Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

DNA replication is a fundamental process in all living organisms. It is a semi- discontinuous process in which the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously as short Okazaki fragments (OF). The initiation of DNA synthesis requires DNA polymerase α (Pol α/primase) in complex with the primase to form a complex of four subunits. Pol α/primase is the only enzyme that can perform de novo DNA synthesis on single-stranded DNA. The catalytic subunit of the primase (PRIM1) synthesizes RNA primers that are approximately nine nucleotides long. The synthesized RNA primers are then passed intramolecularly to the polymerase active site (POLA1), which is thought to be mediated by the C-terminal domain of the primase large subunit (PRIM2-C) to synthesize dNTPs of approximately 20 nucleotides. The aim of this project was to optimize the expression and purification of Pol α/primase. The insect codon optimized POLA1 was C-terminally Strep tagged and transposed into the baculovirus genome. The other subunits of Pol α/primase, POLA2, PRIM1 and PRIM2 were cloned and expressed in E. coli cells. The cell lysates from Sf9 insect cells and E. coli cells were then mixed and purified by immunoaffinity chromatography and size-exclusion chromatography. This helped us achieve a pure Pol α/primase containing all the four subunits with a good total yield. The identity of all the protein bands were verified by mass spectroscopy. Furthermore, the protein demonstrated primer extension activity on multiple primer/template substrates. We also characterized the effect of the human replication protein A (RPA) on the DNA polymerization activity of Pol α/primase.

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein-levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1- integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.

No description available.

Flavobacterium columnare, a Gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in a variety of fish hosts but is of particular significance to the catfish industry located in the southeastern United States. Columnaris infections are a leading cause of mortalities in catfish ponds, occurring alone or in conjunction with other diseases. Typical diagnostic methods for columnaris infections involve the use of selective media following the observation of gross signs of disease. A real-time quantitative PCR (qPCR) assay to estimate the quantity of bacteria present in environmental and tissue samples was developed and validated. The genetic variability seen in F. columnare makes detection of isolates from different genomovars (genetic groups) essential to an assay for diagnostic application. Isolates from catfish generally fall into one of two different genomovars, one being virulent to catfish, while the other genomovar is thought to be largely opportunistic. The qPCR assay described herein was designed specifically to detect F. columnare isolates from the two major genomovars most often associated with farm-raised catfish. The assay was shown specific to F. columnare, regardless of genomovar, and demonstrated sensitivity consistent with similar qPCR assays. In addition, the assay provides quantitative information, estimating the bacterial loads in fish tissue and the environment. Two different applications of the assay are presented: (1) Estimate bacterial burden in fish tissue following immersion challenges to identify variation in transmission rates between channel and blue x channel hybrid catfish, and (2) Estimate the environmental burden of F. columnare in catfish ponds over the course of a single calendar year. This assay will provide an invaluable tool for researchers and diagnosticians in expanding our understanding of F. columnare and how it interacts with the host and environment.

The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...

Background Lower respiratory tract infection (LRTI) is a major cause of morbidity and mortality in children under 5 years of age. Bacterial pathogens contribute significantly to this process. Culture of respiratory tract specimens is labour-intensive and slow. Polymerase chain reaction (PCR) is comparatively, a rapid, sensitive method of detecting low levels of nucleic acid for clinically relevant bacteria. This study compares the yield of bacteria obtained from culture and the FTDResp33 multiplex PCR of nasopharyngeal swabs (NPs) during LRTI episodes in children, in the Drakenstein Child Health Study. Methods At each episode of LRTI, 2 NPs were obtained, one for culture and one for PCR testing. Bacterial yields and concordance for the 5 commonest bacteria were compared using frequencies and proportions. Results From 13th August 2012 to 23rd November 2020, there were 859 episodes of LRTI in 434 children [median age 9.2 (IQR 3.8; 18.9) months; 0.2% HIV-infected]. S. pneumoniae, S. aureus, M. catarrhalis, H. influenzae and K. pneumoniae were the predominant bacteria detected by either method. Concordance between culture and PCR for S. pneumoniae, S. aureus, and K. pneumoniae was 84.9%, 89.7% and 86.3% respectively. Culture and PCR for H. influenzae had a concordance of 76.9%. The greatest discordance between culture and PCR was for the detection of M. catarrhalis (34.4%). Median bacterial loads on PCR for all 5 organisms were significantly associated with semi-quantitative culture results (p<0.001 for each). Adjusting for age and hospitalization, children on antibiotics at the time of sampling, had a reduced chance of having a positive culture (OR 0.1; 95% CI 0.1-0.4), and a reduction in PCR yield (OR 0.8; 95% CI 0.4-1.6). Conclusion: Significant concordance existed between PCR and culture for 4 of the 5 common bacteria, affirming PCR as a comparable method of testing to culture.

Indiana University-Purdue University Indianapolis (IUPUI) / During RNA Polymerase II (RNAPII) transcription, a dynamic network of protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and termination. Taking a proteomics approach to study RNAPII transcription can offer a comprehensive view of the regulatory mechanisms mediated by PPIs within the transcription complex. However, traditional affinity purification mass spectrometry (APMS) methods have struggled to quantitatively capture many of the more dynamic, less abundant interactions within the elaborate RNAPII transcription interactome. To combat this challenge, we have developed and optimized a quantitative AP-MS based method termed Disruption-Compensation (DisCo) Network Analysis that we coupled with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to investigate the PPIs that regulate RNAPII transcription. In the first study, TMT-DisCo network analysis was used to analyze how perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5 (DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network. TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII elongation complex. The observed changes in the RNAPII interactome also reveal the distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate complex processes. The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to proceed through elongation, resulting in more arrested RNAPII that requires proteasomal degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/ recycling and toward de novo expression and newly assembled RNAPII, aided by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the 26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a model of the nuclear proteasome functioning as a chaperone during transcription initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex with a hyperphosphorylated RNAPII CTD. / 2024-08-16

<p> This thesis describes the isolation and investigation of a DNA-dependent RNA polymerase from the extreme halophile Halobacterium cutirubrum.</p> <p> The enzyme system was analyzed under conditions of very high ionic strengths which are characteristic of the internal salt concentrations of extreme halophiles and at much lower ionic strengths found in conventional bacterial systems. The enzyme was found to have activity in a wide range of salt concentrations when attached to its DNA template in the form of a DNA-Membrane-Protein complex. The enzyme, however, lost the ability to function at high ionic strengths when freed from this complex.</p> <p> The properties of the isolated DNA-dependent RNA polymerase from the halophile were then compared to the properties of the same enzyme isolated from the non-halophilic bacterium, Eschericia coli. Both enzymes were found to have the same approximate molecular weights and to share the same substrate requirements. The enzymes differed, however, in their response to inhibitors specific for RNA synthesis. </p> / Thesis / Master of Science (MSc)

This study focused on isolating important wood decay fungi from two field sites located in Harrison County, MS, and Oktibbeha County, MS. Southern Yellow Pine samples of various types and treatments including: Cu8, CuOm, ACQ, PCP, proprietary organic biocide, and un-treated were collected, and fungal isolates were cultured. DNA was extracted from isolated fungal cultures and amplified by polymerase chain reaction (PCR). The internal transcribe spacer (ITS) region was sequenced, and fungal cultures were identified by comparison to sequences on GenBank using BLAST. A total of 68 fungal isolates were recovered and successfully identified from 196 samples. Thirteen basidiomycete isolates were identified, with Veluticeps fimbriata occurring most frequently. The white-rot ascomycete, Daldinia fissa was also common. Two sequence-specific oligonucleotide probes (SSOP) were designed using Lasergene® PrimerSelect software. Unsuccessful attempts were made to attach poly (dT) tails to the probes in order to cross link the probes to nylon membranes.