Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.     

Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.     

Serotonin,Eating Behavior,and Fat Intake

There is an intimate relationship between nutritional intake (eating) and serotonin activity. Experimental manipulations (mainly neuropharmacological) of serotonin influence the pattern of eating behavior, subjective feelings of appetite motivation, and the response to nutritional challenges. Similarly, nutritional manipulations (food restriction, dieting, or altered nutrient supply) change the sensitivity of the serotonin network. Traditionally, serotonin has been linked to the macronutrient carbohydrate via the intermediary step of plasma amino acid ratios. However, it has also been demonstrated that 5-HT drugs will reduce energy intake and reverse body weight gain in rats exposed to weight increasing high fat diets. 5-HT drugs can also reduce food intake and block weight gain of rats on a high fat cafeteria diet. Some diet selection studies in rats indicate that the most prominent reduction of macronutrient intake is for fat. These data indicate that 5-HT activity can bring about a reduction in fat consumption. In turn, different types of dietary fat can alter brain 5-HT activity. In human studies the methodology of food choice experiments has often precluded the detection of an effect of 5-HT manipulation on fat intake. However, there is evidence that in obese and lean subjects some 5-HT drugs can readily reduce the intake of high fat foods. Data also suggest that 5-HT activation can lead to a selective avoidance of fat in the diet. These effects of 5-HT on the intake of dietary fat may involve a pre-absorptive mechanism and there is evidence that 5-HT is linked to cholecystokinin and enterostatin. These proposals have theoretical and practical implications and suggest possible strategies to intensify or advance fat-induced satiety signals.     

Diarrhea-associated biofilm formed by enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis> and aggregative <Emphasis Type="Italic">Citrobacter freundii</Emphasis>: a consortium mediated by putative F pili

Background  

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.