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Dissertations / Theses on the topic 'Endonucleasi'

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Published: 9 March 2023
Last updated: 10 March 2023

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1

FIRRITO, CLAUDIA. "Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/94656.

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La terapia genica basata sull’utilizzo di vettori integranti è stata già applicata con successo per la cura di varie malattie genetiche come le malattie da accumulo lisosomiale (LSD), la beta-talassemia (β-Thal) e le immunodeficienze primarie (PID). L’immunodeficienza combinata grave legata al cromosoma X (SCID-X1) è una malattia monogenica letale causata da mutazioni del gene codificante la catena comune gamma del recettore per l’interleuchina 2 (IL2RG). I primi studi clinici per la SCID-X1 hanno mostrato il potenziale terapeutico della terapia genica basata su vettori integranti, risultando nella ricostituzione del compartimento linfoide grazie al vantaggio selettivo delle cellule geneticamente modificate. D’altra parte, tali studi hanno evidenziato il rischio di mutagenesi inserzionale dovuto all’integrazione casuale del virus nel genoma della cellula ospite e all’espressione non regolata del transgene, sottolineando la necessità di sviluppare nuove strategie di terapia genica più sicure. In questo lavoro, sfruttando la tecnologia delle Zinc-Finger Nucleasi (ZFN) per indurre una rottura del doppio filamento del DNA in maniera sito specifica e dei vettori lentivirali difettivi per l’integrazione (IDLV) per l’introduzione di un templato donatore, abbiamo impiegato il processo di riparazione del DNA guidata dall’omologia per la correzione delle mutazioni che causano la SCID-X1, ripristinando così la funzione genica e l’espressione fisiologica del gene IL2RG. Mediante l’integrazione di un cDNA correttivo del gene IL2RG a valle del promotore endogeno sia in cellule B linfoblastodi, che esprimono costitutivamente la catena gamma comune, sia in linfociti T da donatori sani, che richiedono IL2RG per la loro sopravvivenza, abbiamo dimostrato la funzionalità e l’attività fisiologica del gene modificato. Abbiamo quindi accoppiato la correzione genica con la selezione delle cellule mediante l’inclusione di una cassetta excidibile di espressione della GFP o della resistenza alla puromicina (PuroR) a valle del cDNA correttivo, al fine di correggere fibroblasti, che normalmente non esprimono IL2R, derivati da pazienti SCID-X1. Abbiamo quindi ottenuto una popolazione di fibroblasti corretti che abbiamo “ riprogrammato” mediante un nuovo vettore di reprogramming che esprime i fattori di trascrizione (SOX2, OCT4, KLf4) e il microRNA cluster 367, generando così una fonte illimitata di cellule staminali pluripotenti indotte (iPSC) geneticamente corrette di interesse terapeutico. L’espressione transiente della Cre-ricombinasi mediante IDLV ha inoltre permesso l’excisione del vettore di reprogramming e della cassetta di selezione, permettendo così l’ottenimento di cellule iPSC corrette, prive di vettore e con un normale cariotipo. Infine, attraverso il differenziamento delle cellule iPSC in progenitori T-linfoidi, un tipo cellulare assente nei pazienti SCID-X1, e l’osservazione di un vantaggio selettivo delle cellule linfoidi derivate dalle iPSC corrette, abbiamo dimostrato la correzione funzionale dell’allele IL2RG mutato. In conclusione questi dati dimostrano la validità della nostra strategia di integrazione sito-specifica che, mediante la correzione e la riprogrammazione cellulare, consente di ottenere cellule iPSC geneticamente corrette, aprendo la strada a nuove opportunità terapeutiche più sicure per il trattamento della SCID-X1.
Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.
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2

Daniels, Lucy Elizabeth. "The SgrAI restriction endonuclease." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393877.

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3

Chevalier, Brett S. "Homing endonuclease mechanism, structure and design /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/4984.

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4

AlMalki, Faizah. "Structural studies on flap endonuclease complexes." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/7293/.

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Flap endonucleases (FENs) are structure-specific enzymes that play critical roles in DNA replication and repair. Three members of the FEN family have been investigated during this project in complexes with DNA substrates and metal ions: bacteriophage T5FEN, hFEN and Trypanosoma brucei FEN. T5FEN wild type and two catalytically inactive versions, D153K and D155K were successfully crystallized in complexes with DNA substrates containing 5' or 3' overhangs. The crystal structure for T5FEN-D153K in complex with a duplex containing 5' overhangs at each end and two Mg2+ ions was solved. The structure of T5FEN-D155K was solved in complex with a duplex containing 3' overhangs and a Ca2+ ion in the active site. In addition, wild type T5FEN was also crystallized with the same 3'-overhang substrate in the absence of metal ions. These structures revealed that the single strand-5' overhang in T5FEN-D153K pushed electrostatically and looped-up before threading through an arch-like structure composed of two helixes located over the active site of the enzyme. This arch is fully ordered in all of these structures. In the active sites of the variant complexes the Lys-153 and Lys155 are visible. The lysine's long side chain allows its ε amino group to occupy similar positions to the metal ion sites in one of the two active site subsites known as Cat1. The ε amino of Lys-153 directly coordinates the scissile phosphate of the DNA substrate. Important residues concerned with the 5' overhang threading are also determined. His-36 rotates by 180° to allow the duplex DNA movement before threading while Tyr-90 and Phe-105 form a gate-like structure after the 5' overhang has threaded. The conserved Arg-86 plays a critical role in 5' overhang transmission during threading process. Lys-83 and Arg-125 are found to interact symmetrically with the DNA backbone in the T5FEN-D155K:DNA complex. A new trans-arch/distal phosphate-binding site composed of Gly- 70 and Lys-71 has been determined in the far side of the arch. These structures of T5FEN also have a binding site for the potassium ion within the H3TH motif coordinated by the main chain carbonyl oxygens of three residues and directly interacted with the DNA phosphate group.
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5

Barzilay, Gil. "Characterisation of human AP endonuclease I (HAP1)." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318791.

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6

Pernstich, Christian. "Protein dynamics of the restriction endonuclease Fokl." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526007.

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7

Stanford, Neil Philip. "DNA cleavage by the EcoRV restriction endonuclease." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299311.

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8

Wentzell, Lois Marie. "DNA communications by the SfiI restriction endonuclease." Thesis, University of Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388002.

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9

Hanson, Mark Nils. "Biochemical characterization of the endonuclease PMR-1 /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488204276532461.

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10

Zhao, Lei. "Characterization of bacterial homing endonuclease I-Ssp6803I /." Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/9214.

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11

Reuter, Monika. "Die Restriktionsendonuklease EcoRII: Primitives antivirales Abwehrsystem der Bakterien oder mehr?" Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2002. http://dx.doi.org/10.18452/13829.

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Bakterielle Restriktions- und Modifikationssysteme (R/M-Systeme) greifen DNA endonukleolytisch an, die nicht die spezifische Markierung der eigenen Wirtszelle trägt. Zu einem R/M-System gehören eine Restriktionsendonuklease und eine DNA- Methyltransferase gleicher DNA-Spezifität. Die biologische Funktion der Restriktionsendonuklease besteht in der Abwehr von fremder, in die Zelle eindringender DNA, z. B? von Virus-Infektionen. Die korrespondierende DNA-Methyltransferase schützt die zelluläre DNA durch sequenz-spezifische DNA-Methylierung vor der endonukleolytischen Wirkung der Restriktionsendonuklease. Die dimeren TypII- Restriktionsendonukleasen erkennen kurze spezifische, unmethylierte Basensequenzen, die sie in Anwesenheit von Mg2+ Ionen an einer definierten Position endonukleolytisch spalten. Die Restriktionsendonuklease EcoRII braucht die koordinierte Wechselwirkung mit zwei Kopien der Sequenz 5 CCWGG, um katalytisch aktiv sein zu können, wobei eine der beiden Sequenzen als allosterischer Effektor wirkt und nicht gespalten werden muß. Die zwei Kopien der 5 CCWGG Sequenz können sowohl auf demselben als auch auf verschiedenen Molekülen lokalisiert sein. Die Interaktion von EcoRII mit verschiedenen DNA-Molekülen ist durch deren Länge und Konzentration, die Interaktion innerhalb eines DNA-Moleküls durch den Abstand zwischen beiden Sequenzen limitiert. Die durch Proteolyse nachgewiesene Zwei-Domänen-Struktur von EcoRII scheint diese besondere Form der Protein-DNA-Wechselwirkung zu ermöglichen. Die C-terminale Domäne von EcoRII stellt eine neue Restriktionsendonuklease (EcoRII-C) dar. Im Gegensatz zum Wildtyp-Enzym spaltet EcoRII-C an singulären 5 CCWGG Sequenzen. Die trunkierte Endonuklease spaltet DNA spezifisch und unabhängig von einem zweiten EcoRII-Erkennungsort. Die Reaktion verläuft deutlich schneller als die des kompletten EcoRII-Proteins. Die N-terminale Domäne bindet spezifisch DNA, attenuiert die endonukleolytische Aktivität von EcoRII und macht das Enzym abhängig von einer zweiten Kopie der Sequenz 5 CCWGG. EcoRII Wildtyp könnte demzufolge ein evolutionäres Intermediat zwischen einer sequenz-spezifischen Endonuklease und einem Protein sein, das spezifisch mit zwei Orten auf der DNA interagiert, wie z. B. Rekombinasen oder Transposasen. Durch die Kombination beider Funktionen könnte EcoRII selbst die Verbreitung der EcoRII-codierenden DNA-Sequenz in neue Populationen, ähnlich einem transponiblen Element, realisieren.
Bacterial restriction and modification systems (R/M-systems) endonucleolytically attack DNA that is not host cell-specifically modified. R/M-systems comprise a restriction endonuclease and a DNA methyltransferase exhibiting the same DNA sequence specificity. The biological function of the restriction endonuclease is the protection of the cell against invading foreign DNA, e. g. virus infection. The corresponding DNA methyltransferase renders cellular DNA resistent against the endonucleolytic action of the restriction endonuclease by sequence-specific DNA methylation. Dimeric type II- restriction endonucleases recognize short, specific, and unmethylated base sequences that they cut at a defined position in the presence of Mg2+ ions. Restriction endonuclease EcoRII requires the co- ordinated interaction with two copies of the sequence 5 CCWGG for catalytic activity. One of these sequences serves as an allosteric activator site and has not to be cleaved. The two copies of the sequence 5 CCWGG can be located as well on the same as on different DNA molecule(s). EcoRII interaction with two sites on different DNA molecules is limited by their length and concentration, EcoRII interaction within one DNA molecule is limited by the distance between the two sites. The two- domain structure of EcoRII figured out by limited proteolysis studies probably allows this particular form of protein-DNA interaction. The C-terminal domain of EcoRII represents a new restriction endonuclease (EcoRII-C). In contrast to EcoRII wild type, EcoRII-C cleaves DNA at single 5 CCWGG sites. The truncated endonuclease cleaves DNA specifically and independent of a second site. The enzymatic reaction passes well more rapid than that of the complete enzyme. The N-terminal domain binds DNA specifically, attenuates the endonucleolytic activity of EcoRII and makes it dependent on a second copy of the sequence 5 CCWGG. Therefore, the current EcoRII could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites on the DNA such as recombinases and transposases. The combination of both functions may enable EcoRII to accomplish its own propagation similarly to transposable elements.
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12

Wood, Katie Maria. "Interactions of the SgrAl restriction endonuclease with oligoduplexes." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407034.

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13

Oates, S. L. "Molecular characterisation of the Trypanosoma brucei flap endonuclease." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/14274/.

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14

Algasaier, Sana. "Mechanistic studies of flap endonuclease-1 (FEN-1)." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19940/.

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15

Luke, P. A. "The EcoRI and EcoRV restriction endonucleases." Thesis, University of Bristol, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372027.

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16

Goddard, Matthew. "The ecology and evolution of selfish genes." Thesis, Imperial College London, 2000. http://hdl.handle.net/10044/1/11419.

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17

Jacobs, D. "Type II restriction-modified systems in Enterobacter aerogenes and Herpetosiphon giganteus." Thesis, University of Bristol, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379680.

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18

Lagerbäck, Pernilla. "Endonuclease II - a GIY-YIG enzyme of bacteriophage T4." Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9410.

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Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding. It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them.
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19

Sukackaitė, Rasa. "Structural and functional studies of the restriction endonuclease BpuJI." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2009. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2009~D_20091215_091842-18511.

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Type II restriction endonucleases recognize specific DNA sequences and cleave DNA at fixed positions within or close to this sequence. BpuJI recognizes the 5’-CCCGT sequence, but in contrast to other enzymes its cleavage site is very variable. This study shows that BpuJI is a dimer in solution and consists of two separate domains. The N-domain binds to the target sequence as a monomer, while the C-domain is responsible for nuclease activity and dimerization. The nuclease activity is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the N-domains. The activated C-domain cleaves DNA near the target site. In addition, it possesses an end-directed nuclease activity and preferentially cuts ~3 nt from the 3’ terminus. This leads to a very complicated pattern of DNA cleavage. Bioinformatics and mutational analysis revealed that the BpuJI C-domain harbours a PD (D/E)XK active site and is structurally related to archaeal Holliday junction resolvases. The crystal structure of the BpuJI N-domain bound to cognate DNA was solved at 1.3 Å resolution. It revealed two winged-helix subdomains, D1 and D2. The recognition of the target sequence is achieved the amino acid residues located on both the HTH motifs and an N-terminal arm. The BpuJI DNA recognition domain is most similar to the nicking endonuclease Nt.BspD6I. The modelling suggests that Nt.BspD6I could share the specificity-determining regions with BpuJI.
II tipo restrikcijos endonukleazės atpažįsta specifines DNR sekas ir kerpa DNR šiose sekose arba šalia jų. BpuJI, atpažįstanti 5’-CCCGT seką, skiriasi nuo kitų fermentų tuo, kad jos kirpimo vieta yra labai variabili. Čia parodoma, kad BpuJI yra dimeras, sudarytas iš dviejų monomerų, kurie turi po du atskirus domenus. BpuJI N domenas atpažįsta taikinį kaip monomeras, o C-domenas pasižymi nukleaziniu aktyvumu ir dimerizuojasi. Apo-fermento nukleazinis aktyvumas yra nuslopintas. N-domenams atpažinus taikinį, aktyvuojamas C-domenas, kuris perkerpa DNR šalia taikinio. Be to, aktyvuotas C-domenas yra nespecifinė nukleazė, linkusi nukirpti ~3 nt nuo buko dvigrandės DNR galo. Taigi, BpuJI DNR karpymo pobūdis yra labai sudėtingas. Bioinformatinė analizė ir kryptinga mutagenezė parodė, kad BpuJI C-domenas turi PD-(D/E)XK struktūrinę sanklodą ir yra panašus į archėjų Holidėjaus jungtis karpančias nukleazes. Išsprendus 1,3 Å skiriamosios gebos BpuJI N-domeno/DNR komplekso erdvinė struktūrą, paaiškėjo, kad šį domeną sudaro du „sparnuotą“ spiralė-linkis-spiralė motyvą turintys subdomenai. BpuJI taikinį atpažįsta aminorūgštys, esančios N-rankoje ir abiejų spiralė-linkis-spiralė motyvų atpažinimo spiralėse. BpuJI N-domenas yra labiausiai panašus į Nt.BspD6I nukleazę, kerpančią vieną DNR grandinę. Nt.BspD6I/DNR komplekso struktūros modelis rodo, kad Nt.BspD6I ir BpuJI taikinį atpažįstantys struktūriniai elementai yra panašūs.
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Walker, David Colin. "Translocation and cytotoxicity of the HNH endonuclease colicin E9." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368143.

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Williams, Shelley Ann. "Reactions of the SfiI restriction endonuclease with DNA duplexes." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326390.

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22

Nichols, Claire. "Structure-Function Studies of SgrAI: An Allosteric Restriction Endonuclease." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/144913.

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23

Walker, Jeremy N. B. "Identification and characterisation of novel restriction endonucleases." Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277215.

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Marshall, Jacqueline Johanna Tabitha. "BcgI and other type IIB restriction endonucleases." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435433.

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25

Guha, Tuhin. "Biochemical characterization of homing endonucleases encoded by fungal mitochondrial genomes." PLoS ONE, 2014. http://hdl.handle.net/1993/31680.

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The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies.
October 2016
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26

Delgadillo, Liberona José Sebastián. "Clonamiento, expresión y purificación de las endonucleasas apurínicas/apirimidínicas TcAP1 y TcAP2 de Trypanosoma cruzi en condiciones nativas." Tesis, Universidad de Chile, 2011. http://repositorio.uchile.cl/handle/2250/131433.

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Memoria para optar al Título Profesional de Médico Veterinario
El agente causal de la enfermedad de Chagas o tripanosomiasis americana, es el parásito hemoflagelado Trypanosoma cruzi. Esta enfermedad es de carácter endémico en América Latina; se estima un promedio de 10-15 millones de personas infectadas y 75-90 millones en riesgo de contraer la enfermedad. T. cruzi posee un ciclo de vida indirecto y se presenta en cuatro formas celulares; epimastigote, forma extracelular replicativa y no infectiva, tripomastigote metacíclico y tripomastigote sanguíneo, formas no replicativas e infectivas y amastigote, forma intracelular replicativa. T. cruzi es capaz de sobrevivir al daño oxidativo del DNA generado por especies reactivas de oxígeno y nitrógeno (ROS/RNS) producidas por su propio metabolismo, así como aquellas producidas en el intestino del insecto vector y en células del hospedero mamífero. Este daño sería reparado mediante la actividad de las endonucleasas apurínicas/apirimidínicas (endonucleasas AP) de la vía de escisión de bases (BER). T. cruzi presenta en su genoma secuencias que codificarían para endonucleasas AP, entre ellas TcAP1, homóloga de APE1 humana y TcAP2, homóloga de APE2 humana y de Apn2 de Schizosaccaromyces pombe. La expresión de estas proteínas podría ser fundamental para la sobrevida del parásito, tanto en el vector triatomino como en el hospedero mamífero. Para un estudio sistemático de las características de estas enzimas, se requiere obtenerlas en el más alto grado de pureza desde el parásito o por técnicas de ingeniería genética. En esta Memoria de Título se clonaron las secuencias génicas que codifican para TcAP1 y TcAP2 en vectores de expresión para células eucariontes, lo que se confirmó mediante secuenciación automática de DNA. Con estos constructos se transfectaron células S2 de Drosophila melanogaster, levaduras Pichia pastoris y epimastigotes de T. cruzi cepa Dm28c. Sorprendentemente, sólo fue posible expresar ambas endonucleasas en este último modelo. La imposibilidad de expresar ambas endonucleasas AP en los modelos P. pastoris y D. melanogaster podría relacionarse a una variedad insuficiente o limitada de ciertos tRNAs, necesarios para la expresión de TcAP1 y TcAP2, con la consecuente disminución de la traducción de los mRNAs codificantes para ambos genes. Probablemente, los tRNAs que presentan anticodones específicos para la traducción de las endonucleasas AP de T. cruzi sólo se encuentran en concentraciones adecuadas en modelos celulares de expresión filogenéticamente cercanos a este protozoario. Mediante cromatografía de afinidad se intentó purificar TcAP1-GFP a partir de homogeneizados de proteínas totales de epimastigotes transfectados que expresaban esta proteína recombinante. Sin embargo no se logró obtener la proteína recombinante en condiciones nativas de forma pura. Se concluye que la utilización de GFP asociado como proteína de fusión a TcAP1 sólo permite la identificación de la proteína recombinante y no una purificación adecuada de la misma
Proyecto Bicentenario Anillo ACT 112, CONICYT, Chile. Proyecto FONDECYT 1090124.
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27

Russell, Anthony George. "Characterization of a novel archaeal RNA endonuclease from Sulfolobus acidocaldarius." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ56609.pdf.

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28

Lu, Jian. "The Kluyveromyces lactis killer toxin is a transfer RNA endonuclease." Doctoral thesis, Umeå : Department of Molecular Biology, Umeå Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1092.

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29

Repanas, Konstantinos. "Structural and functional studies on the LINE-1 retrotransposon endonuclease." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2007. http://hdl.handle.net/1765/10512.

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30

Šilanskas, Arūnas. "Restriction endonuclease-triplex forming oligonucleotide conjugates with controllable catalytic activity." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2012. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2012~D_20120702_082537-22107.

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Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease... [to full text]
Mutacijos, atsiradusios atitinkamuose žmogaus genuose, gali lemti pakitusių baltymų atsiradimą, kurie sukelia įvairias ligas (pvz.: vėžį), klaidingą embriono vystymąsi ar priešlaikinę mirtį. Tokios genetinės ligos gali būti gydomos genų terapijos būdu. Labiausiai vystoma genų terapijos strategija yra paremta homologine rekombinacija, kurios metu DNR seka, naudojama geno taisymui, yra patiekiama in trans. Natūraliai žinduolių ląstelėse homologinė rekombinacija (HR) vyksta žemu rekombinacijos dažniu (10-6). Tačiau yra žinoma, kad dvigrandininio trūkio įvedimas žymiai pagreitina HR (10-1). In vivo eksperimentų atveju dvigrandininio trūkio įvedimas turi būti ypač tikslus, todėl šis metodas reikalauja naujų molekulinių įrankių, kurie būtų itin specifiški ir griežtai kontroliuojami. Šiame darbe mes orientavomės į itin specifiškų ir griežtai kontroliuojamų meganukleazių kūrimą naudojant restrikcijos endonukleazių (REazių)-tripleksą formuojančių oligonukleotidų (TFO) konjugatus. REazių-TFO konjugatuose TFO suteikia specifiškumą prailgintam atpažinimo taikiniui per DNR triplekso susidarymą taip nukreipdamas restrikcijos fermentą prie konkretaus taikinio kur norima įvesti dvigrandininį trūkį. Šiuo tyrimu mes parodėme dvi alternatyvias restrikcijos endonukleazių-TFO konjugatų aktyvumo reguliavimo strategijas, kas leistų šias nukleazes panaudoti in vivo tyrimuose. Tuo tikslu buvo pasirinkti ortodoksiniai restrikcijos fermentai MunI ir Bse634I, kurie mūsų laboratorijoje yra gerai... [toliau žr. visą tekstą]
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31

Preece, Fiona Louise. "Reactions at two DNA sites by the BSPMI restriction endonuclease." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495821.

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32

Keeble, Anthony Howard. "Biophysical analysis of ligand binding to the colicin E9 endonuclease." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251393.

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33

Elliott, Sarah Louise. "E. coli Vsr endonuclease : mechanism and role in mismatch repair." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417442.

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34

Mosbahi, Khédidja. "Interaction of the endonuclease domain of colicin E9 with membranes." Thesis, University of York, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399260.

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35

Erskine, Symon George. "The kinetics of DNA cleavage by the EcoRV restriction endonuclease." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388107.

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36

Lau, Roxanne. "Characterisation of the streptococcal DNA polymerase I flap endonuclease domain." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/19951/.

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37

Meisenberg, Cornelia. "The role of ubiquitylation in regulating apurinic/apyrimidinic endonuclease 1." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:9a6582d4-6fc0-48c9-9c13-6c99e23e66e9.

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Apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair factor involved in the DNA base excision repair (BER) pathway that is required for the maintenance of genome stability. In this pathway, APE1 cleaves DNA at an abasic site to generate a DNA single strand break, allowing for repair completion by a DNA polymerase and a DNA ligase. High levels of APE1 have been observed in multiple cancer types however it is not understood if this contributes to cancer onset and development. What is known is that these cancers tend to display increased resistance to DNA damaging treatments and APE1 is therefore considered a key target for inhibition in the treatment of APE1-overexpressing cancers. Considering the relevance of modulating APE1 levels in disease and cancer treatment, very little is known about how cellular APE1 levels are regulated. Our lab has previously shown that the levels of the BER factors Pol β, XRCC1 and DNA Lig IIIα are regulated by ubiquitylation-mediated proteasomal degradation. The aim of this doctoral thesis was therefore to determine if ubiquitylation also regulates APE1 stability in cells. I present evidence that APE1 is ubiquitylated in cells and have identified the UBR3 E3 ligase that is responsible for this activity. Using mouse embryonic fibroblasts generated from Ubr3 knockout mice, I demonstrate that UBR3 regulates APE1 cellular levels. I furthermore show that a loss of cellular UBR3 leads to the formation of DNA double strand breaks and genome instability.
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38

Dance, Geoffrey Stephen Charles. "Endonucleases involved in mRNA decay in E. coli." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260160.

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39

Bolton, Bryan John. "Class II restriction endonucleases : screening, purification and characterization." Thesis, University of Salford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240035.

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40

Bilcock, Denzil Trevor. "DNA communications by SfiI and other restriction endonucleases." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299592.

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41

Jakimo, Noah Michael. "Genomic nucleic acid memory storage with directed endonucleases." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/98625.

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Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 40-41).
Technologies for long-term recording of cellular pathway activation are constrained by the difficultly to constantly monitor transient signaling events and expression of target genes. To overcome these limitations we designed a recording tool that uses the transcriptional output of a signaling pathway as the input for an engineered genome encoded memory. The mechanism of recording leverages the programmable nature of the bacterial immune system that consists of Clustered Regularly Interspaced Short Palindromic Repeat Sequences (CRISPR), which can recognize and cleave viral DNA using an RNA-guided directed endonuclease. Cuts left by the endonuclease are repaired by an error-prone DNA damage repair mechanism, namely non-homologous end joining (NHEJ), likely to leave mutations at the cut sites. Defining the cut site with pathway-dependent transcription of guide RNA, this genomic region is sequenced to measure pathway activation by the amount of accumulated mutations. To demonstrate a system to monitor cancer metabolism, guide RNA is expressed in mammalian cell culture with a NF-kappaB promoter. To demonstrate a system that can monitor sugar intake in an environment like the gut, guide RNA is expressed in bacteria with an arabinose promoter.
by Noah Jakimo.
S.M.
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42

Gao, Honghai. "Biochemical study of endonuclease V and its application in mutation scanning." Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1181252071/.

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43

Mitchell, Belinda Michon Hall. "Restriction endonuclease analysis of chromosomal DNA from campylobacter and helicobacter organisms." Thesis, Georgia Institute of Technology, 1993. http://hdl.handle.net/1853/25220.

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44

Gorman, Michael Anthony. "Crystal structure of the human DNA repair enzyme AP endonuclease 1." Thesis, Open University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242493.

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45

Makovets, Svetlana. "Regulation of the endonuclease activity of type 1 restriction-modification systems." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/11090.

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Efficient acquisition of the genes (hsdR, M and S) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems, was shown to require a product of an unknown gene hsdC. The hsdC mutant is shown to have a mutation in clpX. ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the hsd genes by conjugation, transformation and P1 transduction. Inactivation of clpX leads to a bigger barrier than a similar mutation in clpP consistent with a chaperone activity of ClpX in the absence of ClpP. The establishment of the modification activity of EcoKI is not dependent on clpX and takes about 12 generations to reach its maximal activity in methylating incoming phage DNA. This lag probably reflects the time necessary to complete the methylation of bacterial chromosomes. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd+ bacterium as the result of replication errors or recombinant-dependent repair. The presence of unmodified target sequences for type I restriction-modification systems on bacterial chromosomes does not influence the survival of hsd+ bacteria due to ClpXP- dependent regulation of the endonuclease activity. HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP and therefore the bacteria show a temporary drop in restriction activity, referred to as restriction alleviation. The delayed detection of restriction activity followed by the establishment of a new specificity can be considered as a case of restriction alleviation. The data obtained support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences on the chromosome, but before completion of the pathway that would result in DNA breakage. It remains unclear how the restriction-modification systems distinguish between unmethylated host and foreign DNA. The latter is degraded while the former is protected from cleavage by ClpXP-dependent proteolysis of HsdR.
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46

Techner, José-Marc. "Sequence Affinity Studies of E301W, A Mutant of Restriction Endonuclease SgrAI." Thesis, The University of Arizona, 2011. http://hdl.handle.net/10150/144972.

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47

Patel, Nikesh. "Mechanistic and structural studies of the helical arch of flap endonuclease." Thesis, University of Sheffield, 2012. http://etheses.whiterose.ac.uk/3135/.

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Fluorescence anisotropy measurements were also carried out in order to determine the dissociation constant of the synthesised oligos bound to FEN enzymes and validate conditions used in the aforementioned competition experiments. These experiments revealed a small dependence on substrate flap length to binding, and showed stimulation of KD on the addition of divalent metal ions. This was likely due to shielding of 7/8 conserved carboxylates that ligand divalent metal ions within the active site of the enzyme. Measurements with hFEN1 mutants, also highlighted the fact that initial binding of substrates is not affected by the helical arch of FEN enzymes. Mutagenesis studies were also carried out. Leucine residues were mutated to proline along the helical arch of human FEN, to disrupt proper alpha helical structure and any disorder-to-order transitions. This gave an extremely deficient phenotype with rates extremely low compared to the wild type enzyme, showing a large significance of the helical arch structure on the activity of the enzyme.
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48

Silva, Rita de C?ssia Barreto da. "Caracteriza??o de uma nova exonuclease identificada em uma biblioteca metagen?mica." Universidade Federal do Rio Grande do Norte, 2014. http://repositorio.ufrn.br/handle/123456789/19374.

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2019-05-27
A abordagem metagen?mica tem permitido o acesso ao material gen?tico de microrganismos n?o cultivados e tem sido usada para identifica??o de novos genes. Apesar da import?ncia dos mecanismos de reparo de DNA para a manuten??o da integridade gen?mica nosso conhecimento sobre mecanismos de reparo de DNA ? baseado em organismos modelo como E. coli e pouco ? conhecido sobre os organismos de vida livre e n?o cultivados. Neste trabalho, a abordagem metagen?mica foi aplicada para descobrir novos genes envolvidos com a manuten??o da integridade gen?mica. Um clone positivo foi identificado por replicar a biblioteca metagen?mica em meio seletivo contendo H2O2. O clone metagen?mico foi capaz de complementar parcialmente a defici?ncia em reparo de DNA de cepas simples e duplo-mutantes de E.coli (recA e xthA nfo, respectivamente) submetidas ao estresse gerado por H2O2 e MMS.A an?lise de sequ?ncia mostrou uma ORF codificando para uma prote?na hipot?tica membro da superfam?lia Exo_Endo_Phos (PF03372) e, a filogenia indicou que a mesma n?o est? inclusa em nenhuma das subfam?lias EEP. Assim, uma nova nuclease foi identificada e experimentalmente caracterizada in vivo e in vitro. Ensaios espec?ficos utilizando a nuclease purificada e oligonucleotideos fluorescentemente marcados revelaram sua atividade 3?-5?exonuclease, em substratos simples e dupla-fita, dependente de Magn?sio e sens?vel a EDTA. Uma vez que este ? o primeiro relato e caracteriza??o de uma enzima obtida a partir de abordagem metagen?mica mostrando uma atividade exonuclease, foi nomeada EXOMEG1
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49

Sandegren, Linus. "Group I Introns and Homing Endonucleases in T-even-like Bacteriophages." Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-211.

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50

Kim, Hyong-ha. "A novel intron-encoded endonuclease derived from the fourth intron of the Chlamydomonas reinhardtii psbA gene /." Digital version accessible at:, 2000. http://wwwlib.umi.com/cr/utexas/main.

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