Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

Introduction: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene.

Correction to: An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat.

[This corrects the article DOI: 10.1186/s13007-019-0500-2.].

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type as compared with a mutant strain that is unable to produce DON.

An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat.

Background: Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications.

Genomic Identification of the TOR Signaling Pathway as a Target of the Plant Alkaloid Antofine in the Phytopathogen Fusarium graminearum.

Antofine, a phenanthroindolizidine alkaloid, is a bioactive natural product isolated from milkweeds that exhibits numerous biological activities, including anticancer, antimicrobial, antiviral, and anti-inflammatory properties. However, the direct targets and mode of action of antofine have not been determined. In this report, we show that antofine displays antifungal properties against the phytopathogen , the cause of head blight disease (FHB).

Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria.

Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design.

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins.

A novel gene cluster in Fusarium graminearum contains a gene that contributes to butenolide synthesis.

The development of expressed sequence tag (EST) databases, directed transformation and a sequenced genome has facilitated the functional analysis of Fusarium graminearum genes. Extensive analysis of 10,397 ESTs, derived from thirteen cDNA libraries of F. graminearum grown under diverse conditions, identified a novel cluster of eight genes (gene loci fg08077-fg08084) located within a 17kb region of genomic sequence contig 1.

Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein.

We evaluated the effect of the T4 bacteriophage gene 32 protein (T4gp32) on in vitro transcription and reverse transcription. T4gp32 doubled the yield of in vitro transcripts obtained with T7 RNA polymerase and increased the yield of cDNA synthesis when used in combination with an RNaseH-deficient Moloney murine leukemia virus [Au: ok] reverse transcriptase. The positive effect could be correlated with the RNA chaperone activity of T4gp32.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system.

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination.

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum.

A novel DNase from the digestive tract of the spruce budworm (Choristoneura fumiferana) has been isolated and characterized. This DNase has two features that distinguish it from other known DNases: (1) it has a pH optimum of 10.5 to 11; (2) it plays an important role in the conversion of the insecticidal crystal protein from Bacillus thuringiensis to the active DNA-free toxin in the larval gut.

Genomic clones encoding 11S globulins in oats (Avena sativa L.).

We have isolated two complete genomic clones, Glav1 and Glav3, encoding 11S globulins (legumins) in oat. The structure of Glav1 deviates from that of the typical legumin gene. This clone possesses an extra intron and an extra exon that is composed entirely of repeats of sequences found elsewhere in the clone.

Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA.

Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA.

A 41-42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes.

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41-42 bp tandemly repeated, partially symmetrical sequence.

Deletion analysis of a zein gene promoter in transgenic tobacco plants.

A zein gene (Z4) promoter containing 886 bp upstream from the transcription start site has been shown previously to be active specifically in the endosperm of transgenic tobacco seeds. To investigate the region required for this tissue-specific activity, deletions of the Z4 promoter were constructed and placed upstream of the beta-glucuronidase (GUS) reporter gene. When these deletions were tested in transgenic tobacco plants, seed-specific GUS activity, which reached a peak between 15 and 19 DAP, was observed for promoters extending from -886 to -174.

Genetic analysis of T-DNA insertions into the tobacco genome.

A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development.

Endosperm-specific activity of a zein gene promoter in transgenic tobacco plants.

Transgenic tobacco plants containing maize gene (Z4) encoding a 23-kd zein protein, which is normally synthesized in the endosperm of maize seeds, were obtained using a modified Ti plasmid vector. Although a polyadenylated transcript homologous to the Z4 gene was present in the seeds of some of these transgenic plants, zein protein could not be detected in any of the plants tested (35 total). To simplify the analysis of the tissue specificity of the Z4 promoter (Z4(pro)) in different organs of transformed tobacco plants, additional transgenic plants containing the chimeric genes Z4(pro)-CAT and Z4(pro)-GUS were produced.