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PubMed2002

Roles of the recJ and recN genes in homologous recombination and DNA repair pathways of Neisseria gonorrhoeae.

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.

Amino Acid Sequence↗
PubMed1990

Introduction of large linear minichromosomes into Schizosaccharomyces pombe by an improved transformation procedure.

The efficiency of transformation of Schizosaccharomyces pombe has been increased 10- to 50-fold over previously reported methods. By using 1 microgram of plasmid, 7.0 x 10(5) transformants are regularly obtained. This increased transformation efficiency is mainly due to the inclusion of the cationic liposome-forming reagent Lipofectin in the protocol. Various parameters affecting transformation of Sc. pombe in the presence of Lipofectin have been examined. Lipofectin can also be used to increase transformation efficiency in Saccharomyces cerevisiae. It is also demonstrated that by using this improved transformation procedure, linear minichromosomes of greater than 500 kilobases can be introduced into Sc. pombe with relative ease. These minichromosomes can replicate as stable linear molecules upon reintroduction into Sc. pombe, demonstrating that Sc. pombe telomeres retain function when reintroduced as naked DNA. The ability of Sc. pombe to admit large DNA molecules indicates that it should be feasible to clone large DNA from other organisms in Sc. pombe.

Chromosomes, Fungal↗
PubMed1990

Optimization of delivery of foreign DNA into higher-plant chloroplasts.

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.

Bacterial Proteins↗
PubMed1987

The early enhancer-promoter of BKV and host range for transformation.

The early genes of the human papovavirus BKV and simian virus 40 (SV40) show a different host range for transformation. The early region of SV40 efficiently transforms human fibroblast cells, whereas the early region of BKV does not. Interchanging noncoding enhancer-promoter sequences around the origin of replication between BKV and SV40 showed that the early enhancer-promoter sequences of BKV and SV40 could substitute for each other, as far as the induction of expression of tumor antigens and morphological transformation is concerned; the efficiency of transformation was influenced by the enhancer-promoter sequences, and the difference in host range for transformation between BKV and SV40 was determined by the early gene products rather than by the enhancer-promoter sequences.

Animals↗
PubMed1994

Development of a transformation system for Trichoderma longibrachiatum and its use for constructing multicopy transformants for the egl1 gene.

An efficient transformation system for the fungus Trichoderma longibrachiatum has been developed. Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying the Escherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker. The transformation frequency was 0.5 to 5 transformants/micrograms plasmid DNA. Transformation normally occurred by tandem integration of the transforming DNA. A high percentage of the transformants were mitotically unstable. The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of the egl1 gene encoding a beta-(1,4)-endoglucanase were obtained. Some of them secrete increased levels of endoglucanase to the culture medium. In addition, the E. coli lacZ gene was expressed in an active form under control of the Aspergillus nidulans gpdA gene promoter.

Base Sequence↗
PubMed2003

An improved transformation protocol for the human fungal pathogen Candida albicans.

Commonly used protocols for the transformation of the dimorphic human fungal pathogen Candida albicans rely on established methods for the yeast Saccharomyces cerevisiae. With respect to transformation efficiency, however, there is a great difference between these two organisms when using the lithium acetate procedure. Here we present a modified version of this protocol for use with C. albicans. Among the different parameters tested, two turned out to be particularly relevant and, when combined, resulted in an up to 10-fold increase in transformation efficiency (400-500 integrative transformants) compared with previous protocols: first, adjusting the heat shock applied to the cells to 44 degrees C for C. albicans instead of 42 degrees C for S. cerevisiae and, second, treating C. albicans cells with lithium acetate in an overnight incubation instead of for 30 min as used for S. cerevisiae. With these modifications, the lithium acetate procedure becomes a very efficient and reliable tool for C. albicans transformation.

Acetates↗
PubMed1999

A single amino acid substitution in the v-Eyk intracellular domain results in activation of Stat3 and enhances cellular transformation.

The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3 subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher numbers of transformants in soft agar and stronger activation of Stat3; levels of Stat1 activation by the two Eyk molecules were similar. A mutation in the sequence Y933VPL, present in c-Eyk, to the v-Eyk sequence Y933VPQ led to increased activation of Stat3 and increased transformation efficiency. However, altering another sequence, Y862VNT, present in both Eyk molecules to F862VNT markedly decreased transformation without impairing Stat3 activation. These results indicate that activation of Stat3 enhances transformation efficiency and cooperates with another pathway to induce transformation.

Amino Acid Sequence↗
PubMed1996

Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum.

Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum. The efficiency of transformation of P. chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. The AMA1 region works as an origin of replication in P. chrysogenum and A. nidulans but not apparently in Acremonium chrysogenum.

Base Sequence↗
PubMed2005

The contourlet transform: an efficient directional multiresolution image representation.

The limitations of commonly used separable extensions of one-dimensional transforms, such as the Fourier and wavelet transforms, in capturing the geometry of image edges are well known. In this paper, we pursue a "true" two-dimensional transform that can capture the intrinsic geometrical structure that is key in visual information. The main challenge in exploring geometry in images comes from the discrete nature of the data. Thus, unlike other approaches, such as curvelets, that first develop a transform in the continuous domain and then discretize for sampled data, our approach starts with a discrete-domain construction and then studies its convergence to an expansion in the continuous domain. Specifically, we construct a discrete-domain multiresolution and multidirection expansion using nonseparable filter banks, in much the same way that wavelets were derived from filter banks. This construction results in a flexible multiresolution, local, and directional image expansion using contour segments, and, thus, it is named the contourlet transform. The discrete contourlet transform has a fast iterated filter bank algorithm that requires an order N operations for N-pixel images. Furthermore, we establish a precise link between the developed filter bank and the associated continuous-domain contourlet expansion via a directional multiresolution analysis framework. We show that with parabolic scaling and sufficient directional vanishing moments, contourlets achieve the optimal approximation rate for piecewise smooth functions with discontinuities along twice continuously differentiable curves. Finally, we show some numerical experiments demonstrating the potential of contourlets in several image processing applications. Index Terms-Contourlets, contours, filter banks, geometric image processing, multidirection, multiresolution, sparse representation, wavelets.

Algorithms↗
PubMed2007

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

Base Sequence↗
PubMed2000

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Journal Article↗
PubMed2006

Development of transformation system of rice based on binary bacterial artificial chromosome (BIBAC) vector.

An Agrobacterium-mediated transformation protocol using binary bacterial artificial chromosome (BIBAC) vector system in rice (Oryza sativa L.) was developed. Calli derived from mature embryos of japonica rice cv. H1493 were used as target tissues. Various aspects in transformation and regeneration processes including callus induction and culture, Agrobacterium concentration and duration of co-cultivation, bacterial elimination and transformant selection were examined in order to improve the transformation efficiency. An optimized transformation conditions was established including: using an Agrobacterium strain, LBA4404(HP4404), which carries a super-virulent helper plasmid pCH32, for the infection; a modified N6 medium system for callus induction and culture; pH 5.6 for media in pre-cultivation and co-cultivation; Agrobacterium concentration at OD600 = 1.0 for 3 days co-cultivation and 7 days for a resting period of the infected calli. Based on PCR and Southern blot analysis, it was demonstrated that insert DNA and marker genes carried by BIBAC2 were integrated into the rice genome.

Chromosomes, Artificial, Bacterial↗
PubMed2003

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.

Animals↗
PubMed1997

Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA.

Evidence is presented that linearized plasmid DNA is capable of electrotransforming Lactobacillus plantarum at a frequency 500-fold lower than with the covalently closed circular molecule. When the linearized plasmid was religated prior to transformation the transformation efficiency was < 10-fold higher, suggesting that open circular plasmid was only slightly more efficient in the transformation of Lact. plantarum than linear DNA. This observation has implications for direct cloning into this species since the high background transformation frequency produced by the linear DNA could potentially obscure the recovery of clones. Nevertheless, using positive selection for enhanced chloramphenicol resistance, cloned fragments of Lact. helveticus DNA were obtained using the shuttle vector pGKV110.

Lactobacillus↗
PubMed2001

Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA.

Analysis of the nucleotide sequence downstream from the Xanthomonas oryzae pv. oryzae recA gene reveals two orfs designated orfX and recX. The former has the potential to code for a 5.6 kDa protein of unknown function while the latter encodes for a putative 14.6 kDa protein with homology to RecX from various bacteria. Northern blot analysis and RT-PCR results show that recA-orfX-recX are co-regulated and arranged in an operon. A recX mutant was constructed. The mutant has no obvious growth defects or stress response defects, except that it cannot support high-level expression of recA from an expression vector. Introduction of the plasmid containing recA into the recX mutant resulted in reduced transformation efficiency and all transformants tested had mutations with reduced RecA levels. Moreover, the recX mutant has reduced basal levels of RecA. This has not been observed in other bacteria. When inactivated recX was complemented in trans, both changes were reversed. recX mutation has no effect on the regulation of the recA promoter, suggesting that its effect on the RecA level could be post-transcriptional.

Bacterial Proteins↗
PubMed1976

Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation.

Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a,b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterspecific transfection was at least 100 times lower than homospecific transfection in both r+ and r- recipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.

Bacteriophages↗
PubMed2026

Genome Editing in Solanaceae: Harnessing CRISPR-Cas Technology for Precision Crop Improvement.

Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience.

CRISPR/Cas↗
PubMed1987

Amino-acid substitution at codon 13 of the N-ras oncogene in rectal cancer in a Japanese patient.

The activation of proto-oncogenes in colorectal cancers in Japanese patients was studied using a mouse NIH3T3 cell transfection assay system. Of thirty-five colorectal cancers examined, one rectal cancer showed an unusually high transformation efficiency and, in this rectal cancer, the N-ras oncogene was found to be activated. Nucleotide sequence analysis of the activated N-ras showed a single G----C point mutation at the first letter of codon 13, resulting in the coding of arginine instead of glycine. This amino-acid substitution at codon 13 may be responsible for the efficient induction of transformants of NIH3T3 cells in vitro.

Animals↗