Environmental signals such as photoperiod and temperature provide plants with seasonal information, allowing them to time flowering to occur in favourable conditions. Most ecotypes of the model plant Arabidopsis thaliana flower earlier in long photoperiods and after prolonged exposure to cold (vernalization). The vernalized state is stable through mitosis, but is not transmitted to progeny, suggesting that the vernalization signal may be transmitted via a modification of DNA such as cytosine methylation. The role of methylation in the vernalization response is investigated in this thesis. ¶ Arabidopsis plants transformed with an antisense construct to the cytosine methyltransferase METI (AMT) showed significant decreases in methylation. AMT plants flowered significantly earlier than unvernalized wildtype plants, and the promotion of flowering correlated with the extent of demethylation. The flowering time of mutants with decreased DNA methylation (ddm1) was promoted only in growth conditions in which wildtype plants showed a vernalization response, suggesting that the early flowering response to demethylation operated specifically through the vernalization pathway. ¶ The AMT construct was crossed into two late flowering mutants that differed in vernalization responsiveness. Demethylation promoted flowering of the vernalization responsive mutant fca, but not of the fe mutant, which has only a slight vernalization response. This supports the hypothesis that demethylation is a step in the vernalization pathway. ¶ The role of gibberellic acid (GA) in the early flowering response to demethylation was investigated by observing the effect of the gai mutation, which disrupts the GA signal transduction pathway, on flowering time in plants with demethylated DNA. The presence of a single gai allele delayed flowering, suggesting that the early flowering response to demethylation requires a functional GA signal transduction pathway, and that demethylation increases GA levels or responses, directly or indirectly. ¶ In most transgenic lines, AMT-mediated demethylation did not fully substitute for vernalization. This indicates that part of the response is not affected by METI-mediated methylation, and may involve a second methyltransferase or a factor other than methylation. A second Arabidopsis methyltransferase, METIIa, was characterized and compared to METI. The two genes are very similar throughout the coding region, and share the location of their eleven introns, indicating that they diverged relatively recently. Both are transcribed in all tissues and at all developmental stages assayed, but the level of expression of METI is significantly higher than that of METIIa. The possible functions of METI, METIIa, and other Arabidopsis cytosine methyltransferase genes recently identified are discussed.

Struktūrinės genomikos tyrimams reikalingi gausiai mikroorganizmuose sintetinami rekombinantiniai baltymai dažnai būna netirpūs. Tradiciniai tirpių ir teisingos struktūros baltymų gavimo metodai – sintezė žemoje temperatūroje, skirtingo stiprumo promotoriai, tirpumą didinantys priedai, netirpaus baltymo suliejimas su tirpiu baltymu ar paraleli tikslinio baltymo ir įvairių šaperonų sintezė ne visada padeda. Šios priemonės nepakeičia baltymo vidinio gebėjimo susiklostyti į tirpią ir stabilią struktūrą, ir net pavykus tokį baltymą ištirpinti pradiniuose sintezės ar gryninimo bandymuose jis gali vėl agreguotis. Galingas tirpesnių baltymų variantų kūrimo metodas yra jų evoliucija in vitro, kurios metu iš mutantinių baltymų kolekcijos tirpesni baltymų variantai atrenkami pagal prie jų prijungto reporterinio baltymo funkciją. Baltymams būdinga didžiulė struktūros ir savybių įvairovė, todėl tokiems eksperimentams yra labai svarbu parinkti tinkamomis atrankai savybės pasižymintį reporterį. Kelių ar net keliolikos alternatyvių reporterių bei su jais susijusių atrankos sistemų lygiagretus panaudojimas baltymų evoliucijos eksperimentuose padidintų sėkmės tikimybę. Šiame darbe buvo kuriama tirpesnių baltymų atrankos sistema, kaip reporterinius baltymus naudojanti DNR metiltransferazes. Realizuojant šią užduotį eksperimentiniam patikrinimui buvo pasirinktos penkios skirtingoms klasėms priklausančios DNR metiltransferazės. Įvertinus jų tirpumą bei veiklumą atitinkami genai per lanksčią... [toliau žr. visą tekstą] / Overexpressed recombinant proteins required for structural genomics are often insoluble. Conventional approaches to obtain soluble, correctly folded protein include low-temperature expression, promoters with different strengths, fusion of the protein with a more soluble partner, coexpression of folding catalyst and chaperones. These approaches do not always work, because they fail to modify the intrinsic folding stability and solubility of the protein. Even when a fraction of the protein is obtained in a soluble form during initial expression or refolding experiments, the protein may still aggregate irreversibly during subsequent workup. In vitro evolution is powerful approach to obtain more soluble protein variants. After protein evolution in vitro more soluble proteins variants from library of mutants can be detected by fusion reporter tags. There is a vast diversity of protein structures and their properties, therefore it is very important to choose for such experiments reporter with good characteristic for genetic screen. Parallel use of few or more alternative reporters in protein evolution experiments may enhance possibility to select more soluble protein variants. In this study was created selection system of more soluble proteins, in which five DNA methyltransferases from different classes were used as fusion reporters. Solubility and activity of methyltransferases was assayed and then appropriate genes of five methyltransferases were fused via flexible linker with... [to full text]

Methylenetetrahydrofolate reductase (MTHFR) catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for the remethylation of homocysteine to methionine. A common mutation in MTHFR, 677C → T, which converts an alanine (A) to a valine (V) residue, is associated with increased plasma homocysteine and, consequently, is a risk factor for several vasculopathies. Recently, a second common mutation in MTHFR was reported (1298A → C) which converts a glutamate (E) to an alanine (A) residue in the C-terminal regulatory region. Thus study was designed to assess the in vitro and in vivo impact of this polymorphism. Site-directed mutagenesis of the human MTHFR cDNA was performed to create all four combinations of the two common MTHFR polymorphisms. Enzyme activity was assessed in a bacterial expression system. / Since the 1298A → C polymorphism is due to a mutation in the regulatory domain, a parallel study was performed involving the complete deletion of this region. / For in vivo assessment of the 1298A → C variant, we examined this mutation in three clinical populations. / Betaine-homocysteine methyltransferase (BHMT) is a second remethylation enzyme that converts betaine and homocysteine to dimethylglycine and methionine, respectively. This work represents the first report of sequence variants in the newly-cloned BHMT gene. Using SSCP analysis, three common base changes were identified in a panel of healthy controls. (Abstract shortened by UMI.)

RNA m5C methyltransferases are a group of enzymes that catalyze the transfer of a methyl group to a cytosine nucleotide of RNA. Only two of these enzymes have been well characterized: Fmu from E. coli and Trm4p from S. cerevisiae. YNLO22c is one of three ORFs identified in S. cerevisiae that have homology with both known and putative RNA m5C methyltransferases, but its encoded protein, YNLO22p, has not been confirmed to have enzyme activity. Verifying that YNLO22c encodes an RNA m5C methyltransferase will require adequate amounts of soluble YNLO22p for enzyme assays. A bacterial expression plasmid for YNLO22c was developed, but the result was insoluble protein. Therefore, several methods known to improve protein solubility were tested to develop a system in which a sufficient amount of soluble YNLO22p could be produced. Results of this study found that coexpression of YNLO22c with chaperone proteins can provide sufficient quantities of soluble YNLO22p. / Department of Biology

No description available.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xviii, 157 p.; also includes graphics (some col.). Includes bibliographical references (p. 125-157). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 144-158).

Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.

Fusobacterium are Gram-negative anaerobic bacteria that colonize a variety of eukaryotes including cattle and humans. In humans, Fusobacterium coordinates the central architecture of the oral biofilm by expressing an abundance of outer membrane adhesins that mediate bridging between early and late colonizing bacteria. While Fusobacterium are mostly considered commensal microorganisms, they can also become an opportunistic pathogen that spreads throughout the human body and promote the development of oral and extra-oral infections and diseases including colorectal cancer. Importantly for this work, many Fusobacterium species and strains are recalcitrant to genetic manipulation, the majority of which has led to hindrance in the study of their biology, molecular mechanisms, and pathogenesis. The genetic intractability of Fusobacterium is an obstacle for the development of future treatments for diseases associated with these anaerobic bacteria. Therefore, the creation of tools to enhance genome editing in target species is crucial to understand the molecular mechanisms driving Fusobacterium infections. This dissertation exploits innate and adaptive defense systems present in Fusobacterium for their use as molecular tools for genome editing. Accordingly, we first investigated restriction-modification systems with a focus on the role of DNA methyltransferases and endonucleases in host defense and genetic recalcitrance in several strains of Fusobacterium through bioinformatic and biochemical approaches. Altogether, over 15 DNA methyltransferases were characterized. Most notably, we identified and characterized two type II DNA methyltransferases that are capable of methylating plasmid DNA by treating with purified enzymes in-vitro and coexpression approaches in Escherichia coli strains, enabling an statiscally improved transformation efficiency via electroporation in F. nucleatum. Also contained in this dissertation is the first detailed description of CRISPR-Cas adaptive immunity systems present in Fusobacterium strains. Most of the discovered CRISPR-Cas systems in Fusobacterium belong to Class 1 systems. Nonetheless we identified Type II-A and Type VI-C Class 2 systems. The discovery of Cas9 and Cas13c effectors respectively from these systems will be crucial in the development of a new generation of genome-editing tools in Fusobacterium. The studies included in this dissertation provide the framework for overcoming Fusobacterium genetic recalcitrance by the implementation of host mimicking techniques. By utilizing restriction-modification system enzymes and the adaptive immunity CRISPR-Cas systems, we will gain a better understanding of how Fusobacterium modulates infections and diseases, and ultimately explore the potential of novel therapeutic treatments. / Doctor of Philosophy / The oral cavity has one of the most diverse and largest microbial populations, where microorganisms are capable of colonizing hard surfaces of the teeth and the soft tissues of the oral mucosa. A fundamental member of the oral microbiome is Fusobacterium, a Gram-negative bacterium which coordinates the oral biofilm formation by interacting with other microorganisms. In recent studies, Fusobacterium has been associated with oral and extra-oral infections and diseases including periodontitis, preterm birth, Lemiere syndrome, inflammatory bowel disease and colorectal cancer. Importantly, many Fusobacterium species and strains are challenging to study due to their inability to uptake exogenous DNA and lack of genetic tools, which has hindered the study of their biology, molecular mechanisms and pathogenesis. The challenges in the genetic manipulation of Fusobacterium present a significant obstacle for the development of future treatments for diseases associated with these bacteria. Therefore, the creation of tools to expand bacterial transformation of exogenous DNA and genome editing to more than just one Fusobacterium species is crucial to understand how Fusobacterium is causing these infections. This dissertation explores the presence and utilization of defense systems, which defend bacteria from phage attack, as an alternative to improve Fusobacterium genetics. Accordingly, we first studied a set of over 15 enzymes that recognize a specific DNA pattern and add a methyl group (DNA methyltransferases) to specific nucleotides in several strains of Fusobacterium. We discovered that two of these enzymes improve Fusobacterium's ability of importing and genomically incorporating exogenous DNA after an electric discharge permeabilizes the bacterial membrane. Furthermore, for the first time we have described the composition of CRISPR-Cas bacterial defense systems, that detect invading DNA from viruses and provide protection to Fusobacterium strains. These systems have previously been successfully used as genetic tools to achieve genome editing. Thus, their further characterization is warranted to create novel molecular tools in Fusobacterium. Altogether, these discoveries will lead to a better comprehension of Fusobacterium biology in infections and diseases, while exploring novel therapeutic strategies.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 13, 2010). Includes bibliographical references.