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Homology-facilitated plasmid transfer in Haemophilus influenzae.

The 8 kbp plasmid pAT4 transformed Haemophilus influenzae Rd cells at low frequencies. Transformation was increased up to 100 times, however, when the recipient cells carried a DNA segment in either their chromosome or in a resident plasmid that was homologous to at least part of plasmid pAT4. Linearized plasmid DNA molecules did not transform cells without DNA homology; they efficiently transformed homology recipients, but only when the cuts had been made in the region of shared homology. In most cases examined the circular donor plasmid had been reconstituted from the transforming DNA; in some cases the reconstituted plasmid carried a mutation initially present in the recipient chromosome, provided the transforming plasmid had been linearized in the region of shared homology. Plasmid reconstitution was not observed in recA1 cells. We conclude that homology-facilitated plasmid transformation (transfer) is similar to that reported for Bacillus subtilis and Streptococcus pneumoniae.

DNA Restriction Enzymes↗

Evidence that a cytoplasmically located version of a v-erbB-encoded protein can transform both fibroblasts and erythroblasts.

We previously isolated an avian erythroblastosis virus, AEV-GEE35, in which the complete extracellular and transmembrane domains of the v-erbB oncoprotein were replaced with sequences from the gag and env proteins. The GEE35 virus was capable of transforming both fibroblasts and erythroblasts as efficiently as wild-type v-erbB. Analysis of the v-erbB proteins encoded by GEE35 revealed two proteins of similar molecular weights of approximately 130,000 Da. One of these proteins was an N-linked glycosylated membrane protein, whereas the other was a cytoplasmic protein. Biochemical characterization of these two proteins revealed that the transmembrane protein has the v-erbB domain outside the cell, such that it no longer had access to its tyrosine kinase substrates. This implies that it is the cytoplasmically located v-erbB-encoded protein that is responsible for the efficient transforming ability of this virus.

Alpharetrovirus↗

Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines.

Lactobacillus plantarum NC13, a strain derived from the L. plantarum strain L137 isolated from a traditional fermented food produced in the Philippines, contains 9 of the 15 plasmids in the parental strain. To construct a shuttle vector between L. plantarum and Escherichia coli for genetic manipulation of L137 and its derivatives, recombinant plasmids were prepared by using the 9-plasmid DNA mixture and an E. coli vector, pBluescript II SK+. The resultant recombinant plasmids were re-transferred to L. plantarum NCL21, an NC13-derived strain cured of 3 of the 9 plasmids, and 3 recombinant plasmids were obtained. The smallest plasmid, pRN14, contained a small cryptic plasmid, pLTK2, which is one of the plasmids in L. plantarum L137. Thus, the complete nucleotide sequence of pLTK2 was determined. The pLTK2 is 2295 bp in length, and has a major open reading frame of 951 bp. An encoded sequence of 317-amino acids showed extensive similarity with genes encoding replication protein (repA). A putative replication origin in pLTK2 also showed high homology to those of other gram-positive bacterial plasmids that replicate by the rolling circle mechanism. The shuttle vector pRN14 contained the erythromycin resistance gene and the ColE1 and pLTK2 replication origins. Transformation of L. plantarum strains with pRN14 by electroporation was optimized to give a transformation efficiency of 2 x 10(4) transformants/ mug plasmid. Plasmid pRN14 was stably maintained in strain NCL21, as well as in L. casei K95-5.

Journal Article↗

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers.

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.

Biolistics↗

Generalized Haar DWT and transformations between decision trees and neural networks.

The core contribution of this paper is a three-fold improvement of the Haar discrete wavelet transform (DWT). It is modified to efficiently transform a multiclass- (rather than numerical-) valued function over a multidimensional (rather than low dimensional) domain, or transform a multiclass-valued decision tree into another useful representation. We prove that this multidimensional, multiclass DWT uses dynamic programming to minimize (within its framework) the number of nontrivial wavelet coefficients needed to summarize a training set or decision tree. It is a spatially localized algorithm that takes linear time in the number of training samples, after a sort. Convergence of the DWT to benchmark training sets seems to degrade with rising dimension in this test of high dimensional wavelets, which have been seen as difficult to implement. This multiclass multidimensional DWT has tightly coupled applications from learning "dyadic" decision trees directly from training data, rebalancing or converting preexisting decision trees to fixed depth boolean or threshold neural networks (in effect parallelizing the evaluation of the trees), or learning rule/exception sets represented as a new form of tree called an "E-tree", which could greatly help interpretation/visualization of a dataset.

Journal Article↗

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production.

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.

Arachidonic Acid↗

Transformation of Lactobacillus helveticus subsp. jugurti with plasmid pLHR by electroporation.

Lactobacillus helveticus subsp. jugurti was transformed with plasmid, pLHR (8.5 kilobases), by electroporation. The plasmid, pLHR, consists of a cryptic plasmid, pLJ1, from L. helveticus subsp. jugurti, the Escherichia coli vector pBR329, and the erythromycin resistance gene of pAM beta 1 from Enterococcus faecalis. Maximum transformation efficiency of 1.3 x 10(4) transformants per microgram of DNA was obtained by exposure to a pulse with an exponential decay waveform at 4 kV/cm with 25 microF capacitance. The presence of glycine in the growth medium was essential for transformation. Plasmid DNA isolated from transformants had not undergone detectable rearrangements or deletions. In addition, it was found that L. helveticus subsp. jugurti has a restriction and modification system.

Culture Media↗

Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule.

We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.

3T3 Cells↗

Rapid Agrobacterium-mediated transformation and high-efficiency regeneration of finger millet (Eleusine coracana) for crop improvement.

Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens-mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5 mg L⁻1 6-benzylaminopurine (BAP), 1.5 mg L⁻1 kinetin, 0.1 mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2 mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30-32% were achieved in PR-202 and VL-376, respectively, by optimising infection and co-cultivation conditions, including reduced MS salt strength and pre-incubation of Agrobacterium. Molecular analyses, including PCR and Southern blot hybridisation, confirmed stable T-DNA integration in independent lines, while segregation analysis of T₁ progenies demonstrated Mendelian inheritance of the transgene. In addition, CRISPR/Cas9 constructs targeting EcCKX2 were successfully introduced via Agrobacterium, demonstrating the suitability of this system for genome engineering applications. Overall, this optimised SAM-based protocol provides a rapid (45-50 days), efficient, and reproducible platform for stable genetic transformation in finger millet and establishes a strong foundation for transgenic research and future genome editing studies in this underutilized crop.

Eleusine↗

Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants.

We have developed a new Agrobacterium-mediated transformation method for the low-frequency-regenerating pear (Pyrus communis L.) cvs. Silver bell and La France. Leaf sections derived from in vitro shoots were initially used for the transformation procedure. Under optimum transformation conditions, which included culture and selection on 30 mg/l kanamycin (Km) combined with 500 mg/l sulbenicillin, a 3.2% transformation efficiency was obtained for cv. Silver bell, but no transformants of La France were obtained because of the very low regeneration frequency. Axillary shoot meristems were then examined as potential explants for La France. Selection in 5 mg/l Km and 375 mg/l carbenicillin resulted in transformed shoots being produced at an efficiency of 4.8%, and the apparent white Km-sensitive shoots were not formed during a 2-year subculture on micropropagation medium containing 50 mg/l Km. Therefore, transformations using axillary shoot meristems may be an alternative method for pear cultivars recalcitrant to regeneration from leaf sections.

Agrobacterium tumefaciens↗

Flow cytometric testing of green fluorescent protein-tagged Lactobacillus rhamnosus GG for response to defensins.

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 10(4) transformants per microg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP+ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.

Anti-Infective Agents↗

The suppression of SH3BGRL is important for v-Rel-mediated transformation.

The v-rel oncogene is the most efficient transforming member of the Rel/NF-kappaB family of transcription factors. v-Rel induces avian and mammalian lymphoid cell tumors and transforms chicken embryo fibroblasts in culture by the aberrant regulation of genes under the control of Rel/NF-kappaB proteins. Here we report that the expression of SH3BGRL, a member of the SH3BGR (SH3 domain-binding glutamic acid-rich) family of proteins, is downregulated in v-Rel-expressing fibroblasts, lymphoid cells, and splenic tumor cells. Chromatin immunoprecipitation experiments demonstrated that v-Rel binds to the sh3bgrl promoter in transformed cells. Coexpression of SH3BGRL with v-Rel in primary splenic lymphocytes reduced the number of colonies formed by 76%. Mutations in the predicted SH3-binding domain of SH3BGRL abolished the suppressive effect on v-Rel transformation and resulted in colony numbers comparable to those formed by v-Rel alone. However, mutations in the predicted EVH1-binding domain of SH3BGRL only had a modest effect on suppression of v-Rel transformation. This study provides the first example of a gene that is downregulated in v-Rel-expressing cells that also plays a role in v-Rel transformation.

Amino Acid Sequence↗

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation.

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.

Acetyltransferases↗

The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain.

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

3T3 Cells↗

Epstein-Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus.

Epstein-Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807-2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+ LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.

Animals↗

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the gram-positive methylotroph Brevibacterium methylicum by electrotransformation.

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved.

Biotechnology↗

Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation.

In this paper, the influence of various parameters on plasmid transformation by electroporation of Staphylococcus epidermidis Tü3298 was investigated. Cell growth conditions, various concentrations and forms of plasmid DNA, field strength, pulse duration and media for electroporation and regeneration were tested. In order to obtain optimal transformation efficiency, the cells were incubated for 30 min with DNA before pulsing. With the optimized procedure, other staphylococcal species such as S. aureus, S. staphylolyticus and S. carnosus were transformed with an efficiency up to 3 X 10(5) transformants per micrograms pC194 plasmid DNA.

DNA, Bacterial↗

A mutant cell line derived from NIH/3T3 cells: two oncogenes required for in vitro transformation.

EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.

Animals↗