The eukaryotic Puf proteins bind 3' untranslated region (UTR) sequence elements to regulate the stability and translation of their target transcripts, such regulatory events are critical for cell growth development. Several global genome analyses have identified hundreds potential mRNA targets Saccharomyces cerevisiae proteins; however, only three these been characterized thus far. After direct testing nearly 40 candidate mRNAs, we established two as true Puf-mediated decay in yeast, HXK1...

The most recent work toward compiling a comprehensive database of adenosine‐to‐inosine RNA editing events suggests that the potential for is much more pervasive than previously thought; indeed, it manifest in 100 million located primarily within Alu repeat elements human transcriptome. Pairs inverted repeats are found substantial number genes, and when transcribed, they form long double‐stranded structures serve as optimal substrates enzymes. A small subset edited has been shown to exhibit...

RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-to-inosine editing). Commonly used methodologies quantify levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations these methods lead small number clones characterized comparison the mRNA molecules original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method...

Providing undergraduate life-science students with a course-based research experience that utilizes cutting-edge technology, is tractable for students, and manageable as an instructor challenge. Here, I describe multi-week lesson plan laboratory-based course the goal of editing genome budding yeast, <em>Saccharomyces cerevisiae</em>. Students apply knowledge regarding advanced topics such as: CRISPR/Cas9 gene editing, DNA repair, genetics, cloning. The requires to master skills...

A-to-I RNA editing is a process where adenosine (A) nucleotides are deaminated by an enzyme, ADAR1, to become inosines (I) in select transcripts. can affect the sequence of encoded protein as well regulation RNA. ADAR1p150 isoform also plays role regulating innate immunity and its expression upregulated during inflammation, while ADAR2's not influenced inflammation. ADAR1 ADAR2 have both overlapping distinct targets. We induced p150 injecting mice with lipopolysaccharide (LPS) then measured...

The sex of an animal impacts glucose sensitivity, but little information is available regarding the mechanisms causing that difference, especially during acute inflammation. We examined sex-specific differences in role P2Y 2 receptor (P2Y R) flux with and without LPS challenge. Male female wild-type R knockout mice -/- ) were injected or saline tolerance tests (GTT) performed. R, insulin receptor, GLUT4 transporter gene expression was also evaluated. Female had reduced fasting plasma females...

ABSTRACT Calcium-dependent activator protein for secretion 1 (CAPS1) facilitates the docking and priming of synaptic dense core vesicles. A conserved hairpin structure in CAPS1 pre-mRNA allows an post-transcriptional adenosine-to-inosine RNA editing event to alter a genomically-encoded glutamate glycine codon. Functional comparisons isoforms primary hippocampal neurons show that elevation edited presynaptic vesicle clustering turnover. Conversely, non-edited slow evoked release, increase...

A-to-I RNA editing is a process where adenosine (A) nucleotides are deaminated by an enzyme, ADAR1, to become inosines (I) in select transcripts. can affect the sequence of encoded protein as well regulation RNA. ADAR1 also plays role regulating innate immunity and upregulated during inflammation. Current data on effects increasing limited, most studies completed only male mice. We interested expanding include female animals. Lipopolysaccharide (LPS) was used induce inflammation increase...

CAPS1 is a priming factor, involved in the regulated release of hormones, peptides and neurotransmitters from large dense‐core secretory granules synaptic vesicles. Tran‐scripts encoding are modified by an adenosine‐to‐inosine (A‐to‐I) RNA editing event which genomically‐encoded glutamate (GAG) converted to glycine (GIG) codon. This non‐synonymous amino acid change located within essential carboxyl‐terminal domain protein mediates interaction with peptide hormone‐containing vesicles,...

Undergraduate students in an upper‐level molecular biology laboratory class used their training biochemistry, cell and to design perform CRISPR/Cas9 genome editing on yeast. After introduction DNA repair the lecture, took task experiment using Cas9 generate auxotrophic yeast mutants. They readily available bioinformatics databases retrieve sequence CRISPR‐associated sgRNA target gene of interest. cloned genes for sgRNAs into a commercially vector, then transformed it with template...

CAPS1 RNA under‐goes a site‐specific adenosine‐to‐inosine editing event that alters genomically‐encod‐ed glutamate (GAG) to glycine (GIG) codon within the encoded Calcium‐dependent activator protein for secretion 1 (CAPS1) protein. The isoforms generated from edited and non‐edited tran‐scripts have differential effects on synaptic vesicle distribution, release recycling. Increasing expression of negatively affects evoked release, leads more diffuse distribution vesicles increases spontaneous...

Nucleotide P2Y 2 receptor (P2Y R) plays important roles in inflammation. The leukocyte‐endothelium interaction is essential for leukocyte recruitment at the site of tissue damage or injury. Little known about role R regulation vivo interaction. This study tested hypothesis that mediates using cremaster muscle. Leukocytes were labeled with Rhodamine 6G injected via tail vein. Leukocyte rolling and stable adhesion analyzed a semi‐automatic tracking method. activation by UTP increased (EC 50 =...