Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex.

Specific Binding of Alzheimer's Aβ Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aβ42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aβ42 is detectable in the plasma, a phenomenon thought to result from Aβ becoming more aggregated in the brain and less Aβ42 and Aβ40 being transported from the brain to the plasma via the CSF.

Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG) repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex.

Z-Form Adoption of Nucleic Acid is a Multi-Step Process Which Proceeds through a Melted Intermediate.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of innate immune response proteins. Zα domains stabilize this higher-energy conformation by making specific interactions with the unique geometry of Z-DNA/Z-RNA. However, the mechanism by which a right-handed helix contorts to become left-handed in the presence of proteins, including the intermediate steps involved, is poorly understood.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components.

Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.

Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts.

Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis.

Riboswitches are a class of RNA motifs in the untranslated regions of bacterial messenger RNAs (mRNAs) that can adopt different conformations to regulate gene expression. The binding of specific small molecule or ion ligands, or other RNAs, influences the conformation the riboswitch adopts. Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) offers an approach for probing this structural isomerization, or conformational switching, at the level of single mRNA molecules.

Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain.

The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11, and this complex is essential to ribosome function. Previous cocrystal structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the structure. Here, we report a new crystal structure of the free GAC that is essentially identical to the L11-bound structure, which retains many common sites of divalent ion occupation.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg, Ca, or Sr together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure.

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding.

The tertiary structure of the GTPase center (GAC) of 23S ribosomal RNA (rRNA) as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking and by a triloop that forms within its three-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown to require Mg ions in equilibrium experiments.

Formation of Tertiary Interactions during rRNA GTPase Center Folding.

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg(2+) ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg(2+)-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals.

A PCR-Based Technique for Detection of Neotyphodium Endophytes in Diverse Accessions of Tall Fescue.

A previously described polymerase chain reaction (PCR)-based method used for detection of Neotyphodium coenophialum in tall fescue detected Neotyphodium endophytes in some, but not all, infected plants from a geographically diverse sample. In the study reported here, a different set of primers, based on intervening sequences of the tubulin 2 gene, were prepared and used for PCR. PCR with these primers yielded the expected 444 base pair amplification product with DNA from 104 of the 106 infected accessions tested.

First Report of Choke, Caused by Epichloe typhina, on Orchardgrass in Oregon.

During July 1997, Epichloe typhina (Pers.:Fr.) Tul.

Improved extraction and HPLC methods for ergovaline from plant material and rumen fluid.

Ergovaline, the main ergopeptine alkaloid produced in tall fescue infected with Acremonium coenophialum, is known to cause tall fescue toxicosis. Current methods in use for quantifying ergovaline in plant material have several disadvantages, including large solvent volumes and long analysis time. We report here improvements in extraction and cleanup and the high-pressure liquid chromatographic methods.

Carbonic anhydrase II deficiency syndrome in a Belgian family is caused by a point mutation at an invariant histidine residue (107 His----Tyr): complete structure of the normal human CA II gene.

Carbonic anhydrase II (CA II), which has the highest turnover number and widest tissue distribution of any of the seven CA isozymes known in humans, is absent from the red blood cells and probably from other tissues of patients with CA II deficiency syndrome. We have sequenced the CA II gene in a patient from a consanguinous marriage in a Belgian family and identified the mutation that is probably the cause of the CA II deficiency in that family. The change is a C-to-T transition which results in the substitution of Tyr (TAT) for His (CAT) at position 107.

Effects of human carbonic anhydrase III (CA III) on synovial and muscle fibroblast glycosaminoglycan metabolism.

We investigated the ability of CA III, isolated from adult human skeletal muscle, to regulate cell growth and glycosaminoglycan (GAG) formation in connective tissue cells derived from various human tissues. Unlike muscle, dermal, and cartilage fibroblasts, synovial connective tissue cells were substantially activated by CA III and showed enhanced hyaluronic acid (HA) synthesis. Cell culture experiments showed that CA III induced a 2- to 11-fold increase in [14C]HA synthesis by human synovial fibroblasts (SF) in a dose-dependent manner (P less than 0.

A widespread silent polymorphism of human carbonic anhydrase III (31 Ile in equilibrium Val): implications for evolutionary genetics.

During amino acid sequence studies of carbonic anhydrase (CA) III, purified from a pool of human skeletal muscles, and electrophoretically undetectable (silent) variation was found at residue 31 which was either valine and/or isoleucine. To distinguish a simple allelic polymorphism from more complex models involving gene duplication, 11 separate CA III samples were purified from individuals of different age and racial backgrounds. Peptide mapping by high performance liquid chromatography and sequencing indicated that four were homozygous for 31-Val, three homozygous for 31-Ile and four were apparent heterozygotes.

Fungi isolated from flue-cured tobacco sold in Southeast United States, 1968-1970.

Flue-cured tobacco leaves, from low- and middle-stalk positions, offered for sale in each of two markets, within each of five tobacco types, were evaluated for moisture content (MC) and filamentous fungi during August through October in 1968, 1969, and 1970. Alternaria alternata, Penicillium cyclopium, Aspergillus niger, Aspergillus repens, and Aspergillus flavus were most frequently isolated from cultured tissue. Other filamentous fungi that grew from the tissue included species from four genera of field fungi and seven species of storage fungi.

Antifungal spectra of actinomycetes isolated from tobacco.

Five species (28 strains) of actinomycetes isolated from tobacco were tested for antagonism against 12 species of storage and field fungi associated with tobacco. Two strains of Streptomyces albus were antagonistic against all test fungi. The actinomycetes grew more rapidly, produced more pigment, and had more pronounced antibiotic activity when grown at 36 C than at 28 C.