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Jean-Christophe Breitler Eveline Dechamp Claudine Campa Leonardo Augusto Zebral Rodrigues Romain Guyot Pierre Marraccini Hervé Etienne 《Plant Cell, Tissue and Organ Culture》2018,134(3):383-394
Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps. 相似文献
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84.
Camila Simioni Vanzin Vanusa Manfredini Ana Eveline Marinho Giovana Brondani Biancini Graziela Schmitt Ribas Marion Deon Angela Terezinha de Souza Wyse Moacir Wajner Carmen Regla Vargas 《Gene》2014
High blood levels of homocysteine (Hcy) are found in patients affected by homocystinuria, a genetic disorder caused by deficiency of cystathionine β-synthase (CBS) activity, as well as in nutritional deficiencies (vitamin B12 or folate) and in abnormal renal function. We previously demonstrated that lipid and protein oxidative damage is increased and the antioxidant defenses diminished in plasma of CBS-deficient patients, indicating that oxidative stress is involved in the pathophysiology of this disease. In the present work, we extended these investigations by evaluating DNA damage through the comet assay in peripheral leukocytes from CBS-deficient patients, as well as by analyzing of the in vitro effect of Hcy on DNA damage in white blood cells. We verified that DNA damage was significantly higher in the CBS-deficient patients under treatment based on a protein-restricted diet and pyridoxine, folic acid, betaine and vitamin B12 supplementation, when compared to controls. Furthermore, the in vitro study showed a concentration-dependent effect of Hcy inducing DNA damage. Taken together, the present data indicate that DNA damage occurs in treated CBS-deficient patients, possibly due to high Hcy levels. 相似文献
85.
X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the 3T2 conformation with a tendency towards the E2 form, the substituents on C-1, C-4, and P-5 are linked bisectionally, and the acetoxyl groups on C-2 and C-3 are respectively attached axially and equatorially. Based on the X-ray crystallographic and 1H-n.m.r.-spectral data, favored conformations of P-in-ring analogs of aldopentofuranose peracetates in solution are discussed. 相似文献
86.
Monensin interferes with the determination of the mesodermal cell line in embryos of Patella vulgata
Wiel M. Kühtreiber Eveline H. van Til Cees A. M. van Dongen 《Development genes and evolution》1988,197(1):10-18
Summary In embryos of the equally cleaving marine gastropod Patella vulgata, the mesodermal stem cell is determined during the interval between the fifth and sixth cleavage by means of cellular interactions between one of the four vegetally located macromeres with the overlying animal micromeres. Shortly before and during this interaction phase an extracellular matrix (ECM) is present between the interacting cells. In this study the glycosylation-perturbing ionophore monensin was used to investigate the possible morphogenetic significance of the ECM. Incubation of 32-cell-stage Patella embryos in 10–6 M monensin results in radialized embryos in which none of the four macromeres interacts with the overlying animal micromeres. None of the macromeres is determined, therefore, to form mesoderm in such embryos. Trochophore larvae reared from these embryos retain their radial symmetry, as is indicated by the presence of four shell glands and four blastopore- or stomodeum-like invaginations in these larvae. The monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the blastomeres to stretch and interact with the micromeres. Changes in intracellular ionic concentrations may also be involved. 相似文献
87.
Yeast-like cells with a conidiogenesis of the annellidic type were obtained by culturing a strain of Fonsecaea pedrosoi, the principal agent of chromomycosis, under very acidic conditions (pH 2.5). These annellides resemble those of such pathogenic black yeasts as Exophiala jeanselmei. 相似文献
88.
Summary The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 m) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (–20°C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes. 相似文献
89.
Milan Eveline Pípolo Malheiros Estella Starling Fischman Olga Colombo Arnaldo Lopes 《Mycopathologia》1997,137(3):153-157
In the last decade the number of systemic yeast infections has increased significantly. Although Candida albicans is the most
frequently isolated yeast from clinical specimens, the emergence of non-albicans species has clearly been a recent concern.
As a consequence, there is a greater need for rapid and accurate methods for yeast identification. The aim of this study was
to evaluate the performance of the AUXACOLOR system (Sanofi Diagnostics Pasteur) for the identification of clinically relevant
yeasts, as compared with the conventional method. Yeast isolates (n = 97) belonging to 12 species were identified by the commercial
system and the classic method. Correct identifications were obtained by using AUXACOLOR system in 79.4% of the isolates tested.
Misidentification occurred in 5.2% of the strains and 15.5% were not identified due to a failure in the manufacturer's data
base. In order to improve its accuracy, there is a need for expanding the database or revamping the tests included in the
system.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
90.