ABSTRACT
Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.
Subject(s)
Agrobacterium tumefaciens/genetics , Arabidopsis/genetics , Genome, Plant/genetics , Arabidopsis/microbiology , DNA Breaks, Double-Stranded , DNA Repair , DNA, Bacterial/genetics , DNA, Plant/genetics , Flowers/genetics , Flowers/microbiology , Gene Knockout Techniques , Gene Transfer Techniques , Genes, Reporter , Genetic Vectors , Mutation , Plant Roots/genetics , Plant Roots/microbiology , Plant Tumors , Plants, Genetically Modified , Recombination, Genetic , Transformation, GeneticABSTRACT
The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.
Subject(s)
DNA-Binding Proteins/genetics , Nicotiana/enzymology , Nicotiana/genetics , Transposases/genetics , Base Sequence , Cell Nucleus/metabolism , Green Fluorescent Proteins/metabolism , In Vitro Techniques , Microscopy, Fluorescence/methods , Models, Genetic , Molecular Sequence Data , Plants/genetics , Plants, Genetically Modified , Protein Binding , Protoplasts/metabolism , Recombinant Proteins/genetics , TemperatureABSTRACT
New weapons to fight cancer are constantly needed. Among chemotherapeutics, anti-cancer metal-drugs have enjoyed a long and successful history since the discovery of the benchmark cisplatin. Advances in metal-drug discovery have motivated chemists to build plethora of complex structures. Among them, a novel area is emerging. This article presents a survey of the metal-N-Heterocyclic Carbenes (Ag(I), Au(I), Pd(II) and Cu(I)-NHCs) as potential anti-cancer agents. Most of the metal-NHCs considered display higher cytotoxicities than the reference metallo-drug cisplatin. Some of them are even selective for particular cell lines. Their mechanisms of action at the cellular level are further discussed, showing that the nature of the metal is of great importance. All these promising results demonstrate that this approach deserves more attention and work.
Subject(s)
Antineoplastic Agents/pharmacology , Heterocyclic Compounds/pharmacology , Methane/analogs & derivatives , Organometallic Compounds/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Heterocyclic Compounds/chemistry , Humans , Methane/chemistry , Methane/pharmacology , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistryABSTRACT
The proximal region of the high-molecular-weight glutenin promoter of the Dx5 gene (PrHMWG-Dx5) carries an atypical bifactorial endosperm box containing two cis-acting elements, namely a G-box like motif followed by a prolamin-box motif (Pb1). Transient expression assays in maize endosperm indicate that a promoter fragment containing at least the G-box like element is necessary and sufficient for maximal expression of the HMWG-Dx5 promoter. In transformed maize, we have shown that a 89 bp sequence bearing the bifactorial endosperm box behaves like a functional cis-acting unit. Its repetition in tandem confers a strong specific additive effect specifically in endosperm tissue. In contrast, the fusion of the activation sequences 1 (as-1) and 2 (as-2) of the cauliflower mosaic virus (CaMV) 35S promoter with HMWG-Dx5 derived promoter sequences deregulates its activity in transformed maize. By gel mobility shift assays we have demonstrated that the G-box like motif may alternatively bind two protein groups which have the same DNA-binding affinities as the transcription factors of either the Opaque2 (O2) family and/or the ASF-1 family.
