In contrast, it is now reported that cell clones derived from nonterminally differentiated 3T3 T stem cells are highly resistant to neoplastic transformation induced by physical and chemical carcinogens. Differentiation-induced resistance to neoplastic transformation is evident in both in vitro and in vivo transformation assays and can be stably expressed for greater than 50 population doublings. These results establish that resistance to neoplastic transformation can be regulated in mammalian stem cells by the process of nonterminal differentiation.

Animals↗
PubMedDate not supplied

Dominant selection system for use in Cryptococcus neoformans.

Present transformation systems for Cryptococcus neoformans depend on complementation of auxotrophic mutants. We have developed a dominant selection system for transformation of wild-type strains of cryptococci in which resistance to the antibiotic hygromycin B is used as the selectable marker. A heterologous fusion gene construct was created by attaching the putative promoter sequence and start site from a cryptococcal actin gene to a truncated hygromycin B phosphotransferase gene from E. coli. Biolistic transformation with this construct resulted in cryptococci resistant to hygromycin B, and transformation efficiencies approached approximately 500 transformants per microgram DNA. The construct was found to exist in transformants as both extrachromosomal and integrative forms. The transformants with integrated constructs were stable both in vitro and in vivo, and constructs were recoverable from most transformed cells using a plasmid rescue technique. This is the first dominant selection system for use in C. neoformans, and it should prove useful for molecular studies with this important pathogenic yeast.

Actins↗
PubMed2025

A genotype-independent and highly efficient Agrobacterium-mediated soybean genetic transformation system.

A stable and efficient transformation system is crucial for functional genomics and trait improvement in soybean. This study developed a tissue culture based genetic transformation system incorporating dual selection (Spectinomycin and RUBY). This system significantly enhances transformation efficiency, shortens the transformation cycle, and demonstrates broad genotype independence, providing a powerful tool for soybean research and breeding.

Glycine max↗
PubMed1989

Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation.

Epstein-Barr virus (EBV) efficiently transforms B lymphocytes to perpetual proliferation. The EBV laboratory strain P3HR-1 is transformation-incompetent and lacks a DNA segment that includes the EBV nuclear antigen 2 (EBNA-2) gene and a portion of the EBNA leader protein (EBNA-LP) gene. These two genes are expressed in transformed B lymphocytes. Recombinant transformation-competent EBVs were produced by transfecting P3HR-1-infected cells with a cosmid containing the DNA deleted in P3HR-1. Deletion of 105 nucleotides from the middle of the EBNA-2 gene had no discernible affect on transformation. Two larger EBNA-2 deletions abolished transformation but did not affect EBNA-2 nuclear localization. Two naturally occurring EBV variants (EBV types 1 and 2) differ extensively in their growth-transformation phenotype and in their EBNA-LP, EBNA-2, and EBNA-3A, -3B, and -3C genes. Recombinant P3HR-1 carrying EBV-1 EBNA-2 has many of the EBV-1 in vitro growth-transforming effects; recombinant P3HR-1, isogenic except for EBV-2 EBNA-2, has many of the EBV-2 growth-transforming effects including slow emergence of transformants, growth in tight clumps with few surrounding viable cells, and early sensitivity to dilution with fresh medium. Thus, EBNA-2 is an essential molecule in lymphocyte growth transformation by EBV and a major determinant of the differences between EBV-1 and EBV-2 in lymphocyte growth transformation.

Antigens, Viral↗