Search results for "Cloning"
showing 10 items of 498 documents
Highly efficient construction of infectious viroid-derived clones
2019
[Background] Viroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence. At present, those dimers are generated by self-ligation of monomeric cDNA, a strategy that presents several disadvantages: (i) low efficiency, (ii) it is a non-oriented reaction requiring tedious screenings and (iii) additional steps are required for cloning into a binary vector for agroinfiltration or for in vitro RNA production.
Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine
2017
Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-β-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expres…
Variation in gene expression within clones of the earthworm Dendrobaena octaedra
2017
Gene expression is highly plastic, which can help organisms to both acclimate and adapt to changing environments. Possible variation in gene expression among individuals with the same genotype (among clones) is not widely considered, even though it could impact the results of studies that focus on gene expression phenotypes, for example studies using clonal lines. We examined the extent of within and between clone variation in gene expression in the earthworm Dendrobaena octaedra, which reproduces through apomictic parthenogenesis. Five microsatellite markers were developed and used to confirm that offspring are genetic clones of their parent. After that, expression of 12 genes was measured…
Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani
2003
The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA Thr and tRNA Pro genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of cae…
Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the correspondi…
1997
p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 4…
Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes
2004
Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Fatty acid hydroperoxide lyase (HPO lyase) is a cytochrome P450 acting on fatty acid hydroperoxides in many organisms. The expression of green bell pepper HPO lyase in the yeast Yarrowia lipolytica is described for the first time. HPO lyase activity from yeast extract and whole yeast cells is measured and aldehydes production from yeast extract and whole yeast cells is compared. 1200 U/L reaction medium were obtained after 96 h of culture on olive oil rich me…
Molecular markers linked to breeding system differences in segregating and natural populations of the cereal aphid Rhopalosiphum padi L.
1999
The aphid Rhopalosiphum padi shows coexistence of sexual and asexual populations, providing an opportunity to study the evolution of breeding system variation in the context of theories on the origin and maintenance of sex. However, assessments of the distribution of sexual and asexual lineages of this aphid are complicated by the difficulties in rapidly characterizing their breeding system. To facilitate this task and to gain insight into the genetic relatedness between sexual and asexual genotypes, molecular markers linked to breeding system differences were recently developed. In this study, we have successfully converted a random amplified polymorphic DNA (RAPD) marker associated with l…
Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobac…
2007
International audience; Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in …
Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.
2000
AbstractPosttranscriptional gene silencing (PTGS) in plants results from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, d…
An isoleucine-leucine substitution in chloroplastic acetyl-CoA carboxylase from green foxtail (Setaria viridis L. Beauv.) is responsible for resistan…
2002
The cDNAs encoding chloroplastic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) from three lines of Setaria viridis (L. Beauv.) resistant or sensitive to sethoxydim, and from one sethoxydim-sensitive line of Setaria italica (L. Beauv.) were cloned and sequenced. Sequence comparison revealed that a single isoleucine-leucine substitution discriminated ACCases from sensitive and resistant lines. Using near-isogenic lines of S. italica derived from interspecific hybridisation, we demonstrated that the transfer of the S. viridis mutant ACCase allele into a sethoxydim-sensitive S. italica line conferred resistance to this herbicide. We confirmed this result using allele-specific polymerase chain rea…