The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. majority of proteins that have been studied are derived from animals. Here, we characterize member distinct class plant in detail. A cDNA clone encoding protein Arabidopsis was isolated by SouthWestern screening. protein, termed ZAP1 (Zinc-dependent Activator Protein-1), encoded single copy gene, which expressed to similar levels root and flower, somewhat lower level stem low leaf siliques....

Summary Zinc‐finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double‐strand breaks (DSBs) at a specific locus. These DSBs may result in site‐specific mutagenesis or homologous recombination the repair site, depending on DNA pathway that is used. promising techniques genome engineering were evaluated Arabidopsis plants using Agrobacterium ‐mediated floral dip transformation. A T‐DNA containing target site ZFN pair, was shown to be active yeast,...

A method for mass production of triploid carp was developed by the cold treatment eggs after fertilization. Triploidy demonstrated cytophotometry and chromosome preparation techniques. Of haploid embryos, 100% showed morphological abnormalities died soon hatching. Haploid embryos maternal paternal origin occurring in course were distinguished using a genetic marker. Among morphologically normal larvae, frequency triploidy 100%, viability larvae juvenile fish did not differ from diploid...

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch cells was studied by transmission electron microscopy (TEM) and confocal laser scanning (CLSM) after expression targeting fluorescent protein into amyloplasts. Both techniques demonstrated the presence protrusions emanating from A-type granules-containing amyloplasts B-type these evaginations. Moreover, CLSM recordings interconnection protrusions, suggesting possible role interplastid...

Summary Previously, we showed that ZFN ‐mediated induction of double‐strand breaks ( DSB s) at the intended recombination site enhanced frequency gene targeting GT ) an artificial target locus using Agrobacterium floral dip transformation. Here, designed zinc finger nucleases for s in natural protoporphyrinogen oxidase PPO gene, which can be conveniently utilized experiments. Wild‐type Arabidopsis plants and expressing were transformed via transformation with a repair T‐ DNA incomplete...

Abstract Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into KU-dependent classical (c-NHEJ) more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving poly (ADP-ribose) polymerases (PARPs). However, in absence these factors, cells still seem able to adequately maintain genome integrity, suggesting presence other b-NHEJ...

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via

In recent years, several tools have become available for improved gene-targeting (GT) in plants. DNA breaks at specific sites activate local repair and recombination, including recombination with ectopic sequences leading to GT. Large-scale transformation the template can be avoided by pre-insertion of genome liberation sequence-specific nucleases (in planta GT procedure). Here, we tested whether release was required Plants were transformed constructs encoding a CRISPR/Cas nuclease...

DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little known about presence of orthologs other species, or how and when it was domesticated. We studied plants propose model domestication ancestral gene angiosperms.Using specific BLAST searches genomic EST libraries, we found that DAYSLEEPER-like genes (hereafter called SLEEPER genes) are unique to angiosperms. Basal angiosperms as well grasses (Poaceae)...

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, cell wall synthesis and/or of starch. We isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function RGPs. Sequence comparisons showed existence two classes RGP proteins, designated RGP1 RGP2. Glucosylation activity RGP2 from was studied. After separate expression Rgp1 Rgp2 Escherichia coli or yeast...

Summary Previously, it has been shown that a trimer of 22 bp fragment the promoter seed‐specific pea lectin gene confers high expression in seed. Here is reported this contains binding site for cloned basic domain/leucine zipper (bZIP) proteins TGA1a and Opaque‐2 (02). Gel shift assays, DNasel footprinting methylation interference assays using purified were performed to determine whether additional sites are present psl promoter. Within 469 upstream region only one other TGAla was found,...

To elucidate the molecular mechanisms responsible for seed-specific gene expression in plants, promoter of pea lectin (psl) gene, encoding an abundant seed protein, was used as a model. Leaf and nuclear proteins bound to region psl containing three overlapping TGAC-like motifs, which have been shown be binding site basic/leucine zipper proteins, including TGA1a. A trimer 22-bp promoter, coupled heterologous minimal conferred low reporter root, stem, leaf high transgenic tobacco. Expression...

Abstract The widespread occurrence of sex is one the most elusive problems in evolutionary biology. Theory predicts that asexual lineages can be driven to extinction by uncontrolled proliferation vertically transmitted transposable elements (TEs), which accumulate because inefficiency purifying selection absence and recombination. To test this prediction, we compared genome‐wide TE load between a sexual lineage parasitoid wasp Leptopilina clavipes same species rendered Wolbachia ‐induced...

In order to study the role of AtKu70 and AtKu80 in Agrobacterium -mediated transformation gene targeting, plant lines with a T-DNA insertion or genes were functionally characterized. Such lacked both subunits, indicating that heterodimer formation between is needed for stability proteins. Homozygous mutants phenotypically indistinguishable from wild-type plants fertile. However, they hypersensitive genotoxic agent bleomycin, resulting more DSBs as quantified comet assays. They had lower...

Summary Starch granule size is an important parameter for starch applications in industry. granules are formed amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery likely to be similar all plastids. Essential roles FtsZ proteins plastid division land plants have been revealed. forms the so‐called Z ring which, together with inner outer rings, brings about constriction of plastid. It has shown that modulation expression level may...

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNA-guided Cas9 endonuclease usually delivered into plant cells as DNA construct encoding and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations. In addition, constructs can integrate host genome, causing mutations complicating regulatory approval. Instead DNA, here we deliver...

Abstract Background DAYSLEEPER is a domesticated transposase that essential for development in Arabidopsis thaliana [Nature, 436:282–284, 2005]. It derived from hAT-superfamily transposon and contains many of the features found coding sequence these elements 2005, Genetics , 158:949–957, 2001]. This work sheds light on expression this gene localization its product protoplasts planta . Using deletion constructs, important domains protein were identified. Results predominantly expressed...

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from bacterium plant cells, where it randomly integrates into genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of specific genomic locus homologous recombination (HR) also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by...

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It considered effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, widely used mice, potential becoming routine many species. Nevertheless, epigenetic status targeted allele remains largely unexplored. Using GT-modified lines model plant Arabidopsis thaliana, we show that DNA methylation profile locus...