N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some …

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

Isolating novel phages using Streptomyces azureus, which produces antibiotic thiostrepton, as a host, and characterizing the genomes may help us to find new tools that could be used to develop antibiotics in addition to contribute to the databases of phages and specifically, Streptomyces phages. Streptomyces phages Alsaber, Omar, Attoomi, Rowa, and ZamZam were isolated using during this study. They were isolated from enriched soil and sequenced by Illumina sequencing method. They were isolated from three different geographical regions. They are siphoviridae phages that create small clear plaques with a diameter of approximately 0.5-1 mm, except for Rowa which has cloudy plaques, and they have varied sizes of their heads and tails. ZamZam was not characterized at this time. The sequencing shows that they are circular genome with 3' sticky overhang and various genomes' sizes with high percentage of GC content with the average of 66%. Alsaber was classified under sub-cluster BD3, while Omar was categorized under sub-cluster BD2. They share the same cluster of Cluster BD. Rowa was placed in Cluster BL and Attoomi is currently a singleton that does not fit into an established cluster. Alsaber yields 76 putative genes with no tRNA, Omar 81 putative genes with 1 …

Since phages use the host resources to replicate themselves after infection, the different sizes of the phage genome should influence the replication rate. We, therefore, hypothesized that the smaller genomes should burst the cell faster than the larger ones. As well, the shorter genomes would have greater burst sizes because they should replicate faster. Here, we obtained 16 phages of various genome length. All phages were isolated on Streptomyces griseus and available in our phage bank at the University of North Texas. We performed one-step growth studies for the 16 phages, as well as determined the host doubling time from its growth curve. The results show that S. griseus grown in nutrient broth has a doubling time of 5 hours and 22 minutes. This doubling time is used as a guideline for the phage growth studies. Because the filamentous nature of the host caused several difficulties during the experiment, we isolated single cells by sonication and centrifugation. After the cell number was determined by viable cell count, the cells were infected with each type of phage using a multiplicity of infection (MOI) of 0.5. The results show that phages' burst times range between 45 (±0, standard error) and 420 (±30) …

Bacteriophages are viruses that specifically infect bacteria. When a phage infects a bacterium, it attaches itself to the surface of the bacteria and injects its DNA into the intracellular space. The phage DNA hijacks the cellular machinery of the bacteria and forces it to produce phage proteins. Eventually, the bacteria cell bursts or lyses, releasing new phage. The bacteria act as a host for phage reproduction. The ability for a phage to infect multiple bacterial species is known as host range. In siphoviridae bacteriophages, host range is thought to primarily be determined by proteins at the tip of their tail fibers. These proteins act as anti-receptors to specific receptors on the surface of bacteria. In siphoviridae Gram-positive infecting phages, the genes that code these proteins are typically located between the tape measure protein gene and the endolysin gene. It is hypothesized that phages that have similar anti-receptor proteins will have similar host range. In this study, the host ranges of 12 BD1 bacteriophages were tested on 9 different Streptomyces species. In these 12 phages, the genes between the tape measure protein gene and endolysin gene were compared. The 12 phages had high levels of variability in these genes. Five genes …

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is one of a group of bacteria known as cyanotrophs that exhibit the unique ability to grow on toxic cyanide as the sole nitrogen source. This ability has previously been genetically linked to a conserved cluster of seven genes (Nit1C), the signature gene (nitC) coding for a nitrilase enzyme. Nitrilases convert nitriles to ammonia and a carboxylic acid. Still, for the Pf11764 NitC enzyme (Nit11764), no in vivo substrate has been identified, and the basis of the enzyme's requirement for cyanide growth has remained unclear. Therefore, the gene was cloned and the enzyme was characterized with respect to its structure and function. These efforts resulted in the unique discovery that, aside from its nitrilase activity, Nit11764 exhibits nuclease activity towards both DNA and RNA. This ability is consistent with computer analysis of the protein providing evidence of a preponderance of amino acids with a high probability for RNA binding. A Nit11764 knock-out mutant was shown to exhibit a higher sensitivity to both cyanide (KCN) and mitomycin C, both known to induce chromosomal damage. Thus, the overall conclusion is that Nit11764, and likely the entire Nit1C gene cluster, functions as a possible repair mechanism for overcoming …