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PubMed1990

Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells.

Previous studies have demonstrated that the HPV-16 E7 gene product encodes the major transforming activity of the virus in rodent cell systems. In this study we have generated a series of point mutations affecting the region of HPV-16 E7, which shows homology to adenovirus E1a conserved domain (CD)1. In conjunction with previously described mutants in the region of E7 with similarity to E1a CD2 and SV40 LT, we have investigated three known activities of the E7 protein; transformation, association with the cellular RB protein and induction of cellular DNA synthesis. The results show that RB binding correlates with the ability of E7 to induce cellular DNA synthesis and mediate cell transformation. In addition an unidentified function of E7, which is necessary for transformation of NIH3T3 cells, but does not affect RB binding or the ability to induce cellular DNA synthesis, has also been demonstrated. This study therefore identifies two separate regions of the E7 gene necessary for transformation of established cells. One of these, in the region of E7 which shows similarity to E1a CD2 and LT, is required for RB binding and DNA synthesis. The other region important for transformation, in the N-terminus of E7, is separable from the RB binding/DNA synthesis function.

Amino Acid Sequence↗
PubMed1986

Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation.

Electroporation has recently been shown to have advantages over the commonly used method of calcium phosphate precipitation for obtaining DNA-mediated transformation of certain types of cells. Although mouse erythroleukemia cells and other cells of hematopoietic origin are not transformed at useful frequencies by calcium phosphate-DNA precipitation methods, we obtained high frequencies of transformation (approximately 10(-5)) of these cells with electroporation. Even higher transformation frequencies (approximately 10(-3)) were obtained with human fibroblasts. Another advantage of electroporation was found when analysis of Southern blots of DNA from 243 transformed erythroleukemia cell colonies indicated that, under appropriate conditions, about 79% of the transformed cells had the exogenous DNA integrated in single copies at single sites. Under conditions of higher DNA and lower cell concentrations using fibroblasts, cotransformation was obtained with two plasmids that confer HAT or G418 resistance when integrated into cellular DNA. About 23% of the transformed cells developed both types of resistance. We describe a simple, inexpensive apparatus for carrying out electroporation.

Animals↗
PubMed2006

An optimal range of transcription potency is necessary for efficient cell transformation by c-Rel to ensure optimal nuclear localization and gene-specific activation.

c-Rel is overexpressed in several B-cell lymphomas and c-rel gene overexpression can transform primary chicken lymphoid cells and induce tumors in animals. Although c-Rel is generally a stronger transcriptional activator than its viral derivative v-Rel, its oncogenic activity is significantly weaker. Among the mutations acquired during c-Rel's evolution into v-Rel are deletion of c-Rel's transactivation domain 2 (cTAD2) and mutations in cTAD1. Given the critical role of the Rel TADs in cell transformation, we investigated how mutations in c-Rel's cTAD1 and cTAD2 contribute to its oncogenicity and that of v-Rel. Mutations in cTAD2 noticeably increased c-Rel's transforming activity by promoting its nuclear localization and gene-specific transactivation, despite an overall decrease in kappaB site-dependent transactivation potency. Conversely, substitution of vTAD by cTAD1 increased v-Rel's transactivation and transforming efficiencies, whereas its substitution by the stronger cTAD2 compromised activation of mip-1beta but not irf-4 and was detrimental to cell transformation. These results suggest that the Rel TADs differentially contribute to gene-specific activation and that an optimal range of transcription potency is necessary for efficient transformation. These findings may have important implications for understanding how Rel TAD mutations can lead to a more oncogenic phenotype.

Amino Acid Sequence↗
PubMed1995

Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation.

Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation. The transformation efficiency (transformants per microgram plasmid DNA) decreased with increases of size of the DNA. However that of 2.0 x 10(3) transformants per microgram of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp. The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA). The transformation efficiency as high as 9.3 x 10(4) transformants per microgram of purified plasmid pUB110 was obtained, using a cell concentration of 7.6 x 10(10) cells/ml and DNA concentration of 4 micrograms/ml in buffer containing 0.3 M sucrose, 1 mM CaCl2, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 mus with a single squared pulse at 0 degrees C. The gene expression for antibiotic resistance after electroporation was completed within 1.5 h. The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis.

Bacillus subtilis↗
PubMed2006

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.

Animals↗
PubMed1994

Effect of different antibiotics on efficiency of transformation of bacteria by electroporation.

We noted large differences in the transformation efficiency of Escherichia coli when different methods were used for transformation. To analyze these observations, the ampicillin and tetracycline resistance plasmid, pBR322, was transformed into HB101 and DH5 alpha E. coli using calcium chloride or electroporation, and the clones containing the plasmids were selected under various conditions. Electroporation yielded around 5 x 10(7) transformants per microgram of plasmid DNA when ampicillin was used for selection, but only 5 x 10(5) transformants per microgram when tetracycline was used. A calcium-chloride method of transformation showed no differences between the two antibiotics. The loss of efficiency of electroporation in the presence of tetracycline was also seen with three tetracycline-related antibiotics and could be blocked by chelating agents. It was also overcome by a 3-h preincubation of the bacteria before exposure to antibiotic. D-Cycloserine showed a selective effect on electroporated bacteria, but Polymyxin-B, chloramphenicol or phosphonomycin did not. The mechanism of inhibition by tetracycline-related antibiotics is not known, but the results show that the recovery of electroporated bacteria may be significantly decreased if these antibiotics are used.

Ampicillin↗
PubMed2006

Efficient genetic transformation system for the ochratoxigenic fungus Aspergillus carbonarius.

Aspergillus carbonarius is a potent ochratoxin A producer that has been found in products such as grapes, coffee, spices, and cocoa. Ochratoxin A has nephrotoxic effect, and it has been classified as a possible carcinogenic substance for humans. Here we describe for the first time a transformation system for A. carbonarius, providing an important step towards its genetic manipulation. Conidia were transformed to acquire hygromycin B resistance using the AGL-1 strain of Agrobacterium tumefaciens. Genetic transformation was evaluated growing A. tumefaciens cells in induction medium containing or not acetosyringone prior to co-cultivation. The mean transforming efficiencies in IM+AS and IM-AS conditions were 62.2 and 44.5 transformants per 10(5) conidia, respectively. The hph gene was random integrated into the genome of A. carbonarius. Fungal sequences flanking the insertion site could be amplified by TAIL-PCR.

Agrobacterium tumefaciens↗
PubMed1992

Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants.

Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days. Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per microgram of plasmid DNA. A ribosomal protein gene (rpL29) conferring cycloheximide resistance also transformed conjugating cells. The transformation efficiency of the plasmid containing only the rpL29 gene was increased by insertion of an rDNA replication origin and by cotransformation and preselection with an rDNA vector. These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena.

Animals↗
PubMed1995

A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA.

We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.

Amino Acid Sequence↗
PubMed1991

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors.

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10 to 10 transformants per mug of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.

Journal Article↗
PubMedDate not supplied

Presence of mrr- and mcr-like restriction systems in coryneform bacteria.

Efficient transformation of Brevibacterium flavum MJ233C and Corynebacterium glutamicum ATCC 31831 (up to 5.0 x 10(7) transformants/microgram DNA) depends on the source of plasmid DNA. The transformation efficiencies of B. flavum MJ233C and C. glutamicum ATCC 31831 increased nearly 10(3)-fold when plasmid DNA was isolated from the recipient strain itself or from a damdcm Escherichia coli mutant, as compared with DNA passed through a modification-proficient E. coli strain. These results suggest the presence of a methyl-specific restriction system in certain strains of coryneform bacteria. In addition, electroporation conditions were optimized.

Brevibacterium↗
PubMed2001

Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification.

Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells.

Animals↗
PubMed2005

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus.

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5' portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg(-), Leu(-)), obtaining three good Arg(+) transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.

Animals↗