A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E.

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters.

Social defeat stress induces genome-wide 5mC and 5hmC alterations in the mouse brain.

Stress is adverse experience that require constant adaptation to reduce the emotional and physiological burden, or "allostatic load", of an individual. Despite their everyday occurrence, a subpopulation of individuals is more susceptible to stressors, while others remain resilient with unknown molecular signatures. In this study, we investigated the contribution of the DNA modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), underlying the individual differences in stress susceptibility and resilience.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis.

Sacroiliac innervation.

Purpose: To investigate the innervation pattern of the sacroiliac region, especially with regard to the sacroiliac joint (SIJ). Dorsal SIJ innervation was analyzed and described. Our main hypothesis was that nerves reach the SIJ dorsally, passing ligamental compartments, as this would explain dorsal SIJ pain.

5-hydroxymethylcytosine is dynamically regulated during forebrain organoid development and aberrantly altered in Alzheimer's disease.

5-hydroxymethylcytosine (5hmC) undergoes dynamic changes during mammalian brain development, and its dysregulation is associated with Alzheimer's disease (AD). The dynamics of 5hmC during early human brain development and how they contribute to AD pathologies remain largely unexplored. We generate 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients.

Reduction of dioxygen to water by a Co(NO) complex with a 2,2'-bipyridine backbone.

We report a Co-based complex for the reduction of O to HO utilizing decamethylferrocene as chemical reductant and acetic acid as a proton donor in methanol solution. Despite structural similarities to previously reported Co(NO) complexes capable of catalytic O reduction, this system shows selectivity for the four-electron/four-proton reduction product, HO, instead of the two-electron/two-proton reduction product, HO. Mechanistic studies show that the overall rate law is analogous to previous examples, suggesting that the key selectivity difference arises in part from increased favorability of protonation at the distal O position of the key intermediate Co(iii)-hydroperoxide, instead of the proximal one.

Electrocatalytic CO Reduction to Formate with Molecular Fe(III) Complexes Containing Pendent Proton Relays.

Previously, we reported an iron(III) complex with 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(2,4-ditertbutyl-phenol) as a ligand (Fe(dhbpy)Cl, ) as catalytically competent for the electrochemical reduction of CO to formate (Faradaic efficiency FE = 68 ± 4%). In mechanistic experiments, an essential component was found to be a pre-equilibrium reaction involving the association of the proton donor with the catalyst, which preceded proton transfer to the Fe-bound O atoms upon reduction of the Fe center. Here, we report the synthesis, structural characterization, and reactivity of two iron(III) compounds with 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(2-methoxy-4-methylphenol) (crebpy[H], Fe(crebpy)Cl, ) and 6,6'-([2,2'-bipyridine]-6,6'-diyl)bis(4-(-butyl)benzene-1,2-diol) (catbpy[H], Fe(catbpy), ) as ligands, where pendent -OMe and -OH groups are poised to modify the protonation reaction involving the Fe-bound O atoms.

Epigenetic Regulations in Neuropsychiatric Disorders.

Precise genetic and epigenetic spatiotemporal regulation of gene expression is critical for proper brain development, function and circuitry formation in the mammalian central nervous system. Neuronal differentiation processes are tightly regulated by epigenetic mechanisms including DNA methylation, histone modifications, chromatin remodelers and non-coding RNAs. Dysregulation of any of these pathways is detrimental to normal neuronal development and functions, which can result in devastating neuropsychiatric disorders, such as depression, schizophrenia and autism spectrum disorders.

The Dynamic Partnership of Polycomb and Trithorax in Brain Development and Diseases.

Epigenetic mechanisms, including DNA and histone modifications, are pivotal for normal brain development and functions by modulating spatial and temporal gene expression. Dysregulation of the epigenetic machinery can serve as a causal role in numerous brain disorders. Proper mammalian brain development and functions depend on the precise expression of neuronal-specific genes, transcription factors and epigenetic modifications.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, , Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast , Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance.

Stimulation of RNA Polymerase II ubiquitination and degradation by yeast mRNA 3'-end processing factors is a conserved DNA damage response in eukaryotes.

The quality and retrieval of genetic information is imperative to the survival and reproduction of all living cells. Ultraviolet (UV) light induces lesions that obstruct DNA access during transcription, replication, and repair. Failure to remove UV-induced lesions can abrogate gene expression and cell division, resulting in permanent DNA mutations.

DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast.

Systemic response to DNA damage and other stresses is a complex process that includes changes in the regulation and activity of nearly all stages of gene expression. One gene regulatory mechanism used by eukaryotes is selection among alternative transcript isoforms that differ in polyadenylation [poly(A)] sites, resulting in changes either to the coding sequence or to portions of the 3' UTR that govern translation, stability, and localization. To determine the extent to which this means of regulation is used in response to DNA damage, we conducted a global analysis of poly(A) site usage in Saccharomyces cerevisiae after exposure to the UV mimetic, 4-nitroquinoline 1-oxide (4NQO).

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology.

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1.

Unravelling the means to an end: RNA polymerase II transcription termination.

The pervasiveness of RNA synthesis in eukaryotes is largely the result of RNA polymerase II (Pol II)-mediated transcription, and termination of its activity is necessary to partition the genome and maintain the proper expression of neighbouring genes. Despite its ever-increasing biological significance, transcription termination remains one of the least understood processes in gene expression. However, recent mechanistic studies have revealed a striking convergence among several overlapping models of termination, including the poly(A)- and Sen1-dependent pathways, as well as new insights into the specificity of Pol II termination among its diverse gene targets.

Money, sex, and drugs: a case study to teach the genetics of antibiotic resistance.

The goal of the work reported here was to help students expand their understanding of antibiotic resistance, the Central Dogma, and evolution. We developed a unit entitled "Ciprofloxacin Resistance in Neisseria gonorrhoeae," which was constructed according to the principles of scientific teaching by a team of graduate students, science faculty, and instructors. A variety of activities and assessments were used, including a case study, short lectures, and group problem-solving.

Regulation of a eukaryotic gene by GTP-dependent start site selection and transcription attenuation.

Guanine nucleotide negatively regulates yeast inosine monophosphate dehydrogenase (IMPDH) mRNA synthesis by an unknown mechanism. IMPDH catalyzes the first dedicated step of GTP biosynthesis, and feedback control of its expression maintains the proper balance of purine nucleotides. Here we show that RNA polymerase II (Pol II) responds to GTP concentration.

Perturbative theory and modeling of electronic-resonance-enhanced coherent anti-Stokes Raman scattering spectroscopy of nitric oxide.

A theory is developed for three-laser electronic-resonance-enhanced (ERE) coherent anti-Stokes Raman scattering (CARS) spectroscopy of nitric oxide (NO). A vibrational Q-branch Raman polarization is excited in the NO molecule by the frequency difference between visible Raman pump and Stokes beams. An ultraviolet probe beam is scattered from the induced Raman polarization to produce an ultraviolet ERE-CARS signal.

Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase.

Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes.

Measurement of nitric oxide concentrations in flames by using electronic-resonance-enhanced coherent anti-Stokes Raman scattering.

We have measured nitric oxide (NO) concentrations in flames by using electronic-resonance-enhanced coherent anti-Stokes Raman spectroscopy (ERE-CARS). Visible pump and Stokes beams were tuned to a Q-branch vibrational Raman resonance of NO. A UV probe beam was tuned into resonance with specific rotational transitions in the (v"=1,v'=0) vibrational band in the A(2)Sigma(+)-X(2)Pi electronic transition, thus providing a substantial electronic-resonance enhancement of the resulting CARS signal.

Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model.

Initiation of transcription by RNA polymerase II (RNAP II) on Saccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40-120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most protein-coding genes, small nuclear RNA gene transcription starts predominantly at a single position.

High-resolution broadband N2 coherent anti-Stokes Raman spectroscopy: comparison of measurements for conventional and modeless broadband dye lasers.

We have performed high-resolution N2 coherent anti-Stokes Raman spectroscopy (CARS) measurements using a modeless dye laser (MDL) as the Stokes beam source to determine the effects of a reduction in mode noise on the accuracy and precision of the method. These results are compared with previous research that employed a conventional broadband dye laser (CBDL) as the Stokes beam source. A new spectral-fitting procedure was developed to avoid starting-point bias in the least-squares fitting results, which possibly had altered the previous measurements.