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黃雅誼 and Nga-yi Queenie Wong. "DNA engineering utilizing thymidylate synthase A (thyA) selection system in Escherichia coli." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31226851.
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Wong, Nga-yi Queenie. "DNA engineering utilizing thymidylate synthase A (thyA) selection system in Escherichia coli /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?
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Daum, Marilyn. "Geneplanner : a prototype of an expert system to assist with chemical DNA gene synthesis planning /." Online version of thesis, 1989. http://hdl.handle.net/1850/10576.
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Kepple, Kevin V. "Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208620.
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Comeaux, Jay Louis. "Transfer of plasmids by genetically-engineered Erwinia carotovora." Thesis, Virginia Tech, 1989. http://hdl.handle.net/10919/45933.
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The ability of a genetically-engineered Erwirzia carotovora subsp. carotovora (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or Pseudomonas fluorescens was tested on filters, within soil microcosms, and in planta. Ecc was engineered by chromosomal insertion of a disarmed endo-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10-2 transconjugants per donor (TPD) and to P. fluorescens at a frequency of 2.4 X 10-5 TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10-4 and 2.0 X 10-4, while matings on potato slices yielded frequencies of 4.7 X 10-2 and 2.3 X 10-2. In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10-3 and 8.4 X 10-5 TPD.
Master of Science
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Dambros, Régia Maria Feltrin. "Amplificação, clonagem e expressão de proteína recombinante do vírus da doença de Aujeszky em sistema de baculovírus para utilização em programa de controle e erradicação." Universidade do Estado de Santa Catarina, 2006. http://tede.udesc.br/handle/handle/966.
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Aujeszky s disease (AD) is an infect-contagious illness that causes serious economical damages to the producer and the swine industry. Aiming to develop mechanisms and to improve technologies that are faster, more sensitive and more specific for diagnosis and for use in free areas or in AD s eradication programs, the sequence codifier of the glycoprotein E (gE) of Aujeszky s disease virus (ADV) was amplified, cloned and expressed. Through genetic engineering the sequence of the gE gene was propagated in an host organism. The gE was amplified by the technique of polimerase chain reaction (PCR), cloned in the vector pGem®-T Easy and transformed in competent cells of Escherichia coli, DH-5a . The clone obtained was sub-cloned in the expression plasmid pFastBac 1, which contains the promoter gene of the polyhedrin. The obtained subclone was analyzed inside for certification of its correct plasmid orientation with the restriction endonucleases BamH I and EcoR I. The sub-clone with the correct orientation had its DNA extracted and used for transposition inside the bacmid (recombinant baculovirus and helper plasmid with competent DH10Bac cell). Colonies with inserted gE were selected by the phenotype of the colony, which expresses white color when cloned. White recombinant colonies had their DNA extracted and used for cotransfection in insect cells Trichoplusia ni (BTI-Tn5B1-4). The recombinant-gE baculovirus was inoculated in cultured cells and expressed the recombinant gE by PCR and Western blotting . The recombinant-gE baculovirus containing only the gE gene of the VDA will be used for antigen and monoclonal antibodies production, which will aid in the development of a more sensitive, specific and safer for the use in VDA free regions
A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa graves prejuízos econômicos ao produtor e à agroindústria suinícola. Com o objetivo de desenvolver insumos e aprimorar tecnologias que sejam mais rápidas, sensíveis e específicas de diagnóstico para uso em regiões livres ou em erradicação da DA, a seqüência codificadora da glicoproteína E (gE) do vírus da doença de Aujeszky (VDA) foi amplificada, clonada e expressada. Através da engenharia genética a seqüência do gene da gE foi propagada em um organismo hospedeiro. A gE foi amplificada pela técnica da reação em cadeia da polimerase (PCR) , clonada no vetor pGem®-T Easy e transformada em células competentes de Escherichia coli, DH-5a . O clone obtido foi subclonado no plasmídeo de expressão pFastBac 1, o qual possui o sítio promotor do gene da poliedrina. O subclone obtido foi analisado para certificação de sua orientação correta dentro do plasmídeo com as endonucleases de restrição BamH I e EcoR I. O subclone com a orientação correta teve seu DNA extraído e usado para a transposição dentro do bacmid (baculovírus recombinante e plasmídeo helper em célula competente DH10Bac ). As colônias com inserto gE foram selecionadas pelo fenótipo da colônia, a qual expressa cor branca quando clonada. As colônias brancas recombinantes tiveram seu DNA extraído e usado para a co-transfecção em células do inseto Trichoplusia ni (BTITn5B1- 4). O baculovírus gE-recombinante ao ser inoculado em cultivo celular, expressou a gE recombinante, comprovada pela técnica de PCR e Western blotting . O baculovírus gE-recombinante contendo apenas o gene da gE do VDA será utilizado para produção de antígeno e de anticorpos monoclonais, o que auxiliará no desenvolvimento de um teste de diagnóstico mais sensível, específico e mais seguro para uso em áreas livres do VDA
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MURATA, YOKO. "Sintese e caracterizacao do hormonio tireotrofico humano recombinante (rec-hTSH) contendo uma sub unidade beta quimerica (rec-hTSHbeta-CTEP hCGbeta)." reponame:Repositório Institucional do IPEN, 1995. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10432.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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MATHOR, MONICA B. "Estudos da expressao genica mediante utilizacao de queratinocitos humanos normais transduzidos com o gene do hormonio de crescimento humano .Possivel utilizacao em terapia genica." reponame:Repositório Institucional do IPEN, 1994. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10410.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Cousseau, Philippe. "Traitement d'image et traitement graphique en genie genetique : application a l'analyse d'images autoradiographiques et a la representation de cartes de molecules d'adn recombinant." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13037.
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La premiere application concerne la lecture automatique des images autoradiographiques de gel d'electrophorese bidimensionnelle de proteines. La seconde application porte sur la realisation d'un logiciel interactif d'aide au dessin assiste par ordinateur, specifiquement dedie a laconstruction et a la representation graphique de cartes de molecules d'adn recombinants
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Jones, Mary Ellen. "Politically Corrected Science: The Early Negotiation of U.S. Agricultural Biotechnology Policy." Diss., Virginia Tech, 1999. http://hdl.handle.net/10919/29868.
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This social history of science policy development emphasizes the impact on the agricultural community of federal policies regarding release of recombinant DNA (rDNA) organisms into the environment. The history also demonstrates that the U.S. Coordinated Framework for Biotechnology Regulation (1986) is based principally in political criteria, not solidly based in science as its proponents claimed. The power struggle among policy negotiators with incompatible belief systems resulted in a political correction of biotechnology. I also demonstrate that episodes in the rDNA controversy occur in repetitive and periodic patterns.
During the 1980s, the first rDNA microbial pesticide, Ice-Minus, struggled through a policy gauntlet of federal agency approval processes, a Congressional hearing, and many legal actions before it was finally released into the environment. At the height of the controversy (1984-1986), the Reagan Administration would admit no new laws or regulations to slow the development of technologies or hinder American international competitiveness. At the same time, Jeremy Rifkin, a radical activist representing a green world view, used the controversy to agitate for social and economic reform. Meanwhile, a group of Congressional aides who called themselves the "Cloneheads" used the debate to fight for more public participation in the science policy-making process.
Conflicting perspectives regarding biotechnology originated, not in level of understanding of the science involved, but in personal perspectives that were outwardly expressed as political group affiliations. The direction of federal biotechnology policy was influenced most successfully by politically best-positioned individuals (what I call a "hierarchy effect") who based decisions on how biotechnology harmonized with their pre-existing beliefs. The success of their actions also depended on timing.
Historical events during the rDNA controversy followed the same periodic pattern--gestation, threshold, crisis/conflict, and quasi-quiescence--through two consecutive eras--the Containment Era (1970s) and the Release Era (1980s). These periods are modeled after Fletcher's stages through which ethical issues evolve (1990). However, an agricultural perspective on the debate reveals that such stages also occur in finer detail on repeating, overlapping, and multi-level scales. Knowledge of this periodicity may be useful in predicting features of future episodes of the rDNA controversy.Ph. D.
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Houston, Peter Louis. "Biochemical characterization of genetic recombination proteins /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Jenner, Kris Harlan. "The study of inherited diseases using recombinant DNA technology." Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670385.
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Francis, Murray A. "Characterisation of DNA damage inducible responses and repair in human cells using recombinant adenovirus vectors /." *McMaster only, 2000.
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Tang, Shiue-Cheng. "Genetic engineering of non-beta-cells for regulated insulin secretion." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04072004-180222/unrestricted/tang%5Fshiue-cheng%5F200312%5Fphd.pdf.
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Selva, Erica Marie. "Mismatch Repair Acts As a Barrier to Homeologous Recombination in Saccharomyces cerevisiae: A Dissertation." eScholarship@UMMS, 1996. https://escholarship.umassmed.edu/gsbs_diss/61.
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Homeologous recombination refers to genetic exchanges between DNA partners containing similar but not identical DNA sequences. Heteroduplex intermediates in such exchanges are expected to contain multiple DNA mismatches at positions of sequence divergence and hence are potential targets for mismatch correction. Thus recombination of this type is of particular interest in understanding the role of DNA mismatch correction on recombination fidelity. Previous studies that examined this question in prokaryotic systems suggested that mismatch repair acts as a barrier to recombination between diverged sequences. The central hypothesis of this thesis is that mismatch correction acts as a barrier to homeologous recombination in yeast. The objectives of these studies was to elucidate the role of mismatch correction in homeologous recombination as a means of dissecting its mechanism in eukaryotic organisms.
To examine homeologous genetic events in yeast, I developed an in vivo assay system to evaluate recombination between diverged DNA sequences. A homeologous gene pair consisting of Saccharomyces cerevisiae SPT15 and its Schizosaccharomyces pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene. Each gene carried a different inactivating mutation, rendering this starting strain Spt15-. Recombinants that regenerated SPT15function were identified by genetic selection and the rates of recombination in different backgrounds were compared.
The homeologous mitotic recombination assay was utilized to test the role of S. cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. In strains wild type for mismatch repair, homeologous recombination was reduced 150-180 fold relative to homologous controls, indicating that multiply mispaired sequences act in cis as part of an inhibitory mechanism. In the upstream orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17-fold and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1deletion mutant did not exhibit elevated homeologous recombination in either case.
Next, I investigated whether mismatch correction acts as a specific or general obstacle to homeologous recombination by blocking one or many exchange pathways. To answer this question, I performed structural analysis on numerous recombinant products from each strain to determine the percentage of products that fell into a given class (crossovers or gene conversions). Each percentage was then multiplied by the overall rate to arrive at a rate of recombination for individual events. Typically 90-100% of homologous and homeologous recombinant products could be accounted for, either as crossovers or gene conversions. Recombination for all classes of products was inhibited when divergent sequences were present, indicating that homeology blocks formation of both crossovers and gene conversions. Sequence analysis of a limited number of homeologous recombinants indicated that transfer of DNA occurred in continuous stretches and that endpoints fell within regions of 3-11 base pairs of perfect homology. Mutations in the mismatch repair genes MSH2 or MSH3that elevate the overall rate of homeologous recombination produced similar rate increases in formation of each recombinant class. This suggests that mismatch correction proteins block multiple types of homeologous recombination events. Taken together, these results support the hypothesis that homeologous and homologous recombination occur by the same (or similar) pathways, with mismatch repair superimposed as an extra level of control over the fidelity of the process.
I also investigated whether this homeologous recombination system would be useful in a genetic screen to identify novel genes or new alleles of genes known to increase exchanges between diverged DNA sequences. Hyperhomeologous recombination mutants were isolated following ethylmethane sulfonate mutagenesis of yeast that harbored the spt15 homeologous duplication. Preliminary characterization of these mutants demonstrated that some of these isolates yielded phenotypes that were consistent with mutations in mismatch repair genes verifying the utility of this method to identify such mutants. To improve the use of this system for a mutant screen, I developed a second generation homeologous duplication using URA3. These new starter strains are expected to be important for efficient isolation and characterization of hyperhomeologous recombination mutants.
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Bandmann, Nina. "Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification." Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4318.
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Eksteen, Jeremy Michael. "Construction of recombinant Saccharomyces cerevisiae strains for starch utilisation." Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/52745.
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Thesis (MSc)--University of Stellenbosch, 2002.ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are usually used for the production of whisky. One of the first steps in the production of whisky is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is performed to disrupt and solubilise the starch granules to make them more accessible for enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and then saccharified by glucoamylase and a debranching enzyme. Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in existing industrial fermentations because of their low ethanol tolerance, slow growth rate, catabolite repression, poorly characterised genetics and lack of GRAS (Generally Regarded As Safe) status. This study is divided into two sections. The aim of the first section was to clone a gene (LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid, YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and terminator (PGKh) expression cassette. This recombinant plasmid was designated pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid assays revealed that the recombinant yeast secreted active a-amylase into the medium. The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C. The aim of the second part of the study was to construct recombinant strains of S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase (SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids expressing the individual coding sequences under the control of the PGK1 p-PGK1.,- expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively; two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were transformed into a laboratory strain of S. cere visiae , L1278b. By homologous recombination, each plasmid was integrated into the yeast genome at the ura3 locus. S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of a-amylase activity when assayed for amylolytic activity in a liquid medium. This recombinant strain resulted in the most efficient starch utilisation in batch fermentations, consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation. The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette gave the highest levels of glucoamylase activity.' These results confirmed that co-expression of a-amylase and/or glucoamylase synergistically enhance starch degradation. This study paves the way for the development of efficient starch-degrading strains of S. cerevisiae for the production of whisky, beer and biofuel ethanol.
AFRIKAANSE OPSOMMING: Styselbevattende landbougewasse kom wydverspreid voor as die substraat vir die produksie van brandstofetanol, drinkbare spiritualië of bier, enkelselproteïen en hoëfruktose graanstroop. Styselbevattende gewasse, soos mielies, rog, gars en koring, word gewoonlik vir die produksie van whisky gebruik. Die eerste stap in die produksie van whisky is om die stysel by temperature bo 1DOOG te kook. Hierdie jelatinisasie stap word uitgevoer om die styselkorrels te versteur en vloeibaar te maak sodat hulle meer toeganklik vir ensimatiese hidrolise is. Na dié kookproses word die stysel deur o-arnilases vervloei en dan deur glukoamilases en 'n vertakkingsensiem versuiker. Lipomyces kononenkoae en Saccharomycopsis filuligera skei hoogs effektiewe a-amilases en glukoamilases uit, wat dit twee van die effektiefste rou-stysel-afbrekende giste bekend, maak. L. kononenkoae en S. fibuligera kan egter nie in reeds bestaande industriële fermentasies gebruik word nie, as gevolg van hulle lae etanoltoleransie, stadige groeitempo, katabolietonderdrukking, swak gekarakteriseerde genetika en gebrek aan ABAV (Algemeen Beskou As Veilig) status. Hierdie tesis is in twee afdelings verdeel. Die doel van die eerste deel was om 'n geen (LKA2) wat vir 'n nuwe, unieke styselafbrekende ensiem kodeer, te kloneer, 'n tweede a-amilase (Lka2p) van L. kononenkoae. LKA2 is in 'n multikopie plasmied, die gis episomale plasmied, YEp352, onder beheer van die fosfogliseraatkinasepromotor- en termineerder-kasset (PGK1 p-PGK1 r), gekloneer. Hierdie rekornbinante plasmied is pJUL3 genoem en in 'n laboratoriumras van Saccharomyces cerevisiae, L:1278b, getransformeer. Plaat- en vloeibare-ensiem toetse het getoon dat die rekombinante gis aktiewe a-amilase in die medium uitskei. Die optimum pH vir Lka2p is 3.5, is en die optimum temperatuur 60oG. Die doel van die tweede deel van die studie was om rekombinante rasse van S. cerevisiae te konstrueer wat a-amilases en/of glukoamilases uitskei. Die individuele gene is toe in 'n gis-integreringsplasmied, Ylp5, onder beheer van die PGK1p-PGK1,ekspressiekasset, gekloneer. Twee inheemse giste is op grond van hulle vermoë om stysel te benut geselekteer, L. kononenkoae en S. filuIigera, as geen donors. Agt konstrukte bevattende die L. kononenkoae se a-amilasegene, LKA 1 en LKA2, en S. filuligera se a-amilasegeen (SFA 1) en glukoamilasegeen (SFG1), moes gekonstrueer word: vier _enkel-kasset plasmiede wat die individuele koderende sekwense onder beheer van die PGK1 p-PGK1, ekspressiekasset uitdruk, wat onderskeidelik plPLKA 1, pIPLKA2, plPSFA 1 en plPSFG1 lewer; twee dubbel-kasset plasmiede (wat beide LKA 1 en LKA2 onder beheer van die PGK1 p-PGK1,ekspressiekasset, en SFA 1 en SFG1 met hulle onderskeie inheemse promotors en termineerders) uitdruk, wat onderskeidelik pIPLKA1/2 en plPSFAG lewer, en twee enkel-kasset plasmiede wat SFA 1 and SFG1 met hulonderskeie inheemse promotors en termineerders, en wat onderskeidelik pSFA 1 en pSFG1 lewer. Die onderskeie konstrukte is in 'n laboratoriumras van S. cerevisiae, L1278b, getransformeer. Deur middel van homoloë rekombinasie, is die onderskeie plasmiede in die ura3-lokus van die gisgenoom geïntegreer. S. cerevisiae L1278b, getransformeer met plPLKA 1/2, LKA 1 en LKA2 onder die beheer van die PGK1 ~PGK1 ïekspressiekasset, het die hoogste vlakke van a-amilase aktiwiteit gelewer toe dit vir amilolitiese aktiwiteit in vloeibare medium getoets is. Hierdie rekombinante ras het stysel die effektiefste benut, nl. 80% van die stysel en 'n opbrengs van 6 gIL etanol na 156 ure in lotfermentasies. Die ras wat SFG1 onder beheer van die PGK1~PGK1ïekspressiekasset uitdruk, het die hoogste vlakke van glukoamilase-aktiwiteit gelewer. Hierdie resultate bevestig dat die gesamentlike uitdrukking van a-amilase- en/of glukoamilase-ensieme styselafbreking sinergisties . bevorder. Hierdie studie baan die weg vir die ontwikkeling van 'n effektiewe styselfermenterende ras van S. cerevisiae wat moontlik gebruik kan word vir die produksie van whisky en biobrandstofalkohol.
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Schoombie, Johannes Loubser. "Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/23688.
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Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs.Dissertation (MSc)--University of Pretoria, 2012.
Biochemistry
unrestricted
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Huen, Shing-yan Michael. "A mechanistic study of lambdaphage-mediated recombination in E. coli." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B35321854.
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Hartzell, Brittany M. "DNA manipulation and characterization for nanoscale electronics." Ohio : Ohio University, 2004. http://www.ohiolink.edu/etd/view.cgi?ohiou1108051644.
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Grip, Stefan. "Artificial spider silk : recombinant production and determinants for fiber formation /." Uppsala : Dept. of Biomedical Sciences and Veterinary Public Health and Dept. of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/2008100.pdf.
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Chen, Yan, and 陳岩. "Recombinant adenovirus and adeno-associated virus mediated BMP2 and BMP4 gene therapy for new bone formation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31244038.
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Ndabambi, Nonkululeko. "Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies." Thesis, University of the Western Cape, 2004. http://etd.uwc.ac.za/index.php?module=etd&.
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The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.
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Davis, Ashley Stuart. "Aspects of rice transformation using direct DNA uptake." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258367.
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Thanvanthri, Gururajan Vasudevan. "Enhancing xylose utilisation during fermentation by engineering recombinant Saccharomyces cerevisiae strains." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/18705.
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Dissertation (DPhil)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess.
AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is. Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe. Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en Afrika) te verminder. Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word, maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die alkoholbedryf gemaak. Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam, Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk, het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis. Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader: (i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk te wees. Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.
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Ciciotte, Steven. "Characterization of CRE Recombinase Expression in Erythroid Tissues of Transgenic Mice." Fogler Library, University of Maine, 2005. http://www.library.umaine.edu/theses/pdf/CiciotteS2005.pdf.
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Huen, Shing-yan Michael, and 禤承恩. "A mechanistic study of lambdaphage-mediated recombination in E. coli." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B35321854.
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Mukre, Prakash. "Hardware accelerator for DNA code word searching." Diss., Online access via UMI:, 2008.
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Thesis (M.S.)--State University of New York at Binghamton, Thomas J. Watson School of Engineering and Applied Science, Department of Electrical and Computer Engineering, 2008.Includes bibliographical references.
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Dahl, Fredrik. "Selector Technology : For Multiplex DNA Analysis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5921.
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Aboluion, Niema Ali. "The construction of DNA codes using a computer algebra system." Thesis, University of South Wales, 2011. https://pure.southwales.ac.uk/en/studentthesis/the-construction-of-dna-codes-using-a-computer-algebra-system(d0ee33ce-c640-407d-868c-ba5eafa81909).html.
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Coding theory has several applications in Genetics and Bioengineering. This thesis concentrates on a specific application from Computational Biology. This concerns the construction of new DNA codes which satisfy certain combinatorial constraints, using an alphabet of four symbols. The interest in these codes arises because it is possible to synthesise short single strands of DNA known as oligonucleotides. The codes can be useful in the design of these oligonucleotides. For example, the codes are used in DNA computing, as bar codes in molecular libraries and in microarray technologies. The computer algebra system Magma, which deals successfully with coding theory computation, is applied initially to the construction of DNA codes sat- isfying a GC-content constraint and a minimum Hamming distance constraint. The constraints are specified to avoid unwanted hybridizations and to ensure uniform melting temperatures. Additionally, another constraint, known as a reverse-complement constraint, is added to further prevent unwanted hybridiza- tions. This additional constraint is studied using involutions in a permutation group. Codes constructed in this thesis are derived from linear codes over GF(4) and Z4 and additive codes over GF(4). Previous approaches to the construction of these codes are extended in several ways. Longer codes are constructed, the examination of cyclic and extended cyclic codes is more comprehensive, and cosets of codes are considered. In addition, attention is paid to the mapping from field or ring elements to the DNA nucleotides; different mappings can give different lower bounds. Further improvements have been made after the tech- niques of shortening and puncturing are applied to the table of best codes, and also by searching for codes in the tables that have an all-ones vector in their dual. The use of a database of best known linear codes is also considered. In many cases codes are obtained which are larger than the best codes currently known. In the case of codes of length greater than twenty, linear DNA codes have not been constructed previously and so all codes obtained are the best known re- sults. Generator polynomials are given for the codes constructed. Coset leaders are also given in cases where cosets of linear codes are used. Thus it is possible for the reader to construct the codes without repeating the work presented in the thesis. Additionally, files of codewords are available online when the codes constructed are the best known codes and have fewer than 50000 codewords.
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Forget, Anthony L. "Homologous Recombinational DNA Repair: from Prokaryotes to Eukaryotes: a Dissertation." eScholarship@UMMS, 2004. https://escholarship.umassmed.edu/gsbs_diss/68.
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The error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial recA, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51.
Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein.
These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next.
In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes.
In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.
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Nguyen, Doan C. "Immunoglobulin Gamma Subclasses and Corresponding Fc Receptors in Rhesus Macaques: Genetic Characterization and Engineering of Recombinant Molecules." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_diss/111.
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Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions.
We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.
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Bower, James Earl. "Concerted evolution of the rDNA ITS1 in the Anopheles punctulatus group." Access electronically, 2008. http://ro.uow.edu.au/theses/122.
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Fick, Wilhelmina Christina. "Characterisation of promoter sequences in a Capripoxvirus genome." Master's thesis, University of Cape Town, 1992. http://hdl.handle.net/11427/25623.
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Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.
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Yuniaty, Alice. "The extent and importance of DNA methylation in plants /." [St. Lucia, Qld.], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18698.pdf.
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Zhang, Shuyu. "The Relaxosome protein MobC of plasmid R1162 promotes DNA strand separation at the origin of transfer /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Kam, Ka-man. "Expression analysis of Hoxb5 in enteric neurons and generation of Tamoxifen inducible Cre mice for neuronal Hoxb5 signaling perturbation." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290574.
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Staley, Christopher A. "Characterization of the 5̕ untranslated region ( 5̕ UTR) of the alcohol oxidase I (AOX I) gene in Pichia pastoris." Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/646.
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The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.
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Saxon, Herbert. "The molecular biology of orchids : transformation by Agrobacterium Tumefaciens and DNA fingerprinting." Virtual Press, 1995. http://liblink.bsu.edu/uhtbin/catkey/941575.
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The work reported here was done at the Wheeler Orchid Collection and Species Bank and the Department of Biology at Ball State University. We have developed a research teaching program with two applied research goals: genetically transforming and DNA fingerprinting orchid tissue. As part of their molecular biology education, students have investigated the genetic transformation of orchids for mitigating viral symptoms and the identification of unknown orchids by DNA fingerprinting. In a second application of the technology, DNA fingerprinting has been used to determine evolutionary relationships and to quantify genetic diversity among orchids.This dissertation details the background and need for this project and the research that was done to start it. As the early work has, developed and students have added their contributions, the data have developed into two papers formatted for submission to scientific journals. They are included as results.The first is a project designed to insert exognenous DNA into orchid tissue. The soil microbe Agrobacterium tumefaciens causes crown-gall tumors to develop in its plant hosts by inserting DNA into their cells which then controls the biosynthesis of development-controlling hormones. A. tumefaciens which has been disarmed has been routinely used to bioengineer dicotyledonous plants but its use has been rare on monocotyledons. In this paper, we report that A. tumefaciens transformed embryonic orchid tissue and caused alteration in its normal developmental course.The second paper details the DNA fingerprinting of tissue from Aplectrum hymale, a terrestrial orchid native to this climate. Three populations of A. hymale have been sampled and DNA extracted from the tissue samples. RAPD primers were used to prime PCR amplifications of random sequences of the DNA and the amplified DNA was visualized by gel electrophoresis. Loci of the resulting bands were treated as potentially multiallelic gene loci and heterozygosity between and within subpopulations was calculated. We report that the three populations could be partially differentiated by this procedure and that the two populations located nearest to each other yielded the least between -ubpopulation heterozygosity. We report very high levels of genetic diversity between individuals within small subpopulations in spite of the fact that these subpopulations are considered to be primarily clonal in reproductive nature.Department of Biology
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Kam, Ka-man, and 甘嘉敏. "Expression analysis of Hoxb5 in enteric neurons and generation of Tamoxifen inducible Cre mice for neuronal Hoxb5 signalingperturbation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290574.
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Johnson, Sabrina D. "Characterization of the Pichia pastoris alcohol oxidase I promoter." Scholarly Commons, 2003. https://scholarlycommons.pacific.edu/uop_etds/575.
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The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.
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Boyko, Oleksandr, and University of Lethbridge Faculty of Arts and Science. "The versatile role of homologous recombination in plant cell : repair of DNA damage, stress-directed genome evolution and foreign DNA integration." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2008, 2008. http://hdl.handle.net/10133/724.
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Homologous recombination represents a DNA repair pathway. Its role in a plant cell is not limited to double strand break repair. It also extends to genome evolution via rearranging of DNA sequences, and has an important application in foreign DNA integration in the plant genome. Our study demonstrated that effects exerted by stress on homologous recombination and genome stability are not restricted to the exposed generation. The progeny of plants exposed to stress exhibited elevated spontaneous homologous recombination, changes in DNA methylation and higher tolerance to stress. These heritable changes are mediated by an unknown stress-inducible epigenetic signal. Furthermore, we demonstrated that using factors that enhance homologous recombination can improve the efficiency of genetic transformation by Agrobacterium. We have developed and patented a plant growth medium enhancing homologous recombination and significantly increasing the transformation frequency. The role of several other chemicals for the improvement of transformation was also evaluated.xxi, 246 leaves : ill. ; 29 cm. --
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Conradie, E. C. (Elizabeth Cornelia). "Promotor engineering in Saccharomyces cerevisiae for transcriptional control under different physiological conditions." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/16512.
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Dissertation (PhD)--University of Stellenbosch, 2005.ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”.
AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.
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DeLuca, Adam Peter. "Computational methods for efficient exome sequencing-based genetic testing." Diss., University of Iowa, 2013. http://ir.uiowa.edu/etd/4607.
Full textAbstract:
Exome sequencing, the process of sequencing the set of all known exons simultaneously using next-generation sequencing technology, has dramatically changed the landscape of genetic research and genetic testing. The incredible volume of data produced by these experiments creates challenges in: 1) annotating the affects of observed variants, 2) filtering to remove noise, 3) identifying plausible disease-causing variants, and 4) validating experimental results. Here we will present a series of bioinformatic tools and techniques intended to address these challenges with exome sequencing and associated validation experiments.
First, we will present the Automated Sequence Analysis Pipeline (ASAP), a tool for the efficient and automated management, detection and annotation of Sanger sequencing-based genetic testing and variant validation. This pipeline is extended to annotate exome-sequencing derived variants.
Exome sequencing experiments produce a great number of variants that do not cause a patient's disease. One of the biggest challenges in exome sequencing experiments is sorting through these false positives to discover the true disease-causing variants. We have developed several techniques to aid in the reduction of these errors. The techniques described include: 1) the construction of a catalog of systematic errors by reprocessing thousands of publically available exomes, 2) a tool for the filtering of variants based on family structure and disease assumptions, and 3) a tool for discovering regions of autozygosity from the exomes of several affected patients in consanguineous pedigrees.
Classes of variants that are undiscoverable using current analysis techniques gives rise to false negatives in exome sequencing experiments. We will present a tool, the Retrotransposon Insertion Detector for Exomes (RIDE) that uses the characteristic anomalies present in sequence alignments to detect the insertion of repetitive elements.
The process of identifying a the cause of a patient's disease using exome sequencing data has been equated to finding a needle in a stack of needles. Only through the proper annotation of variants and the reduction of the error rates associated with exome sequencing experiments can this task be achieved in an efficient manner.
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Hu, Xiao Ping. "Identification and characterisation of novel cellulolytic genes using metagenomics." Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_9293_1308049102.
Full textAbstract:
Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study.
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Gulino, Angela Marie. "Insulin secretion dynamics of recombinant hepatic and intestinal cells." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/28220.
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Caliando, Brian James. "Targeted destruction of intracellular DNA using a CRISPR-based genetic device that can be carried indefinitely in the host genome." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/99051.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 101-104).
Environmental release of synthetic DNA resulting from the disposal of spent microbial biocatalyst potentially represents an ecological risk to the environment or a financial risk to biotechnology firms, who might have their intellectual property stolen as a consequence. Thus, a genetically-encoded device that is capable of degrading DNA in a controlled manner would be a valuable and enabling tool. To that end, we have constructed a modular, switchable, genetically-encoded E. coli device for the controlled destruction of user-specified DNA targets in vivo that is based on the organism's native type-IE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) DNA interference (DNAi) pathway. The optimized DNAi device is comprised of two components: a chromosomally-integrated actuator element, which encodes the minimal set of CRISPR-associated (cas) genes required for DNAi activity, and a reprogrammable targeting plasmid, which encodes the CRISPR array specifying the DNA target(s). The device is stable in the OFF state, with >98% of cells retaining a low-copy DNA target over the course of an 8-hr experiment. Upon DNAi activation, the target plasmid is lost from all but 1 in 10⁸ cells and there is a corresponding >10,000-fold decrease in the abundance of the target DNA sequence as recovered by PCR. When the device is targeted to the host genome instead of a plasmid, activation also results in the self-destruction of the host, killing all but -1 in 10⁸ of cells in the ON state but with no appreciable effect on cell viability in the OFF state. Further characterization has also revealed that when DNAi activity is maintained in the OFF state, the overall maintenance cost to the host strain is exceedingly low; the device remains functionally stable over hundreds of cell generations in continuous culture, has little-to-no impact on host growth or plasmid stability, and doesn't interfere with ectopic over-expression of other proteins. The DNAi device is therefore a powerful tool that can potentially be combined with other genetically engineered systems to create safer and more secure forms of biotechnology.
by Brian James Caliando.
Ph. D.
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Stuckey, Samantha Anne. "Gene targeting at and distant from DNA breaks in yeast and human cells." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/51721.
Full textAbstract:
Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.
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Pillai, Suresh Divakaran. "Ecology and genetic stability of Tn5 mutants of bean rhizobia in Sonoran desert soils." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184823.
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Five transposon Tn5 mutants of bean rhizobia (Rhizobium leguminosarum b.v. phaseoli) and the wild type strain were used in ecological studies to evaluate the efficacy of transposon Tn5 as a phenotypic marker in rhizobia for ecological studies in two Sonoran desert soils. All mutants possessed chromosomal insertions of the transposable element. Survival of each mutant strain was compared to that of the wild type strain under non stress, moisture stress and temperature stress conditions in Pima silty clay loam and Brazil to sandy loam. The genetic stability of Tn5 in terms of transposition of the element within the chromosome and the Tn5 coded antibiotic resistant phenotype was determined in cells recovered throughout the survival period. Under non stress conditions, the viable Tn5 mutant population decreased in size. Two mutants showed significantly (p < 0.01) lower populations than the wild type at the end of 30 days in the silty clay loam. In the sandy loam, four of the five mutant populations were significantly lower than the wild type. Tn5 was genetically stable in both soils. Under moisture stress conditions, the decline of the Tn5 mutant and wild type populations corresponded to a decline in soil moisture content. The finer textured soil afforded more protection to the cells than the coarse textured soil. There were no indications of Tn5 instability under moisture stress. In both soils under temperature stress, sizes of all populations declined rapidly and after 12 days, the mutant cells when screened using the Tn5 coded markers were significantly less in numbers than the wild type indicating a loss of Tn5 coded antibiotic resistance phenotype. There were no significant differences in numbers between wild type and mutant cells when screened using only the intrinsic markers. DNA:DNA hybridizations confirmed that the lack of Tn5 coded antibiotic resistance phenotype was probably not due to a deletion or transposition of the element. Under non stress conditions Tn5 is a useful ecological marker, but each Tn5 mutant has to be evaluated independently under specific environmental conditions to determine the efficacy of Tn5 as an ecological marker.
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Beaulieu, Julie. "Exploration of high-density oligoarrays as tools to assess substantial equivalence of genetically modified crops." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97904.
Full textAbstract:
Since the early 1990s, the concept of substantial equivalence has been a guiding principle of the Canadian Food Inspection Agency and Health Canada's regulatory approach toward products of plant biotechnology destined for the food and livestock feed markets. To assess substantial equivalence in terms of chemical composition, genetically modified (GM) plants are compared to conventional counterparts at the level of macro- and micro-nutrients, allergens and toxicants. Such targeted comparative analyses are limited in their scope and their capacity to detect unintended changes in chemical composition. There is a need to develop more effective testing protocols to improve the substantial equivalence assessment of GM crops. The objective of this thesis was to explore high-density oligoarrays as tools to assess substantial equivalence of Roundup Ready(TM) soybean. Three conventional and two GM soybean varieties were selected according to the similarity of their performance in field trials. Total RNA was extracted from first trifoliate leaves harvested from soybean plants grown in a controlled environment until the V2 stage. To annotate the 37 776 soybean probesets present on the multi-organism Soybean Affymetrix GeneChip(TM), consensus sequences were aligned with TIGR Soybean Gene Index tentative consensus sequences using BLASTN. After redefining the chip description file to exclude non-soybean probesets, the effects of three different normalization methods (Robust Multichip Average (RMA), Microarray Analysis Suite (MAS 5.0) and Model-Based Expression Index) were compared and Significance Analysis of Microarrays (SAM for R-Bioconductor) was applied to detect differential gene expression between conventional and GM soybean varieties. Eleven candidate genes were selected for further studies.