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1
Elliott, Gillian Daphne. "The cloning and sequencing of mumps virus." Thesis, Queen's University Belfast, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356913.
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Kourdi, Zerikly Malek. "Cloning, sequencing and analysis of spirotetronate biosynthetic gene clusters." Thesis, University of Warwick, 2011. http://wrap.warwick.ac.uk/38545/.
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Spirotetronate antibiotics are natural products that have been shown to have a broad spectrum of biological activities, including antiviral, antibacterial, antitumor and antimalarial activities. Two representatives of this group are quartromicins and chrolactomycin, which are produced by Amycolatopsis orientalis and Streptomyces sp. 569N-3 respectively. Based on other related natural products with known biosynthetic pathways, the biosynthetic routes for quartromicins and chrolactomycin were proposed and several strategies for locating the putative gene clusters directing the biosynthesis of these natural products were used. An FkbH-like protein was proposed to be specifically involved in the biosynthetic incorporation of a glycerol-derived three-carbon unit into the tetronate moieties of quartromicins and chrolactomycin. It was selected as a target to locate the biosynthetic gene clusters of tetronate natural products. After aligning sequences of known FkbH-like proteins, a set of degenerate oligonucleotide primers for amplification of fkbH-like genes was designed and tested. Fragments of fkbH-like sequences were amplified from several available strains and sequenced. Several genomic libraries were created and screened by PCR using the degenerate fkbH primers. After validating the library, clones containing the putative biosynthetic gene cluster were identified and sequenced. The obtained sequence data was then assembled and analysed. Coding sequences were identified, protein functions assigned and a biosynthetic route was proposed for the biosynthesis of quartromicins and chrolactomycin.
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Vipond, I. Barry. "Cloning, sequencing and expression of Newcastle disease virus genes." Thesis, University of Newcastle Upon Tyne, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315647.
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Self, Susan Jane. "Cloning, sequencing and expression cytochrome C2 from Rhodospirillum rubrum." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46542.
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Antic, Dragana. "Molecular genetics of hantaviruses: Cloning, sequencing, expression, and morphogenesis." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/7653.
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Molecular characterization of the Seoul 80-39 virus, a prototype of the Seoul serotype, was carried out by cloning and sequencing. The large (L) genomic RNA segment is 6530 nucleotides long. The viral complementary-sense RNA contains a single open reading frame (ORF) from the AUG codon at nucleotide positions 37-39 to the UAA stop codon at nucleotide positions 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 D) which likely corresponds to the L protein detected in purified viral particles (Elliot et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). The M RNA segment is 3651 nucleotides long. A single ORF was detected in the viral complementary-sense RNA. This ORF could encode a polypeptide of 1133 amino acids which is cotranslationally processed into viral glycoproteins G1 and G2. The S genomic RNA segment was characterized from the 3$\sp\prime$ end to nucleotide 1332. One long ORF was detected from the AUG codon at nucleotide positions 43-45 to the UAA stop codon at nucleotide positions 1330-1332. This ORF has a capacity to encode a 48 kD protein which corresponds to the viral nucleocapsid (N) protein. A comparison of coding properties of the tripartite genomes of various hantaviruses allowed construction of the evolutionary tree for the Hantavirus genus. The second part of the study was focused on the maturation of Hantaan 76-118 virus glycoproteins G1 and G2 virus morphogenesis. Glycoproteins G1 and G2 form a complex. Experiments with the glycosylation inhibitor brefeldin A indicate that complex formation between the two glycoproteins occurs in the ER. Resistance of both G1 and G2 to endoglycosidase D suggests a structure with more than five mannose residues. Processing of a high mannose structures ((Man)$\sb8$(GlcNAc)$\sb2$ to (Man)$\sb7$(GlcNAc)$\sb2$ or (Man)$\sb6$(GlcNAc)$\sb2$) may take place in the cis-Golgi by the enzyme $\alpha$-mannosidase I. Virus budding was observed only in Hantaan virus-infected cells, in the trans-Golgi network. When the Golgi apparatus was destroyed by brefeldin A treatment, assembly of virus particles was not observed. The third project was to express nucleocapsid proteins of Seoul 80-39 virus and Hantaan 76-118 virus using baculovirus recombinants. Analysis of recombinant virus-infected Sf9 cell lysates by SDS-PAGE showed that N proteins were expressed in high levels and were identical to virion associated N proteins in size and antigenicity. (Abstract shortened by UMI.)
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Cekic, Caglar. "Cloning, Expression And Sequencing Of Citrate Synthase From Thermoplasma Volcanium." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/1260432/index.pdf.
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In this study first time, we have cloned and sequenced the citrate synthase gene from a thermoacidophilic archaeon Thermoplasma (Tp.) volcanium (Optimum growth temperature of Tp.volcanium is 60oC and optimum pH is 2.0.). For cloning we have followed a PCR based approach. Amplification of citrate synthase gene from chromosomal DNA of Tp.volcanium yielded a product of 1476 bp containing an open reading frame of 1161 bp comprising the structural gene. After ligation of the PCR amplicon to pDrive vector through AU complementation, recombinant plasmids were transferred into E.coli TG-1 competent cells. Out of three recombinants, E.coli pDriveCS-31 was selected for further characterization by restriction mapping and DNA sequencing. Southern Blotting and Hybridization using the membrane blot of pDriveCS-31 plasmid and DIG-labeled PCR amplified citrate synthase gene probe, also confirmed the cloning of Tp.volcanium citrate synthase gene in E.coli. Clustal W Version 1.82 was used for alignment of aminoacid sequence of Tp.volcanium citrate synthase with that of other archaeal, bacterial and eukaryotic citrate synthases. The highest sequence similarity (87%) was found between Tp.volcanium and Tp.acidophilum enzymes. Despite low sequence homology (18%) with the pig enzyme, of the 11 residues implicated in catalytic activity of the pig citrate synthase 9 were conserved in the Tp.volcanium enzyme.
Heterologous expression of this citrate synthase gene in E.coli has been achieved under the control of its promoter sequences. The recombinant enzyme (386 aa) has been purified to homogeneity by affinity chromatography on Reactive Red 120 column. The subunit molecular size was estimated as 43 kDa. The purified enzyme followed classical Michaelis-Menten kinetics. The Km values of 5.15 μ
M and 5.60 &
#956
M, and Vmax values of 1.74 &
#956
moles/ml/min and 1.60 &
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moles/ml/min were calculated from Lineweaver-Burk plots for acetyl-CoA and oxaloacetate, respectively. The recombinant enzyme was thermostable and retained about 80% of the activity at 85oC after 1 hour.
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Lloyd, Amanda Lian. "Cloning, characterisation and sequencing of promoters of Helicobacter pylori 4187E." University of Western Australia. Microbiology Discipline Group, 2005. http://theses.library.uwa.edu.au/adt-WU2005.0112.
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Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress
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Xu, Jing. "Cloning and sequencing of genes that control lung branching morphogenesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0002/MQ28788.pdf.
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Lloyd, Amanda Lian. "Cloning, characterisation and sequencing of promoters of Helicobacter pylori 4187E /." Connect to this title, 2004. http://theses.library.uwa.edu.au/adt-WU2005.0112.
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Bright, Jeremy R. "Cloning, sequencing and expression of glucose dehydrogenase from Thermoplasma acidophilum." Thesis, University of Bath, 1991. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292614.
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Sutherland, Katharine J. "Thermoplasma acidophilum citrate synthase : cloning and sequencing of the gene." Thesis, University of Bath, 1990. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278281.
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Filho, JoÃo Garcia Alves. "Cloning, sequencing and partial characterization of Vatairea Macrocarpa lectin related genes." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=8149.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoNo presente trabalho à feita a descriÃÃo de trÃs genes distintos que codificam lectinas ou proteÃnas relacionadas à lectina de Vatairea macrocarpa. As sequÃncias foram obtidas pela amplificaÃÃo de DNA genÃmico e cDNA de folhas utilizando primers semi-degenerados construÃdos a partir da informaÃÃo da sequÃncia de aminoÃcidos da lectina VML depositada no GenBank. O resultado do sequenciamento revelou a presenÃa de trÃs contigs. O contig1 corresponde à lectina VML desde que se assuma que a lectina depositada VML contenha heterogeneidades ou ambiguidades decorrentes na degradaÃÃo de Edman. A traduÃÃo dos contigs 2 e 3 mostram identidade de sequÃncia de 77% quando comparadas com VML. As sequÃncias, apesar de apresentar regiÃes conservadas, mostram diferenÃas de aminoÃcidos nos sÃtios de N-glicosilaÃÃo, sÃtios de ligaÃÃo a carboidrato e metais alÃm da presenÃa de resÃduos de cisteÃna sugerindo que tais proteÃnas podem ter outras atividades biolÃgicas. A anÃlise da sequÃncia obtida pelo 3â RACE se mostrou complementar ao contig3. Sendo assim, a sequÃncia hÃbrida contig3/contigA possui 2 resÃduos de cisteÃna alÃm de revelar diferenÃas de aminoÃcidos na regiÃo C-terminal quando alinhada com outras lectinas de leguminosas. AnÃlises filogenÃticas revelaram que os contigs observados formam um grupo monofiletico e tem alta similaridade com as lectinas de Sohora japonica e Robinia pseudoacacia, alÃm da proteÃna relacionada à lectina de Cladrastis lutea.
In this paper is made a description of three distinct genes that encode Vatairea macrocarpa lectin and related proteins. The sequences were obtained by amplification of genomic DNA and cDNA of leaves using semi - degenerate primers constructed from the information of the amino acid sequence of VML lectin deposited in GenBank. The result of sequencing rev eals the presence of three different genes, called contig 1, 2 and 3 . The VML lectin corresponds to contig1 long as one assumes that the lectin contains heterogeneities deposited VML or ambiguities arising in the Edman degradation . The translation of cont igs 2 and 3 show sequence identity of 77% compared to VML. Sequences, despite having conserved regions show differences in amino acid N - glycosylation sites, carbohydrate binding sites and metals and the presence of cysteine residues suggests that these pro teins may have other biological activities . The analysis of the sequence obtained by 3 'RACE proved complementary to contig3. Thus, the sequence contig3/contigA hybrid has two cysteine residues in addition to revealing differences in amino acid C - terminal region when aligned with other legume lectins. Phylogenetic analysis revealed that the observed contigs form a monophyletic group and has high similarity with lectins from Robinia pseudoacacia Sohora japonica and, in addition to the lectin - related protein Cladrastis lutea .
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Ferguson, Christine Anne. "Cloning, sequencing and functional analysis of the chicken tyrosine gene promoter." Doctoral thesis, University of Cape Town, 1996. http://hdl.handle.net/11427/26630.
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The differentiation of melanocytes from multipotential neural crest cells is an ideal system for studying the processes underlying lineage determination in development. Tyrosinase is a key enzyme in melanin biosynthesis and the activation of the tyrosinase gene is characteristic of differentiated melanocytes. In order to study the mechanisms underlying activation of melanocyte-specific genes during differentiation in chick embryos, a chicken genomic DNA library was screened for tyrosinase-encoding sequences using a mouse tyrosinase cDNA probe. Two identical hybridising clones were identified. Restriction mapping and sequencing revealed that both clones contained a 4.3 kb genomic DNA fragment, CTYR4.3, that included 2125 nt of the 5' flanking region, the first exon and part of the first intron of the chicken tyrosinase gene. The 5' flanking sequence of CTYR4.3, which is the most extensive to be reported for a lower vertebrate tyrosinase gene to date, was analysed further using computer-aided homology searches and primer extension. Alignment of the promoter sequences of CTYR4.3 with those of the human, mouse, quail and turtle tyrosinase genes revealed two evolutionary conserved regions. These regions may be functionally significant as they contain regulatory elements previously reported to play a role in melanocyte-specific expression of the tyrosinase gene in mammals. These include an initiator region and an associated SP1-binding site, the M-box and an upstream enhancer element, TDE. In addition, other potential transcription factor binding motifs were identified, including an AP-1-binding site, a UV-responsive element and glucocorticoid-responsive elements. Although several TATA box motifs were identified, they were situated more than 200 bp upstream of the transcription start sites mapped by primer extension analysis and therefore are unlikely to function as TFIID-binding sites. Transcription initiation appears to occur at heterogeneous start sites, and given the absence of a functional TATA box, may be mediated via the conserved initiator region and SP1-binding site. To test the ability of the 5' flanking sequence of CTYR4.3 to drive transcription and to begin to assess the functional significance of the various conserved elements, transient transfection assays were carried out. Constructs were generated in which 2.1 kb, 1.1 kb, 0.5 kb and 0.2 kb fragments of the 5' flanking sequence were linked to a luciferase reporter gene. These constructs were introduced into cultures of chicken retinal pigment epithelial cells (RPE), immortalised quail neural crest cells (MQTNC), and human liver cells (Hep G2) by calcium phosphate-mediated transfection. Transfections with all constructs resulted in luciferase activities significantly greater than those that were observed with the promoterless luciferase construct, thus confirming that the 5' flanking sequence of CTYR4.3 does possess promoter activity. However, the level of expression from the various constructs differed markedly in the different cell types. In the tyrosinase-negative Hep G2 cells, low levels of expression were observed with all constructs. In the tyrosinase-positive RPE cells, a high level of luciferase activity was obtained specifically with the smallest (0.2 kb) promoter construct. Since the 0.2 kb promoter fragment does not include the conserved initiator region, SP1-binding site, or M-box, the role of these elements in tissue-specific transcription initiation of the chicken tyrosinase gene is now questionable. These results suggest the existence of transcription regulatory mechanisms that are unique to avians and possibly other lower vertebrates. In contrast to the results obtained for RPE cells, the highest luciferase activity was obtained with the full length 2.1 kb promoter construct in the immortalised quail neural crest-derived cells. These results may have developmental significance since they suggest that the chicken tyrosinase gene promoter is regulated differently in RPE cells and neural crest-derived cells.
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Ozcan, Numan. "Cloning and sequencing of a cellulase gene from Fibrobacter succinogenes SD35." Thesis, University of Aberdeen, 1992. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU546892.
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F.succinogenes is one of the most active bacteria of the rumen in growth-related degradation of recalcitrant forms of cellulose. The bacterium has multienzyme cellulase complex encoded by multiple genes to degrade crystalline cellulose. A genomic library of F.succinogenes SD35, constructed in the EMBL3, was screened for cellulolytic activity and 2.7kbp DNA fragment expressing a endoglucanase (SD-END-1) was further subcloned into pUC18 and expressed in E.coli DH5 alpha. DNA hybridisation (dot blot) experiments revealed that this gene (sd-end-1) is different from the endoglucanase encoding gene in either Ruminococcus albus SY3 or F.succinogenes BL2. The 2.7 kbp DNA fragment carrying the sd-end-1 with an ORF of 1167 bp was fully sequenced. The alignment of the amino acid sequence of SD-END-1 (388 amino acids) with other multifunctional endoglucanases from F.succinogenes S85 (EG3), R.albus SY3 (EGA) and B.fibrisolvens (End1) revealed less than 25% identity. The molecular weight of SD-END-1 calculated from deduced amino acid sequence, 50,201 Da is in agreement with the molecular weight estimated by CMC-SDS-PAGE (49 kDa). Specific activity of SD-END-1 calculated by CMC, lichenan, xylan and avicel were 101, 41, 27 and 15mumol reducing sugar/min/mg protein. SD-END-1 has a pH optimum of 6.0 and a temperature optimum of 37.5°C. When the enzyme and substrate were incubated alone at various temperatures for 10 min and then assayed at 39°C for 30 min, 75% of its activity was lost at 60°C. On the other hand, its activity was slightly stimulated in the presence of 4 mM CaCl2 or MgCl2. A rapid and simple method for the isolation of lambda and a new plate for rapid screening of CMC+ recombinant E.coli cells were also developed. In addition, a new CMC-PAGE assay was developed and used as an alternative method to investigate whether SD-END-1 was subject to proteolysis in the E.coli DH5 alpha cells.
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Taylor, Tanya. "Protein purification, sequencing, and cDNA cloning of P68, a sperm surface protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ36745.pdf.
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Park, Sung-Goo. "Cloning and sequencing of the spoIIA locus from four species of Bacillus." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260784.
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Dodsworth, Steven James. "Cloning, sequencing and expression of the Aeromonas salmonicida maltose-inducible porin gene." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359863.
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Ghosh, Ratan Chandra. "Molecular cloning and nucleotide sequencing of sacbrood virus of the honey bee." Thesis, University of Surrey, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267850.
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Perron, Lepage Marie-France. "Cloning and sequencing of an alternatively spliced variant of bovine interleukin-4 /." [S.l.] : [s.n.], 1999. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Deng, Ruitang. "Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487779914825349.
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Aakula, Srikanth. "Cloning and Sequencing of Bovine Na+/K= Atpase α-1 Sub-Unit." TopSCHOLAR®, 2004. http://digitalcommons.wku.edu/theses/520.
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The major focus of this project was cloning and sequencing the α-1 sub-unit of Na+/K+-ATase gene in bovine corneal endothelium. The Na+/K+-ATase, also called the Na+ pump, is a crucial transmembrane protein. By transporting water and ions from and through the cornea into the aqueous humor, it is responsible for maintenance of structural integrity, corneal hydration and thereby transparency of the cornea. The Na+ pump is characterized by a complex molecular heterogeneity that results from differential association of multiple isoforms of both a (the catalytic) and [3 (glycoprotein) sub-units. In the corneal endothelium, α-1 α-2, β-l and β-2 sub-units are expressed. Pathological and mechanical causes can disrupt the endothelial morphology and deregulate the pump function leading to corneal swelling and opacity. Recent works have focused on the study of pump expression, and the influence of different factors on the upregulation of its expression. For example, hypersaline and hyperosmotic treatment significantly increases pump expression.
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Argyle, David John. "Cloning sequencing, expression and demonstration of biological activity of feline recombinant interferon-gamma." Thesis, University of Glasgow, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284707.
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Jones, Dawn L. "The cloning and sequencing of the plant nuclear poly ADP-ribose polymerase gene." Thesis, University of Aberdeen, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262927.
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The project was to clone the gene encoding a nuclear enzyme poly-ADP-ribose polymerase (PARP) involved in the posttranslational modification of nuclear proteins. This modification is important in the regulation of various cellular processes such as cell differentiation, proliferation and in the molecular events involved in the recovery of cells from DNA damage. At the start of this project this enzyme had been well characterized in animal systems but had not as yet been explored in plants. Firstly, I showed the enzyme to be present in higher plant nuclei, by a series of experiments including enzyme assays and western blotting (probed with a polyclonal antibody specific to the protein). Molecular biology approaches were used to isolate the gene encoding the PARP enzyme. These techniques included screening of cDNA libraries, constructed in λZAPII, with both the polyclonal antibody and a gene probe (a restriction digest of a human PARP gene was performed and a 1.4Kb fragment containing the C-terminal region of the gene used as a probe). This identified a number of bacteriophages from which sequence information was obtained (by making and extracting phagemids). Subsequent translation of these nucleotide sequences revealed that one of the sequences (245941) obtained in this manner showed homology to the PARP protein. Degenerate oligonucleotide primers, designed to the conserved C-terminal region of the gene, were also used in an attempt to amplify a 375bp region of the gene thought to contain the PARP signature. PCR products of the expected size (375bp) were obtained from an SST cDNA library. These PCR products hybridized to the gene probe (mentioned earlier). Subsequent subcloning revealed multiple products of 375bp comigrating in the agarose gels. Two different sequences were obtained which were, upon translation, shown not be PARP-like sequence.
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Brand, N. J. "Cloning and sequencing of the gene for valyl-tRNA synthetase from Bacillus stearothermophilus." Thesis, Imperial College London, 1986. http://hdl.handle.net/10044/1/37951.
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Anderson, Marie June. "Complementary DNA cloning, sequencing, and analysis of the Pelargonium flower-break virus genome /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487842372897839.
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Zhang, Xiaohong. "Cloning, sequencing, expression, and inactivation of the aminodehydroquinate dehydratase gene in Amycolatopsis mediterranei S699 /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/8602.
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Booker, Squire J. "Cloning, sequencing, expression, and characterization of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/32144.
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Kumar, Purnima. "Molecular analysis of bacteria associated with chronic periodontitis and periodontal health." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1124221576.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains x, 104 p.; also includes graphics (some col.). Includes bibliographical references (p. 92-104). Available online via OhioLINK's ETD Center
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Wang, Qiong. "Characterization of white spot syndrome virus of penaeid shrimp: Genomic cloning and sequencing, structural protein analyzing and sequencing, genetic diversity, pathology and virulence." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/284292.
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The purpose of this dissertation was to characterize virulence, genomic and protein composition of a newly emerged virus of penaeid shrimp: white spot syndrome virus (WSSV). A partial genomic library, covering approximately 30-50% of the genome, of WSSV isolated from crayfish Orconectes punctimanus, was constructed by digesting viral DNA with endonuclease ClaI and cloning into the system pBluescript-JM109. Three viral inserts of approximately 2.2 kb, 2.8 kb and 6.3 kb, named as QW245, CR44, and QW237 respectively, were sequenced and analyzed. Six geographic isolates of WSSV, from China, India, Thailand, South Carolina, Texas, as well as from crayfish obtained from the US National Zoo in Washington D.C. were compared by electron microscopy (TEM) and SDS-PAGE. All viral isolates contained three major polypeptides of 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the viral isolates. The 19 kDa polypeptide of the crayfish WSSV appeared larger in size than that of the other isolates. Amino acid composition of four of the major structural polypeptides of the South Carolina WSSV was analyzed. The NH₂ terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the SC WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. The genomic composition of the six geographic isolates of WSSV were compared by combining the methods of restriction analysis using nine endonucleases AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI, XhoI and Southern blot hybridization applying three digoxigenin-11-dUTP labeled WSSV genomic probes LN4, C42 and A6. No distinctive difference among five WSSV isolated from penaeid shrimp was detected; differences were observed in the crayfish isolate of WSSV. The virulence of the six geographic isolates of WSSV were compared by per os challenge of Litopenaeus vannamei postlarvae and juveniles, and Farfantepenaeus duorarum juveniles. The Texas WSSV caused higher and more rapid mortalities; the crayfish WSSV caused lower and less rapid mortalities. L. vannamei postlarvae and juveniles were very susceptible to WSSV infection, while Fa. duorarum juveniles showed moderate resistance.
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Luo, Zongshu. "Cloning and sequencing of a storage protein receptor fragment from the corn earworm, Helicoverpa zea." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq24191.pdf.
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McNally, Roisin M. P. "Molecular cloning and nucleotide sequencing of the capsid protein regions of two bovine enterovirus strains." Thesis, Queen's University Belfast, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335430.
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Taylor, Sally Catriona. "Cloning and sequencing pea early browning virus RNA-1 and studies on the 30K gene." Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278116.
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Strong, Christy. "Flavonoid Glucosyltranferases: Cloning and Sequencing of Putative Glucosyltranferases from Citrus paradisi (Grapefruit) Leaves." Digital Commons @ East Tennessee State University, 2005. https://dc.etsu.edu/etd/989.
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Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization.
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Klang, Judith Elisa. "Cloning, Sequencing and Expression of a Porcine Intestinal Peptide Transporter in a Mammalian Cell Line." Thesis, Virginia Tech, 2002. http://hdl.handle.net/10919/30880.
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Absorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported.Master of Science
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Dunne, Patrick W. "Cloning and sequencing of beta-tubulin mutations that inhibit mitotic spindle formation in Aspergillus nidulans /." The Ohio State University, 1988. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487596307359427.
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Gu, Zhengming. "Cloning and sequencing of the phosphoribosylformylglycin-amidine synthase II gene from Lactobacillus casei subsp. casei and attempted cloning of the caprylate-esterase gene." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61201.
Full textAbstract:
Plasmid curing experiments showed that the Caprylate-Esterase (CE) gene of Lactobacillus casei subsp. casei (LLG) is located on the LLG chromosome rather than on a plasmid. The enzyme was purified and shown to be a monomer with an apparent Mr of 32,000 on sodium dodecyl sulfate-polyacrylamide gels. The N-terminal end of the CE protein was microsequenced. A synthesized 29-mer oligonucleotide deduced from L. casei codon usage and the N-terminal peptide sequence was used as probe against an LLG genomic library. Library screening yielded one positive clone (pLLG1435) with an insert of 8 kb. Southern analysis of pLLG1435 showed that the oligonucleotide probe strongly hybridized with the 1.9 kb PstI/SphI fragment found within its insert. Th fragment was subcloned and its DNA sequence was determined and found to be closely related to the phosphoribosylformylglycinamidine synthase II gene of Bacillus subtilis. The gene was completely sequenced.
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Roach, Jared C. "Random subcloning, pairwise end sequencing, and the molecular evolution of the vertebrate trypsinogens /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/8331.
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Yu, Chih-Wen, and 尤志文. "Facile Cloning , Sequencing and Expression of .delta." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/99762604730401065874.
Full textAbstract:
碩士國立臺灣大學
生化科學研究所
81
.delta. 水晶體蛋白是存在於鳥類和爬蟲類眼球中的主要水晶體蛋白。為 了快速選殖定序鵝之.delta.水晶體蛋白基因,於是便利用簡易快速的PCR 技術從鵝眼球水晶體cDNA混合液中,將.delta. 水晶體蛋白基因增殖。然 後將增殖產物接上pUC19載體, 殖入大腸桿菌JM109中繁殖。篩選後,取三 個含有約1.4kb插入基因的菌落予以核酸定序。結果得到一條1,401bp完整 的.de -lta.水晶體蛋白基因,包含了466個胺基酸序列。在與其他鳥類. delta. 水晶體蛋白胺基酸序列比較後,發現有很大的相似性。其分別與鴨 之.delta.1 ,.delta.2水晶體蛋白有97%,94%相似性;與雞之.delta.1,. delta.2 水晶體蛋白有88%,90%相似性;與鴿子之.delta.水晶體蛋白有88% 相似性;而與人之精氨琥珀酸分解酵素只有69%相似性。因藉由陰離子交換 樹脂純化出來的鵝 .delta.水晶體蛋白具有與鴨.delta. 水晶體蛋白相當 的精氨琥珀酸分解酵素活性。而有高相似胺基酸序列的雞與鴿子.delta. 水晶體蛋白卻無此活性。為進一步研究分析,便將得到的鵝.delta.水晶體 蛋白基因接上pQE31表現載體,於大腸桿菌M15中大量表現,並對表現產物特 性予以探討。
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Chang, Kuei-Yun, and 張貴雲. "Identification, sequencing and cloning of Arabidopsis HIT1 gene." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/40776152090017723853.
Full textAbstract:
碩士國立中央大學
生命科學研究所
92
A heat-hypersensitive mutant of Arabidopsis was isolated based on its inability to survive under normally non-lethal high temperature condition. The mutant named hit1(heat-intolerant) is distinguished from wild-type plant via incubation at 37℃ for 4 days. hit1 is unable to survive under such condition for more than 4 days. This program is to sequence genomic DNA and identify the locus on chromosome. Current results suggest that the hit1 locus is on BAC F11F12, within the gene whose login number in the Arabidopsis database is AT1g50500, and the mutation of hit1 is a single nucleotide changing from C to A. Amino acid sequence alignment indicated that AT1G50500 contains conserve domain KOG2180, and is highly similar to the late golgi membrane sorting complex, subunit VPS53. Vps53p exists in the yeast, forming a complex with Vps52p and Vps54p, and the main function of the complex is to transport carboxypeptidase Y (CPY) from trans golgi membrane to prevacuolar / endosomal compartment (PVC). It has been indicated by some documents that CPY and HSP might have similar function in terms of structures. According to these data, it is reasonable to hypothesize that the HIT1 might play a role in transporting HSP in the plant cell. On the other hand, HIT1 could affect water balance in plants. Wild-type plants grow better than hit1 mutant at high mannitol concentrations. Taken together, HIT1 may function in sorting certain proteins whose roles are critical for plants to cope with both heat and osmotic stresses.
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Pey, Nan-Shan Kristy. "Automatic cloning vectors for large scale DNA sequencing." 1991. http://catalog.hathitrust.org/api/volumes/oclc/23717675.html.
Full textAbstract:
Thesis (M.S.)--University of Wisconsin--Madison, 1990.Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 36-42).
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Lang, Walter H. "CDNA cloning and sequencing of Octopus dofleini hemocyanin /." 1990. http://hdl.handle.net/1957/14155.
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LIN, XIANG-YOU, and 林相佑. "cDNA cloning and sequencing of grass carp prolatin." Thesis, 1989. http://ndltd.ncl.edu.tw/handle/20618571571637426953.
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Chang, Chin-Sun, and 張集昇. "Cloning and sequencing polyphosphate kinase-like of Thiosphaera pantotropha." Thesis, 1996. http://ndltd.ncl.edu.tw/handle/84555723189928966892.
Full textAbstract:
碩士國立中山大學
海洋資源學系
84
We have constructed the polyphosphate kinase gene of E. coli into different vectors, then obtained different strains. One strain was controlled by lac promoter and three clones were controlled by tac promoter. The difference of the p-tac controlled strains is that one has a fusion of eight amino acids with vector at the front of polyphosphate kinase, another have original form of polyphosphate kinase protein. By using these stains, we can study the controlled effect of the original polyphosphate kinase promoter of E. coli , the physiological functions of the polyphosphate kinase, and how this enzyme be affected by the environments and nutritions. Meanwhile we used the DNA sequence of polyphosphate kinase to synthesize a pair of primers, to clone a 2.4 kb DNA fragment from T. pantotropha genome library. This DNA fragment had been sequenced, which contained many open reading frames. we subcloned the 2.4kb DNA fragment into six IPTG inducible clones. Among these six clones, two can express a large protein. The sizes of the two proteins expressed are 19 and 20 kd, respectively. The relationship between the 2.4kb DNA and the polyphosphate kinase will be studied in our laboratory.
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Xie, Gu Yun, and 謝汩雲. "Cloning and sequencing of promoter regions from xanthomonas campestris." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/84336163085902782240.
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Shieh, Mih-Yun, and 謝汨雲. "Cloning and sequencing of promoter regions from Xanthomonas campestris." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/66315266601425932785.
Full textAbstract:
碩士國立中興大學
植物學系
82
To understand gene expression and regulation in Xanthomonas, we are interested in isolating promoters for investigatstigation of structures which determine the efficiency of transcription initi- ation. For this purpose we have constructed a family of promoter- proving vectors based on the replication origin of RK2 plasmid. pFY7, one of the constructed plasmid which carried the tetracyc- line resistance gene and promoterless luciferase reporter gene (luxA-luxB) downstream from useful cloning sites, was used for selection of promoter sequences and determination of the trans- criptional activity of cloned fragments in Xanthomonas. In this study, Xanthomonas campestris (Xc) chromosome and plasmid pXV65 DNA were digested with Sau3AI, fragments with sizes about 500 bp were recovered,and then cloned into the vector pFY7 for construc- tion of "promoter banks". The banks were then electroporated into Xc17, scoring for bioluminescence. Twelve clones, eight from Xc17 chromosome DNA and four from plasmid pXV65, with inserts ranging from 50-700 bp were isolated. Only two were able to express luci- ferase after transferring into E. coli.Sequence determination and analysis showed that clone pX727 carried an open reading frame of 29 amino acids with a sequence which had homology to that of the E. coli threonine dehydrogenase. The transcriptional initiation sites directed by these Xc promoters should be determined by pri- mer extension; and then, upstream sequences of the initiation points could be compared and analyzed.
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Wu, Hui-Hua, and 吳惠華. "Cloning and Sequencing of a Trimubin cDNA from Trimeresurus mucrosquamatus." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/11721294897029834395.
Full textAbstract:
碩士國防醫學院
解剖學研究所
83
Trimeresurus mucrosquamatus, a common venomous snake in Taiwan, belongs to the family Viperidae. In previous study we have obtained a partial cDNA clone by immunoscreening from venom gland cDNA library of T. mucrosquamatus. Partial cDNA sequence analysis indicate a strong homology with that of snake venom protein batroxobin. Batroxobin is a thrombin-like enzyme isolated from Bothrops atrox which are also member of the family Viperidae. Batroxobin has a defibrinogenerating effect and is clinically used for the treatment of thrombotic diseases. In this study, a full-length cDNA isolated from T. mucrosquamatus and designated as trimubin. The cDNA sequence and the structure and functional relationships of trimubin cDNA is characterized. The 5''-rapid amplification of cDNA end (RACE) are used in the isolation and construction of the full-length cDNA. Both RACE amplified cDNA and cloned partial cDNA detects a message with a similar size of 1.5 kb. Northern blot analysis by this result suggests that the RACE amplifi ed cDNA and cloned partial cDNA is in the same gene but on the opposite end. The full-length trimubin cDNA consists of 1576 bp with a 5''-noncoding region of 188 bp and a 3''-noncoding segment of 614 bp. Based on the sequence homology comparison and Kozak sequence analysis, the protein translation begins at 189 base and stops at 962 base. The encoding region in between is 771 bases and could code for 257 amino acids with a calculated molecular weight of 28.1 kDa. A typical poly (A) signal, AATAAA is located 15 bases in the upstream of the poly (A) tail. Amino acid sequence comparison shows strong homology with venombin A, ancrod and batroxobin. trimubin is a serine proteinase.
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"Molecular cloning and DNA sequencing of EBV--specific DNase gene." Chinese University of Hong Kong, 1996. http://library.cuhk.edu.hk/record=b5888748.
Full textAbstract:
Ng Dean Yew, Dennis.Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 85-98).
Abstract --- p.i
Acknowledgments --- p.iii
Table of contents --- p.iv
List of figures --- p.vii
List of tables --- p.ix
List of abbreviation --- p.x
Chapter Chapter 1 --- Introduction
Chapter 1.1. --- History --- p.1
Chapter 1.2. --- Classification and structure of Epstein-Barr Virus --- p.2
Chapter 1.3. --- Genomic organization of EBV --- p.3
Chapter 1.4. --- Replication cycle of EBV --- p.5
Chapter 1.5. --- EBV latent and lytic cycle proteins --- p.6
Chapter 1.6. --- Clinical diseases associated with EBV Infection --- p.11
Chapter 1.7. --- Association of EBV and NPC --- p.13
Chapter 1.8. --- EBV serological markers in the diagnosis of NPC --- p.13
Chapter 1.9. --- Sources of EBV-specific DNase --- p.15
Chapter 1.10. --- Characteristics of Epstein-Barr virus alkaline DNase --- p.15
Chapter 1.11. --- Aim of the project --- p.18
Chapter Chapter 2 --- Materials & Methods
Chapter 2.1. --- Molecular cloning --- p.19
Chapter 2.1.1. --- Cell culture --- p.19
Chapter 2.1.2. --- mRNA purification --- p.19
Chapter 2.1.3. --- First strand cDNA synthesis --- p.21
Chapter 2.1.4. --- Polymerase chain reaction (PCR) of cDNA --- p.21
Chapter 2.1.5. --- Purification of PCR product after gel electrophoresis --- p.22
Chapter 2.1.6. --- Ligation of PCR amplified DNase gene into pUC18 Sma/BAP vector --- p.23
Chapter 2.1.7. --- Transformation by electroporation --- p.24
Chapter 2.1.7.1. --- Cell preparation --- p.24
Chapter 2.1.7.2. --- Electroporation procedure --- p.25
Chapter 2.2. --- Extraction ofplasmid DNA --- p.28
Chapter 2.2.1. --- Boiling preparation --- p.28
Chapter 2.2.2. --- Plasmid digestion --- p.29
Chapter 2.3. --- Large-scale purification ofplasmid --- p.29
Chapter 2.4. --- Small-scale purification ofplasmid --- p.32
Chapter 2.5. --- DNA sequencing --- p.33
Chapter 2.5.1. --- Annealing of primer to template DNA --- p.33
Chapter 2.5.2. --- Labelling reaction --- p.34
Chapter 2.5.3. --- Sequencing termination reaction --- p.35
Chapter 2.5.4. --- Prepartion of sequencing gel --- p.36
Chapter 2.5.5. --- Autoradiography of sequencing gel --- p.38
Chapter 2.6. --- Epitope mapping --- p.39
Chapter 2.6.1. --- Processing of EBV- specific DNase peptides --- p.39
Chapter Chapter 3 --- Results
Chapter 3.1. --- Molecular cloning --- p.41
Chapter 3.1.1. --- Cell culture --- p.41
Chapter 3.1.2. --- mRNA purification --- p.42
Chapter 3.1.3. --- PCR amplification --- p.42
Chapter 3. 1.4 --- DNA purification of PCR product --- p.42
Chapter 3.1.5. --- Molecular cloning of PCR amplified DNase gene into pUC18 SmaI/BAP vector --- p.44
Chapter 3.1.6. --- Transformation by electroporation --- p.46
Chapter 3.1.7. --- Extraction of plasmid DNA --- p.48
Chapter 3.1.7.1. --- Boiling preparation --- p.48
Chapter 3.1.8. --- Plasmid digestion --- p.51
Chapter 3.2. --- DNA sequencing --- p.51
Chapter 3.2.1. --- Comparison of B95-8 EBV-speicific DNase gene with gene sequence of EBV in GeneBank --- p.50
Chapter 3.2.2. --- Comparison of 5' end of Raji & B95-8 EBV derived EBV-specific DNase gene --- p.57
Chapter 3.2.3. --- Comparison of the 3'end of the Raji and B95-8 denved EBV-specific DNase gene --- p.63
Chapter 3.2.4. --- Amino acid sequence homology between B95-8 & Raji EBV-specific DNase --- p.64
Chapter 3.2.5. --- Amino acid sequence comparison between the 3' end of the B95-8 EBV DNase protein with that of the Raji EBV DNase protein --- p.62
Chapter 3.3. --- Epitope mapping --- p.67
Chapter 3.3.1. --- Amino acid key --- p.67
Chapter 3.3.2. --- Amino acid sequence of peptides --- p.73
Chapter 3.3.2. --- O.D. readings at 492nm of five histologically proven NPC sera --- p.74
Chapter Chapter 4 --- Discussions
Chapter 4.1. --- Overall strategy --- p.75
Chapter 4 2 --- Significance of EBV-specific DNase as marker for NPC --- p.76
Chapter 4.3. --- Characterization of EBV-specific DNase --- p.76
Chapter 4.4. --- Molecular cloning of PCR amplified gene into PUC18 SmaI/BAP vector --- p.77
Chapter 4.4.1. --- Cell culture --- p.77
Chapter 4.4.2. --- PCR amplification --- p.73
Chapter 4.4.3. --- "Blunting,kinasing and ligation of EBV-specific DNase cDNA into pUC18 vector" --- p.78
Chapter 4.4 .4 --- .Transformation by electroporation --- p.80
Chapter 4.4.5. --- Restriction enzyme digestion of pUC18/EBV-DNase plasmid … --- p.81
Chapter 4.5. --- DNA sequencing --- p.81
Chapter 4.6. --- Epitope mapping --- p.83
Reference --- p.85
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"CLONING, EXPRESSION AND SEQUENCING OF CITRATE SYNTHASE FROM THERMOPLASMA VOLCANIUM." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/1260432/index.pdf.
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Tsai, Wen-Jie, and 蔡文杰. "Gene cloning and sequencing of mouse kidney 15-hydroxyprostaglandin dehydrogenase." Thesis, 1996. http://ndltd.ncl.edu.tw/handle/77004329016326177862.
Full textAbstract:
碩士國立成功大學
生物學系
84
Prostaglandins are a family of biologically potent fatty acids derived fromarachidonic acid. They are ubiquitously present and have actions in almostevery mammalian tissue. The important functions mediated by prostaglandinsinclude platelet aggregation, smooth muscle contraction, growth of tumor cells, inflammatory and immune response. Prostaglandins also act on reproductive system, excretory system and nervous system. Prostaglandins are relatively short-lived in vivo with many of them being biologically inactivated by the enzyme NAD+-dependent 15-hydroxyprostaglandindehydrogenase (15-OH-PGDH). 15-OH-PGDH catalyzes the first step in thecatabolic pathway of prostaglandins. This enzyme oxidizes the 15-hydroxy group to a keto group with subsequent reduction in biological activity of prostaglandins. In this study, human placental 15-OH-PGDH cDNA was used as probes to screen mouse kidney cDNA library. Two 15-OH-PGDH clones were obtained and one of themwas chosen and excised the pBluscript phagemids containing the cDNA inserts using helper phage. After infection with E. coli, the pBluscript containing the cDNA insert was identified by southern blotting and the cDNA was subjected to nucleotide sequencing by dideoxy chain-termination method. The mouse kidney 15-OH-PGDH cDNA was shown to contain 822 bp. Comparing with human placental15-OH- PGDH, mouse kidney 15-OH-PGDH has 43% similarity with human placental enzyme.
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羅祥雲. "Cloning and Sequencing of the Possible Repetitive Sequence in Xiphophorus." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/42189589748406523955.
Full textAbstract:
碩士東吳大學
微生物學研究所
82
From the analysis of the possible relationships between the heritable melanoma formation in the xiphophorine hybrids and the retrovirus, many DNA sequences in xiphophorine genome with homology with the reverse transcriptase gene pol has been found. The proposed characteristics of these DNA sequences were similar with the dispersed repetitive sequences named as retroposon found in the vertebrate. In this srudy, we used these DNA sequences isolated from a cDNA library as a probe to screen the Xiphophorus genomic library. All the clones derived from randomly choosed genomic library gave signals under low stringent condition, but only 3 clones still gave signals when high stringent condition was used. We cloned and sequenced the DNA fragments which gave strong signals in the high stringent condition. These DNA fragments was termed as R-DNA. Surprisingly, the sequence of R-DNA was same as insertion sequence 5 (IS5) found in the bacterial chromosome or replicon. Because IS5 are capable of transposing themselves onto another replicon, the R DNA fragment might be derived from IS5 transposed during cloning. By comparison the characteristics between this R-DNA and the transposed IS5, no distinct similarities had been identified. Furthermore, by using two sequence specific primers, we synthesized the same R-DNA sequence through a PCR reaction directly by using xiphophorine DNA as template, the annealing temperature was 65℃. From these data, the R DNA fragment may be the dispersed repetitive sequence in xiphophorine genome.