Vols 1 and 2 contain 57 papers in the Special Issue of ‘Plant Genomics 2009’ and cover a wide range of topics, highlighting the significant progress that has been made in recent years in our ...understanding of the genetics and genomics of plants’ growth, development, and stress responses. These studies provide valuable insights into the potential applications of genomic tools and technologies for crop improvement and sustainable agriculture, as well as fundamental questions about the evolution and function of plant genes and genomes.
Vols 1 and 2 contain 57 papers in the Special Issue of ‘Plant Genomics 2009’ and cover a wide range of topics, highlighting the significant progress that has been made in recent years in our ...understanding of the genetics and genomics of plants’ growth, development, and stress responses. These studies provide valuable insights into the potential applications of genomic tools and technologies for crop improvement and sustainable agriculture, as well as fundamental questions about the evolution and function of plant genes and genomes.
Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. ...The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 10sup.5 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaClsub.2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.
The implementation of NGS methods into clinical practice allowed researchers effectively to establish the molecular cause of a disorder in cases of a genetically heterogeneous pathology. In cases of ...several potentially causative variants, we need additional analysis that can help in choosing a proper causative variant. In the current study, we described a family case of hereditary motor and sensory neuropathy (HMSN) type 1 (Charcot–Marie–Tooth disease). DNA analysis revealed two variants in the SH3TC2 gene (c.279G>A and c.1177+5G>A), as well as a previously described variant c.449−9C>T in the MPZ gene, in a heterozygous state. This family segregation study was incomplete because of the proband’s father's unavailability. To evaluate the variants’ pathogenicity, minigene splicing assay was carried out. This study showed no effect of the MPZ variant on splicing, but the c.1177+5G>A variant in the SH3TC2 gene leads to the retention of 122 nucleotides from intron 10 in the RNA sequence, causing a frameshift and an occurrence of a premature stop codon (NP_078853.2:p.Ala393GlyfsTer2).
The construction of a genetic circuit requires the substitution and redesign of different promoters and terminators. The assembly efficiency of exogenous pathways will also decrease significantly ...when the number of regulatory elements and genes is increased. We speculated that a novel bifunctional element with promoter and terminator functions could be created via the fusion of a termination signal with a promoter sequence. In this study, the elements from a Saccharomyces cerevisiae promoter and terminator were employed to design a synthetic bifunctional element. The promoter strength of the synthetic element is apparently regulated through a spacer sequence and an upstream activating sequence (UAS) with a ~5-fold increase, and the terminator strength could be finely regulated by the efficiency element, with a ~5-fold increase. Furthermore, the use of a TATA box-like sequence resulted in the adequate execution of both functions of the TATA box and the efficiency element. By regulating the TATA box-like sequence, UAS, and spacer sequence, the strengths of the promoter-like and terminator-like bifunctional elements were optimally fine-tuned with ~8-fold and ~7-fold increases, respectively. The application of bifunctional elements in the lycopene biosynthetic pathway showed an improved pathway assembly efficiency and higher lycopene yield. The designed bifunctional elements effectively simplified pathway construction and can serve as a useful toolbox for yeast synthetic biology.
Keywords: adenine deaminase; APRT; base editing; Cas9; CRISPR; cytosine deaminase; Physcomitrella patens; Physcomitrium patens Summary CRISPR-Cas9 has proven to be highly valuable for genome editing ...in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing. CAPTION(S): Fig. S1 Schematic description of the APT reporter gene and APRT function. Fig. S2 Examples of deletions observed during BE multiplexing. Fig. S3 Examples of multiple cytosines editing or chimerism observed in some clones. Fig. S4 G418 sensitivity of ABE and CBE single and multiplex edited clones after relaxing of the antibiotic selection pressure. Fig. S5 Nature of editing using CBE or ABE for each cytosine or adenine present in the target locus. Fig. S6 Nature of editing using CBE on genes of interest for each cytosine in the target locus. Fig. S7 Sequence of two sgRNAs containing cytosines potentially target of ABE activity and nature of ABE editing using these sgRNAs. Fig. S8 Alignment of APRT sequences from different species and phenotype of the apt P. patens mutants. Fig. S9 View of the P. patens APRT 3D model with amino acids (in blue) that could be modified as single substitutions using CBE or ABE. Fig. S10 Structure of the Pp3c3_13220, Pp3c14_9040 and Pp3c17_3870 targeted genes. Fig. S11 Sequence alignment of VDE from Arabidopsis and Physcomitrella. Fig. S12 Use of the APT gene as a marker of base-editing efficiency. Table S1 List of sgRNAs expression cassettes used in this study. Table S2 Sequences of plasmids used in this study. Table S3 List of PCR primers used in this study. Table S4 Mutation rates of the CBE and ABE systems tested (2-FA direct selection). Table S5 Transfection efficiency of the CBE and ABE systems. Table S6 Mutation rates of the CBE system after preselection on G418. Table S7 Frequency of substitution for cytosines at each position of the eight sgRNAs used in this study. Table S8 Sequences and positions of possible off-target sites for sgRNA1 and sgRNA2. Table S9 List of amino acids modified in the APT gene using the CBE or ABE strategy. Table S10 Consequence of CBE editing in the different edited clones for the three genes of interest. Table S11 Sequence analysis of the APT and Pp3c3_13220 locus in adenine-resistant clones obtained after co-transfection of the ABEv#1 mutant with the CBE system and the two sgRNAs, sgRNArestor and sgRNAPp3c3. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Byline: Anouchka Guyon-Debast, Alessandro Alboresi, Zoe Terret, Florence Charlot, Floriane Berthier, Pol Vendrell-Mir, Josep M. Casacuberta, Florian Veillet, Tomas Morosinotto, Jean-Luc Gallois, Fabien Nogue
Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing ∼1,000 breast cancer samples, we confirmed this association and ...established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.
Flowers are icons in developmental studies of complex structures. The vast majority of 250,000 angiosperm plant species have flowers with a conserved organ plan bearing sepals, petals, stamens, and ...carpels in the center. The combinatorial model for the activity of the so-called ABC homeotic floral genes has guided extensive experimental studies in Arabidopsis thaliana and many other plant species. However, a mechanistic and dynamical explanation for the ABC model and prevalence among flowering plants is lacking. Here, we put forward a simple discrete model that postulates logical rules that formally summarize published ABC and non-ABC gene interaction data for Arabidopsis floral organ cell fate determination and integrates this data into a dynamic network model. This model shows that all possible initial conditions converge to few steady gene activity states that match gene expression profiles observed experimentally in primordial floral organ cells of wild-type and mutant plants. Therefore, the network proposed here provides a dynamical explanation for the ABC model and shows that precise signaling pathways are not required to restrain cell types to those found in Arabidopsis, but these are rather determined by the overall gene network dynamics. Furthermore, we performed robustness analyses that clearly show that the cell types recovered depend on the network architecture rather than on specific values of the model's gene interaction parameters. These results support the hypothesis that such a network constitutes a developmental module, and hence provide a possible explanation for the overall conservation of the ABC model and overall floral plan among angiosperms. In addition, we have been able to predict the effects of differences in network architecture between Arabidopsis and Petunia hybrida.
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