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PubMed1990

A rapid and efficient procedure for transformation of Saccharomyces cerevisiae intact cells with plasmid DNA.

A rapid and efficient yeast transformation procedure has been developed through investigation of factors affecting transformation efficiency. The manipulation of the entire procedure can be done within one and one half hours. High yield of transformants is obtained by: adding calf thymus DNA as carrier DNA; adding PEG4000, carrier DNA and plasmid DNA to cell suspension simultaneously; prolonging heat shock at 42 degrees C from 5 min to 25 min and spreading the transformation mixture directly onto agar plates after heat shock. The pretreatment of yeast intact cells with LiAc can be omitted in this procedure. The transformation rates of four types of plasmid DNA were as follows: pCN60: 3.5-7.2 x 10(4) (for linear pCN60/BamHI: 1.6 x 10(5)); YEp13: 1.7-2.6 x 10(4) (for linear YEp13/BamHI: 8.0 x 10(4)); RC4: 3.7 x 10(4); YIp5/StuI: 7.6 x 10(3). Seven recipient strains transformed by using this procedure all reached the yields of over 10(4) transformants per microgramme of DNA.

Animals↗
PubMed2003

[Sequence and functional analysis of the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422].

Twenty-two DnaA boxes were identified in the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422 based upon the characteristics of consensus sequences. The 21st and 22nd DnaA boxes overlapped 8 base pairs each other reversely. Compared with the oriC database of actinomycetes, similar overlapping DnaA boxes were recognized in several species of Streptomyces and Mycobacterium. These overlapping DnaA boxes were composed of the last two DnaA box (21st and 22nd) in the Streptomyces species, but the of 1st and 2nd ones in the Mycobacterium species. The consensus sequence of the overlapping DnaA box is CTGTGCACAA, one base longer than the normal DnaA box sequence presumably due to the overlapping structure. Although the DnaA boxes exist in the 189 792 bp region only, the 1 188 bp and 793 939 bp regions are also important to the DNA replication. Deletion of the 1 188 bp region may cause absolute loss of DNA replication initiation activity measured by the transformation efficiency of plasmids with truncated oriC. When the 793 939 bp region was truncated, the transformation efficiency reduced about 40%. If the oriC was cloned into a vector with partial flanking region sequences (partial dnaA and dnaN gene sequences), the transformation rate was about 4.3-fold lower than that of the construct containing the oriC region only. However, the transformants were much more similar to the host with respect to the morphology of colony and mycelium. The cis-regulatory functions of the flanking sequences, which may influence the initiation efficiency of the chromosome replication and/or the stability of replicon, are thus suggested.

Actinomycetales↗
PubMed2005

Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica.

Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications.

Basidiomycota↗
PubMed2004

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.

Agrobacterium tumefaciens↗
PubMed2006

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling.

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying beta-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2 approximately 5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T(0) transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T(1) plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T(2) plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that beta-1, 3-glucanase gene was normally expressed in the T(2) plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.

Agrobacterium tumefaciens↗
PubMed2006

Facile transformation of benzocyclobutenones into 2,3-benzodiazepines via 4pi-8pi tandem electrocyclic reactions involving net insertion of diazomethylene compounds.

Efficient transformations of benzocyclobutenones into 2,3-benzodiazepines by a formal insertion of diazomethylene compounds are described. This sequential process includes nucleophilic addition of diazomethylene anion, oxy-anion accelerated o-quinodimethane formation by an electrocyclic ring-opening reaction, and 8pi-electrocyclization in one-pot under remarkably mild conditions. Intermediary oxy-anion plays an important role for the efficient transformations.

Benzodiazepines↗
PubMed2000

At the maize/Agrobacterium interface: natural factors limiting host transformation.

BACKGROUND: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited. Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA/VirG two-component system to induce expression of the virulence genes. RESULTS: Here we show that the root exudate of Zea mays seedlings specifically inhibits virulence gene expression, determine that 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (MDIBOA), which constitutes > 98% of the organic exudate of the roots of these seedlings, is the most potent and specific inhibitor of signal perception in A. tumefaciens-mediated gene transfer yet discovered, and develop a model that is able to predict the MDIBOA concentration at any distance from the root surface. Finally, variants of A. tumefaciens resistant to MDIBOA-mediated inhibition of vir gene expression have been selected and partially characterized. CONCLUSIONS: These results suggest a strategy in which a plant may resist pathogen invasion by specifically blocking virulence gene activation and yet ensure that the 'resistance factor' does not accumulate to levels sufficient to impose toxicity and selection pressure on the pathogen. The data further establish that naturally occurring inhibitors directed against signal perception by the VirA/VirG two-component regulatory system can play an important role in host defense. Finally, selected variants resistant to specific MDIBOA inhibition may now be used to extend the transformation efficiency of maize and possibly other cereals.

Agrobacterium tumefaciens↗
PubMed1993

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library.

Autonomously-replicating, marker-less "helper" plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.

Aspergillus nidulans↗
PubMed2005

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.

Agrobacterium tumefaciens↗
PubMed2002

Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.

Agrobacterium tumefaciens↗
PubMed2006

Soybean (Glycine max) transformation using mature cotyledonary node explants.

Agrobacterium tumefaciens-mediated transformation of soybeans has been steadily improved since its development in 1988. Soybean transformation is now possible in a range of genotypes from different maturity groups using different explants as sources of regenerable cells, various selectable marker genes and selective agents, and different A. tumefaciens strains. The cotyledonary-node method has been extensively investigated and across a number of laboratories yields on average greater than 1% transformation efficiency (one Southern-positive, independent event per 100 cotyledonary-node explants). Continued improvements in the cotyledonary-node method concomitant with further increases in transformation efficiency will enhance broader adoption of this already productive transformation method for use in crop improvement and functional genomics research efforts.

Agrobacterium tumefaciens↗
PubMed1993

Transformation of Kluyveromyces lactis by Electroporation.

The physical and biological parameters involved in efficient transformation of Kluyveromyces lactis by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 10 and 10 transformants per mug of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid.

Journal Article↗
PubMed2006

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.

BACKGROUND: Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. RESULTS: We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7-14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60-70% transformation efficiency. In contrast, using 50 microg of a 12 kb plasmid we obtained a maximum of 25-30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. CONCLUSION: We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

Journal Article↗
PubMed2000

Gene transfer to Clostridium cellulolyticum ATCC 35319.

Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CCE:I, is an isoschizomer of MSP:I (HPA:II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMss1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BSU:FI methylase in vivo or MSP:I methylase in vitro. Plasmids based on the pAMss1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10(2) transformants per microg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10(5) and 10(7) transformants per microg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

Cellulose↗
PubMed2005

Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker.

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.

Antifungal Agents↗
PubMed1985

Processing of donor DNA during Haemophilus influenzae transformation: analysis using a model plasmid system.

A plasmid system was used to investigate the processing of donor DNA during transformation of competent Haemophilus influenzae. Using biochemical and genetic methods, we have determined that portion of a donor plasmid molecule that, on average, can become integrated into a homologous recipient plasmid during transformation. Our results show that (i) transformation efficiency decreases linearly with donor DNA length over the range of 11 to 3.5 kilobase pairs, (ii) transformation efficiency decreases exponentially with size for donor molecules less than 3.5 kilobase pairs in length, and (iii) 5'-end label, but not 3'-end label, can be specifically incorporated into the resident homologous region. We present a model for donor processing during entry that encompasses and explains these observations.

DNA Restriction Enzymes↗
PubMed2001

Germ-line transformation of Arabidopsis lasiocarpa.

In planta transformation methods have opened up the possibility of transforming plant species for which no regeneration protocols currently exist. In this study, the suitability of the germ-line transformation method developed for Arabidopsis thaliana was examined for four taxa in the Brassicaceae that have not been previously transformed: Arabidopsis griffithiana, Arabidopsis lasiocarpa, Arabidopsis petraea and Capsella bursa-pastoris. Numerous transformants were obtained for A. lasiocarpa. Transformation of A. lasiocarpa was confirmed at the phenotypic and molecular levels for stably transformed lines and for backcrossed lines segregating the T-DNA insert. Parameters affecting transformation efficiency of A. lasiocarpa were also explored. As with A. thaliana, sucrose and surfactant in the inoculation medium are required for high levels of transformation, although the suitable concentrations of these are different for A. lasiocarpa. Other components present in earlier versions of the inoculation medium had little effect on transformation efficiency. Vacuum infiltration (rather than simple floral dipping) led to higher rates of transformation and did not seriously affect seed production in A. lasiocarpa. Identification of species susceptible to germ-line transformation will aid in determining the factors important for applying this technology to more recalcitrant species.

Arabidopsis↗
PubMed1986

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.

Cell Membrane↗