The chemical and physico - chemical properties of deoxyribonucleic acid preparations isolated from small amounts of liver and intestinal mucosa of rat (1-10 g.) by five different procedures, have been compared. The first method (29), used for preparation of deoxyribonucleic acid was based on the separation of nuclei from tissue homogenates, followed by extraction and deproteinization of deoxyribonucleic acid with strong salt solutions. The second method (20, 31) consisted of the extraction and deproteinization of nucleic acids by detergent solutions, and separation of ribonucleic acid and deoxyribonucleic acid by fractional precipitation with iso-propyl alcohol. In the third procedure crude deoxyribonucleic acid was isolated from nuclei according to the first method and the crude product was further purified according to the second procedure. The fourth method (32) was based on the disintegration of tissues by high frequency sonic oscillations, extraction of nucleoprotein from the nuclear fragments with strong salt solutions and deproteinization of deoxyribonucleic acid with chloroform -amyl alcohol mixtures. In the fifth method (36, 37) nucleic acids were extracted from tissues by hot, strong salt solutions, ribonucleic acid and deoxyribonucleic acid were separated by alkali treatment and deoxyribonucleic acid was precipitated with concentrated acid solutions. The advantages and shortcomings of the different procedures with respect to yield, purity and macromolecular state of the isolated material have been discussed. An improved technique has been described for the elution of purine and pyrimidine bases from paper chromatograms. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Significant DNA polymerase activity has been found in cytoplasmic preparations from rat intestinal mucosa. The present work involves a partial purification and a study of the general properties of this cytoplasmic enzyme activity. Crude cytoplasmic enzyme was prepared by high-speed centrifugation of the homogenate of washed mucosal scrapings. A strong inhibitor of DNA polymerase was sedimented by the high-speed centrifugation. The bulk of the enzyme activity was unadsorbed on DEAE-cellulose. However, a minor portion of the enzyme was adsorbed, and was eluted with 0.1 M KC1. When crude cytoplasmic enzyme was chromatographed by gel-filtration on Sephadex G-150, a single peak of DNA polymerase activity was detected. By the use of protein markers with known molecular parameters, the molecular weight of the DNA polymerase fraction was estimated to be 101,000. The enzyme required the presence of a DNA template and Mg++ ions. Activity was only slightly enhanced by the addition of dithiothreitol. For maximum activity, the presence of all four deoxy-nucleoside triphosphates were required. Heat-denatured DNA was preferred as primer. The optimum pH for this enzymatic activity was found to be 7.2 in potassium phosphate buffer, and 8.0 in Trisacetate buffer. Time course studies on the enzyme reaction indicated that the reaction was linear with respect to incubation time for at least 30 min. The DNA polymerase activity was stable up to 13 days under temperature conditions of 4°C to -20°C. Glycerol in 20% to 35% (v/v) concentrations was found to have both a stimulatory and a stabilizing effect on the enzyme activity. Ethylene glycol at 20% (v/v) concentration was also found to have a stimulatory effect on the enzyme activity. The enzyme was strongly inhibited in the presence of 0,10 M phosphate ions and activity was drastically reduced in phosphate ion concentrations of 0.20 M and above. The product of the DNA polymerase reaction could be destroyed by DNase, indicating that it was DNA in nature. The purpose of the present work was to determine whether the DNA polymerase activity in the cytoplasmic preparation is actually of cytoplasmic origin, or whether it is due to nuclear contamination. The above results were compared with the results obtained by other workers on the nuclear DNA polymerases. The evidence seems to indicate that the cytoplasmic enzyme activity is not due to nuclear contamination. The nuclear preparation contained several DNA polymerases, while the cytoplasmic preparation contained a single major DNA polymerase activity. This cytoplasmic activity resembled one of the nuclear activities in many respects. The cytoplasmic preparation also contained a minor DNA polymerase activity which may be mitochondrial in origin. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

When Chinese hamster V79 lung fibroblast cells are lysed in solution containing sodium chloride, a non-ionic detergent Triton-X-100, and the DNA intercalating dye propidium iodide, intact loops of DNA can be visualized under the fluorescence microscope as a 'halo' region surrounding the periphery of the nuclear matrix. The size of these halos has a biphasic dependence on the concentration of propidium iodide, suggesting that the DNA is organized into supercoiled loops constrained by attachments to the nuclear matrix. However, the induction of strand breaks in the loops of DNA by ionizing radiation results in loss of supercoiling and a uniform halo size regardless of the concentration of propidium iodide. The goal of this project was to use mathematical procedures and image processing techniques to measure the change in the size of the DNA halo. We suggest that such a quantitative assay will provide a fast and objective means to directly measure the effect of physical and chemical agents on the DNA loops. The image acquisition, processing, and segmentation procedures that are necessary for the development of this quantitative assay are performed with the Fluorescence Image Processing System (FIPS). A gradient-threshold algorithm is implemented to segment the image, which in this case is to separate the DNA halo from the background and nuclear matrix. Damage inflicted by two of the better characterized agents, x-radiation and platinum complexes, are used as a test system for this assay. The results reported here suggest that this assay can detect damage to the DNA loops at a radiation dose of as low as one Gray. The size of the DNA loop is calculated to be about 102µm. The biphasic dependence of the halo size on the concentration of propidium iodide is consistent with published data. Studies on the effect on platinum drugs on DNA loops show a difference in the kinetics of the unwinding and rewinding of the supercoils and the size of the halos at these phases also differ for both cis- and trans-diamminedichloro-platinum(II) treated cells. With our increased knowledge of the genome organization, an objective quantitative assay that can provide some insight to the damage and repair of DNA at the tertiary level of DNA organization should prove to be useful. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [151]-157).

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007. / Donald F. Doyle, Committee Member ; Bridgette Anne Barry, Committee Member ; Dr. Gary B. Schuster, Committee Chair ; Nicholas V. Hud, Committee Member ; Roger M. Wartell, Committee Member.

Orientador: Sergio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. / Made available in DSpace on 2018-08-20T01:25:34Z (GMT). No. of bitstreams: 1 WolfJunior_RobertoBento_M.pdf: 684571 bytes, checksum: dd0c5a5507117b1129be750de6a3c4e9 (MD5) Previous issue date: 2012 / Resumo: O inicio da transcrição gênica é um fenômeno complexo, provavelmente a principal etapa onde ocorre o controle da expressão gênica. A transcrição ocorre pela ligação de proteínas denominadas de fatores de transcrição com sequências específicas do DNA, chamadas de regiões ou seqüências "cis". A interação DNA - proteína pode depender da estrutura do DNA nestas seqüências. A interação dos fatores de transcrição com os sítios no DNA não só depende das bases onde ocorre o contato, mas pode depender também das bases vizinhas e da conformação do DNA. O presente trabalho teve por objetivo investigar a importância da conformação estrutural do DNA dupla fita de seqüências "cis" evolutivamente conservadas, localizadas em regiões promotoras (entre os nucleotídeos -1 e -500) em genes relacionados ao desenvolvimento craniofacial de espécies de mamíferos. Foi analisado se a variação na estrutura do DNA causada por variações genéticas em regiões filogeneticamente conservadas difere da variação na estrutura em regiões menos conservadas. As sequências de DNA com 500 pares de bases das regiões 5' de 22 genes foram obtidas no site Ensembl (http://www.ensembl.org/index.html. Foram utilizadas seqüências de cinco espécies de mamíferos. A estrutura do DNA (secundária) foi estimada pela predição do padrão de clivagem química dos radicais hidroxila do DNA dupla fita, utilizando-se o algoritmo Orchid (http://dna.bu.edu/orchid/). As seqüências das cinco espécies selecionadas foram alinhadas no programa Clustalw (http://www.ebi.ac.uk/Tools/clustalw2/index.html) e as regiões conservadas com entropia 0,1(conservação de 80%) e 0,2 (70%) com tamanho mínimo de 15 pares de bases e sem lacunas (gaps) foram obtidas pelo programa Bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Para avaliação dos valores de predição de clivagem química foi desenvolvido um algoritmo em linguagem Ruby versão 1.91 (anexo 1), onde os valores obtidos correspondiam aos valores absolutos da subtração dos valores de predição de clivagem química das bases que diferiam nas seqüências conservadas e não conservadas entre duas espécies. Comparadas regiões conservadas e não conservadas, não apresentaram diferenças estatisticamente significantes no padrão de clivagem química quando substituímos nucleotídeos na região promotora / Abstract: The initiation of the gene transcription is a very complex phenomenon, probably the principal stage where the control of the gene expression takes place. In general terms the transcription takes place with the connection of proteins called "transcription factors" with specific sequences of the DNA, called of regions or sequences "cis ". The interaction DNA - protein can depend on the configuration of the DNA in these sequences. The interaction of the transcription factors with the site in the DNA depends not only on the bases where the contact takes place, but they can depend also on the nearby bases and configuration of DNA. The aim of this work was to investigate the importance of the structural double band configuration of the DNA of conserved sequences in promoter region of genes (between the nucleotídeos-1 and-500), in genes involved in mammalian craniofacial development .We analyzed if the variation in the structure of the DNA caused by genet ic variations in regions phylogenetically preserved differs from the variation in the structure in less preserved regions. The 500 bp sequences of DNA of the regions 5' of the 22 genes, were obtained of the site Ensembl (http: // www.ensembl.org/index.html). We used sequences of the five species of mammals. The structure (secondary) of DNA was estimated by the prediction hydroxyl radical cleavage of the DNA double strand, by the algorithm Orchid (http: // dna.bu.edu/orchid/). The sequences of five selected species were aligned in the program Clustalw (http: // www.ebi.ac.uk/Tools/clustalw2/index.html)and the regions when 0, 1 (conservation of 80 %) and 0, 2 (70 %) with at least 15 bases and without (gaps) were obtained using the program Bioedit (http: // www.mbio.ncsu.edu/bioedit/bioedit.html). To evaluate the predictive value of chemical cleavage, was developed an algorithm in language Ruby version 1.91 (annex), where they obtained values were corresponding to the absolute values of the subtraction of the values of prediction of fracture chemistry of the bases that were differing in the sequences preserved and not preserved between two species. Comparing conserved and not conserved regions, no statistically significant differences in standard chemical cleavage when substituted nucleotides in promoter region / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed March 27, 2009). Graduate School of Arts and Sciences, Dept. of Physics. Includes bibliographical references (p. 130-157).

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Immunochemical and immunohistochemical techniques were used to determine the role of post-translational modifications in the regulation of DNA polymerase α in Rat-1(tsLA24/RSV) cells. Immunoaffinity purification following sucrose gradient fractionation showed two immunospecific polypeptides of Mᵣ ≃ 185,000 and 220,000 only in those fractions exhibiting DNA polymerase α activity. The Mᵣ ≃ 220,000 polypeptide was shown to be phosphorylated, primarily at serine residues. Incubation of cell lysates with immobilized alkaline phosphatase reduced enzyme activity and subsequent readdition of ATP, but not ATP-γ-S, restored activity suggesting the involvement of an endogenous serine protein kinase. This kinase may be a cAMP dependent protein kinase because prior incubation of the catalytic subunit stimulated DNA polymerase α activity 3-4 fold. In the absence of serum growth factors or pp60ˢʳᶜ, DNA polymerase α activity and semi-conservative DNA replication rates in growth arrested cells were severely depressed. However, both polymerase activity and DNA synthetic rates were subsequently restored by either activation of pp60ˢʳᶜ by temperature shift or by serum addition. DNA polymerase α protein was found primarily in the nucleus of all cells in log phase, growth arrested or subsequently stimulated cultures, independent of whether the cells were replicating DNA. Stimulation by either pp60ˢʳᶜ or serum did not alter DNA polymerase α localization within the cell nor lead to a preferential synthesis of Mᵣ ≃ 220,000 peptide or proteolytic conversion of the Mᵣ ≃ 220,000 peptide to smaller peptides, but did result in phosphorylation of the Mᵣ ≃ 220,000 polypeptide. This phosphorylation was not apparent in serum deprived, growth arrested cells. It is suggested that pp60ˢʳᶜ acts to initiate DNA synthesis through the temporal activation of DNA polymerase α through a mechanism similar to that used by serum growth factors and that phosphorylation by a serine protein kinase serves an important function.

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