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Studies on DNA and DNA polymerases from the intestinal mucosa of ratLeung, Fred Ying Toy January 1968 (has links)
PART I - The base compositions of deoxyribonucleic acid, isolated from whole cells, nuclei, and mitochondria of rat intestinal mucosa were compared. DNA from whole cells or nuclei was fractionated by column chromatography on methylated albumin kieselguhr, MAK. The guanine plus cytosine content of these DNA fractions was determined mainly by the method of heat denaturation, although the methods of acid hydrolysis and equilibrium centrifugation in cesium chloride solutions were also used. The mole % G+C for the DNA fractions from whole cellular extracts ranged between 34.39 and 52.92, while the nuclear DNA fractions showed a range between 37.31 and 50.97. Although the main DNA band which was eluted with 0.6 M NaCl solution had separated into two or three peaks, the detection of a major base compositional class of DNA was not evident. Unfractionated DNA from whole cells or nuclei has a G+C content of 42.2 which corresponds to a midpoint of thermal denaturation, Tm of 86.6° C. DNA isolated from the mitochondria of intestinal mucosa was observed to have a Tm of approximately 85.0° C and a density of 1.702 g/cm³. This density corresponded to the value determined for unfractionated DNA from the whole mucosa cells.
PART II - In initial experiments, DNA polymerase from Escherichia coli was isolated and partially purified by treatment with streptomycin, ammonium sulfate, and by chromatography on DEAE-cellulose. After these introductory experiments, DNA polymerases from the small intestinal mucosa of the rat were studied.
Using suitable assay systems with ¹⁴C-2-dTTP or ¹⁴C-8-dATP, both a replicative and a terminal DNA nucleotidyl-transferase were detected in extracts of nuclei. The replicative enzyme incorporated a labeled precursor into a native or heat denatured DNA primer in the presence of all four complementary triphosphates. The terminal enzyme preferentially incorporated single deoxyribonucleoside triphosphates onto the terminal position of heat denatured DNA primers. Treatment of the DNA products formed in the terminal addition reaction with snake venom phosphodiesterase indicated that the labeled precursors were added to 3'-hydroxy terminal positions of the chains.
A heterogeneous nature of the DNA polymerases from rat intestinal mucosa was indicated by the appearance of three fractions of enzyme activity following DEAE-cellulose chromatography. A distinct peak of terminal-addition enzyme activity was detected by rechromatography on DEAE-cellulose. Gel filtration through Sephadex G-150 or G-200 and sucrose density gradient centrifugation showed that these DNA polymerases varied in molecular sizes. The molecular weights of the DNA polymerase fractions were estimated to be between 2.5 x 10⁴ and 3 x 10⁵ by comparisons with marker proteins. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
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An investigation of the properties of DNase II isolated from bovine intestinal mucosa and the nature of its reaction with DNAKeys, David Stephen January 1978 (has links)
Isolation of DNase II from bovine small intestine by chromatography on DEAE cellulose of a 105,000 xg supernatant solution prepared from an homogenate of the mucosa in Krebs Ringer phosphate buffer appeared to yield two activities, a major activity which was eluted from the column with 20 mM phosphate buffer and a minor activity which was eluted with a potassium chloride gradient. The two DNase II activities differed in their response to increasing ionic strength, pH, sulfate ion concentration and temperature for the hydrolysis of DNA. The major activity degraded native DNA more rapidly than denatured DBA whereas the minor activity degraded both at the same rate. Previous investigators have reported the presence of two DNases lis with different properties in other tissues. In bovine intestinal DNase II, the minor activity, upon rechromatography on DEAE cellulose, eluted in the same position as the major DNase II and it was concluded; that the appearance of the minor DNase II activity was an artifact of the chromatography. It is likely that a small quantity of DNase II was bound to endogenous DNA on the DEAE cellulose column in the 20 mM phosphate buffer and later eluted from the column along with some of the DNA. with the potassium chloride gradient. DNA present in the minor DNase II preparation probably caused the apparent differences in properties of the two DNase lis by interfering in the enzymic reactions.
Intestinal DNase II was partially purified by ion exchange chromatography and gel filtration and had properties similar to DNase lis from other tissues. The enzyme hydrolysed calf thymus DNA endonucleolytically at acid pH in the absence of a divalent metal ion to oligonucleotides with 3'-phosphate and 5'-hydroxyl terminals. The activation energy for the reaction was 19 kcal/mole; that for denaturation of DNase II itself, k3 kcal/mole. Michaelis-Menton kinetics were observed for the reaction of DNase II with Escherichia coli DNA—the Michaelis constant was 2.42 x 10⁻⁷ M DNA-phosphate. The molecular weight of DNase II was estimated to be 41,000 by gel filtration on Sephadex G100.
The early stages of the digestion of DNA by DNase II were investigated by labelling the reaction products with ³²P at their 5'-terminals using polynucleotide kinase and [x-³²P]ATP and at their 3'-terminals using terminal deoxynucleotidyl transferase and [⍺-³²P]ATP.
The mode of cleavage of native DNA by DNase II was determined by comparing the polynucleotide-catalysed incorporation of 32p from
[x-³²P]ATP into native and denatured DNase II reaction products. Since single-strand cleavage of DNA by DNase II released 5'-hydroxyl terminals that were inaccessible to polynucleotide kinase as long as the DNase II reaction products remained double-stranded, incorporation of ³²P into native products was proportional to the number of double-strand cleavages while incorporation of ^P into denatured products was proportional to the number of double-strand cleavages plus single-strand cleavages. It was found that DNase II degraded native DNA primarily by a double-strand cleavage mechanism.
After DNase II catalysed hydrolysis of DNA each of the four bases present in DNA was found at the 5'- and 3'-terminals of the reaction products. Thus DNase II did not have an exclusive preference for one or two bases at either terminal, and likely cleaved a large number of
different base sequences in the DNA.. The most susceptible internucleotide linkage was GpG; the most resistant, CpT. The base specificity at the 5'-terminal changed during the reaction, especially in the initial and terminal phases. In the initial phase the proportion of guanine was elevated and the order of cytosine and adenine was reversed compared to later stages in the reaction. These changes could reflect the presence of a preferred sequence that was selectively degraded and exhausted during the initial phase of the reaction. Different proportions of terminal bases in cleavage products of DNA from diverse species indicated that susceptible sequences occurred with different frequencies in the various DNAs. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Unknown
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Mecanismos de indução de lesões no DNA pela luz UVA e seus efeitos biológicos. / Mechanisms of induction of DNA lesions by UVA light and its biological effects.Teiti Yagura 03 April 2012 (has links)
Irradiamos amostras de DNA com luz UVA em diferentes condições para estudar os possíveis mecanismos envolvidos na indução de lesões de DNA por essa radiação. As lesões de DNA formadas após as irradiações foram quantificadas com enzimas de reparo de DNA que reconhecem e clivam os sítios contendo bases oxidadas e dímeros de pirimidina (CPDs). Complementando essas análises, foram realizados ensaios com anticorpos e HPLC-ED. NaCl e uma maior concentração de DNA são capazes de diminuir a indução de CPDs. Danos gerados por estresse oxidativo são inibidos na presença de azida de sódio e quelantes de metais, indicando o envolvimento de oxigênio singlete e reações de Fenton, na geração dessas lesões. Água deuterada e DNA mais concentrado aumentaram a indução de bases oxidadas. Quanto maior a quantidade de DNA irradiado, mais oxigênio singlete é formado, o que indica um possível mecanismo de fotossensibilização endógeno. / DNA samples were irradiated with UVA light in different conditions for studying the possible mechanisms involved in the induction of DNA lesions by this radiation. DNA lesions formed after irradiation were quantified with DNA repair enzymes, which recognize and cleave the sites containing oxidized bases and pyrimidine dimers (CPDs). Complementing these analyses, tests were performed with antibodies and HPLC-ED. NaCl and more concentrated DNA are capable of reducing the induction of CPDs. Damage caused by oxidative stress is inhibited in the presence of sodium azide and metal chelators, indicating the involvement of singlet oxygen and Fenton reactions, in the generation of these lesions. Deuterated water and more concentrated DNA increased the induction of oxidized bases. The bigger the amount of irradiated DNA, the more singlet oxygen is formed, which indicates a possible endogenous photosensitization mechanism.
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Epigenetic Blocking of an Enhancer Region Controls Irradiation-Induced Proapoptotic Gene Expression in Drosophila EmbryosZhang, Yanping, Lin, Nianwei, Carroll, Pamela M., Chan, Gina, Guan, Bo, Xiao, Hong, Yao, Bing, Wu, Samuel S., Zhou, Lei 15 April 2008 (has links)
Drosophila embryos are highly sensitive to γ-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. Our study indicates that the sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli.
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Biochemical taxonomy by DNA analysisEssop, M Faadiel 19 October 2022 (has links) (PDF)
Defining how closely related a pair or a group of organisms are, is necessary both for the construction of phylogenetic trees, which constitutes the academic science of systematics, and for making practical conservation management decisions, as for example, whether it would result in deleterous genetic consequences (decreased fitness on reproductive capability) if two closely related animals e.g. blesbok, Damaliscus dorcas phillipsi, and bontebok, Damaliscus dorcas dorcas, were allowed to interbreed, in which case active steps would be required to manage the animals in an appropriate way. Reliance on traditional morphological characters to answer such questions are difficult because morphological characters change at a rate which is very poorly correlated with time, whereas the genetic differences which affect management decisions change at a progressive and generally linear rate with time (Wilson et al., 1977). In order, therefore, to try and measure such genetic differences in a more quantitative way, biologists turned to biochemical methods (two to three decades ago) and initially studied differences in protein allozymes, with considerable success (reviewed in Chapter 1). Over the last few years attention has switched to use of differences at the level of DNA, since this is the most direct biochemical measure of genetic variation, being the material of which genes are made (Wilson et al., 1977). Organelle DNA from mitochondria in animals has been widely used since it has a number of advantages compared to nuclear DNA, and is the DNA used in the studies reported here. Mitochondrial DNA accumulates single base point mutations with time, at a rate about 5 to 10 times faster than in nuclear DNA (Brown et al., 1979), which renders it the DNA of choice for comparisons between sub-species, species or genera. This project set out to establish modern biochemical methods of comparative DNA analysis and to apply these to local animal groups and so obtain objective data of both academic interest and of practical value to nature conservation problems. The major academic results are the construction of a molecular phylogeny for a major proportion of the Southern African Bovidae (Chapter 3). The family Bovidae has been one of the most difficult mammalian families to classify and there is no general agreement concerning its classification (Ansell, 1971b). Mt DNA restriction analyses were performed on 14 Southern African bovids and restriction maps constructed independently for all 14 species. Phylogenetic trees were constructed by using both distance and cladistic methods. Cladograms supported a sister status of the impala relative to the Alcelaphini and Connochaetini. Four members of the Tragelaphini remained an unresolved quadrichotomy and this would be consistent with inclusion of the eland within Tragelaphus. Distance dendograms would be consistent with a major radiation at the tribal level at the end of the Miocene. Answers to questions of specific conservation interest have been obtained with respect to several animals where conservation management requires knowledge as to how closely related certain pairs or groups of animals are. The answers relate to: ( 1) bontebok, Damaliscus dorcas dorcas vs blesbok, Damaliscus dorcas phillipsi ( Chapter 4); ( 2) African wild cat, Felis lybica vs domestic cat, Felis catus (Chapter 5) and (3) Kruger vs Addo elephant (Chapter 6). A specific request of Nature conservation authorities was to define the genetic relationship between the bontebok and the blesbok, which has conservation management relevance. The sequence divergence between the blesbok and bontebok confirmed that there is no justification in terms of genetic distance alone for applying separate specific status to the bontebok and the blesbok, but would be an appropriate value consistent with maintaining their present subspecific designation. Another request of Nature conservation authorities was to define the genetic relationship between the African wild cat and the domestic cat, since interbreeding between the two takes place where the range of the African wild cat border on those areas inhabited by man, which might effectively cause the disappearance of F.lybica (Smithers, 1983). Mt DNA restriction maps were constructed for the domestic cat, African wild cat and the European wild cat, Felis sylvestris, which was also included in our analysis. The domestic cat and African wild cat were found to have identical restriction maps emphasizing their close relationship. The domestic cat and African wild cat are therefore likely to have no preferences against inbreeding and the chances of the wild cat phenotype therefore being lost by dilution into the domestic cat is high. The sequence divergence between the African wild cat and European wild cat on the other hand, suggests that the common ancestral mt DNA of these cats existed about 650 000 years ago, indicating a more recent descent than was previously thought. A further request from conservation managers was to show the genetic difference, if any, between the Knysna and Kruger elephant herds. Mt DNA fragment size comparisons were performed on 9 elephants from the Kruger National Park and 5 elephants from the Addo Elephant National Park. All the restriction patterns found in the Addo population could be found in the Kruger population. The Addo population's results was extrapolated to the Knysna population. Therefore, genetically the Knysna population would represent a subset of the larger Kruger population. There is therefore no support from the mitochondrial studies for the Knysna and the Kruger elephant populations to be considered as different subspecies.
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The CIS-Required DNA Sequences for Bacteriophage Mu DNA Transposition and MaturationHarel, Josee 03 1900 (has links)
No description available.
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Interaction of acriflavine with deoxyribonucleohistone /Hardy, Robert Warren January 1965 (has links)
No description available.
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Hair as a source of ancient DNAGilbert, M.T.P., Tobin, Desmond J., Wilson, Andrew S. January 2009 (has links)
No
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TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adductsFielden, J., Wiseman, K., Torrecilla, I., Li, S., Hume, S., Chiang, S., Ruggiano, A., Singh, A.N., Freire, R., Hassanieh, S., Domingo, E., Vendrell, I., Fischer, R., Kessler, B.M., Maughan, T.S., El-Khamisy, Sherif, Ramadan, K. 25 August 2020 (has links)
Yes / Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution. / Supported by the Medical Research Council programme grant (MC_EX_MR/K022830/1) to K.R. J.F. is supported by a CRUK DPhil studentship. K.W. was supported by an MRC studentship and A.R. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF 1109-2017). E.D. is supported by the S:CORT consortium which is funded by a grant from the MRC and CRUK. S.F.E. is funded by a Wellcome Trust Investigator award (103844) and a Lister Institute Fellowship.
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Studies on mammalian DNA ligase IIINash, Rachel Anne January 1996 (has links)
No description available.
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