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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22355号 / 医博第4596号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 岩田 想, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti. / Master of Science in Life Sciences

Made available in DSpace on 2015-03-03T15:19:19Z (GMT). No. of bitstreams: 1 AndreaLO.pdf: 448824 bytes, checksum: 4a360d0db40a607834f5c380870bc7f5 (MD5) Previous issue date: 2009-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress response / O genoma de todos os organismos est? sujeitos a les?es que podem ser causados por fatores end?genos e ambientais. Uma vez no genoma, as les?es podem levar a forma??o e ac?mulo de muta??es, as quais podem ser prejudiciais ao desenvolvimento do organismo. As vias de reparo de DNA s?o um mecanismo que permite o organismo detectar e corrigir essas les?es ou minimizar seus efeitos. V?rias vias de reparo de DNA s?o conhecidas e bem caracterizadas em animais e microorganismos. Em plantas, as vias de reparo ainda n?o s?o bem caracterizadas, mas muitas pesquisas t?m revelado a participa??o de todas as vias conhecidas no reparo do genoma das plantas, sendo os modelos mais estudados Arabidopsis thaliana e Oriza sativa. A via de reparo de interesse deste trabalho ? a via de BER, a qual apresenta v?rias prote?nas atuando no reparo do DNA. Por?m, a classe de prote?nas da via BER focadas s?o as AP endonucleases, respons?veis pela hidr?lise do s?tio AP. Este trabalho teve como objetivo caracterizar dois cDNAs de cana-de-a??car: scARP1 e scARP3, hom?logos a AP endonucleases de A.thaliana e identificados num trabalho de data-mining do projeto SUCEST. Para tanto, foram constru?dos cassetes de super-express?o, contendo o cDNA scARP1 sob o controle do promotor forte 35S. Al?m disso, anteriormente no laborat?rio foi obtido uma planta de icotiana tabacum contendo o cassete de super-express?o (35S+scARP1) em orienta??o anti-senso. Foi analisado o desenvolvimento desta planta, e foram obtidas tamb?m algumas sementes (T2) desta planta. Estas sementes foram germinadas e analisadas quanto ? presen?a do cassete de super-express?o e desenvolvimento. Al?m disso, para estes dois genes foram clonados as regi?es promotoras por PCR walking. Os fragmentos obtidos foram clonados, sequenciados e, analisados por meio do programa PLANTCARE. Os motivos encontrados nessas regi?es dos genes de cana-de-a??car foram comparados com potenciais regi?es promotoras das plantas de Arabidopis, O. sativa e S. bicolor. Estas an?lises mostraram a presen?a de diferentes motivos relacionados ? respostas ao estresse oxidativo

Genetic engineering is important to enhance crop characteristics and certain traits. Genetically engineered crop cultivation brings environmental and ecological concerns with the potential of unwanted transgene escape and introgression. Transgene escape has been considered as a major environmental and regulatory concern. This concern could be alleviated by appropriate biocontainment strategies. Therefore, it is important to develop efficient and reliable biocontainment strategies. Removing transgenes from pollen has been known to be the most environmentally friendly biocontainment strategy. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment. A novel approach for selective male sterility in pollen was developed and evaluated as a biocontainment strategy. Overexpression of the EcoRI restriction endonuclease caused pollen ablation and/or infertility in tobacco, but exhibited normal phenotypes when compared to non-transgenic tobacco. Three EcoRI contained 0% GFP positive pollen, while GFP control plants contained 64% GFP positive pollen based on 9,000 pollen grains analyzed by flow cytometry-based transgenic pollen screening method. However, seven EcoRI events appeared to have 100% efficiency on selective male sterility based on the test-crosses. The results suggested that this selective male sterility could be used as a highly efficient and reliable biocontainment strategy for genetically engineered crop cultivation.

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.

Homing endonucleases (HEs) typically have one of four types of catalytic domains (LAGLIDADG, GIY-YIG, H-N-H, His-Cys), and a DNA-binding region(s) that provides specificity. I-CreII, which is encoded by the psbA4 intron from Chlamydomonas reinhardtii, is unusual in containing two catalytic motifs: H-N-H and GIY-YIG. A previous study showed that I-CreII cleavage leaves 2-nt 3' OH overhangs similar to GIYYIG endonucleases, but that it also has a flexible metal requirement like H-N-H enzymes. Also, alanine substitution of several conserved residues in the GIY-YIG motif and two in the H-N-H motif did not produce a clear catalytic mutant, although some variants had strongly reduced DNA binding. Thus, in order to identify the catalytic motif, I substituted additional amino acids in both domains with alanine, and identified three histidines in the H-N-H motif that are likely to be involved in catalysis. To gain insight into how I-CreII interacts with its ~30-bp homing-site DNA, three types of DNA protection analysis were performed. Hydroxyl-radical footprinting, which reveals regions of tight DNA binding, indicated that I-CreII binds strongly to a region downstream of the cleavage and intron-insertion sites, corresponding to bp 2-10 of exon 5. There was also partial protection around the cleavage site, but only on the top strand, which is consistent with the enzyme's tendency to cleave this strand first. DNase I protection, which can reveal less closely-bound regions of target DNA, gave a larger footprint than hydroxyl-radical protection, with the additional region lying upstream of the cleavage site. These results also suggest that DNA backbone-binding downstream of the cleavage site involves sugars and phosphates, whereas upstream it is mainly with phosphates. DMS protection, which probes guanines on the N-7 position in the major groove, did not provide any evidence of major groove binding (at least not through guanines). DNase I protection could also be performed on the I-CreII variants that had reduced DNA affinity. The N161A variant was instructive in that it showed reduced protection of a T-A bp very close to the cleavage site, providing support for a catalytic role for the H-N-H motif and a possible constraint for modeling. Of the GIY-YIG motif variants, the footprint of the G231E/K245A variant was distinctly useful in that it was preferentially effected downstream of the cleavage site. This result suggested the H-N-H and GIY-YIG motifs are co-linear with their targets in the homing site. Structural modeling of the GIY-YIG domain of I-CreII using the solved I-TevI domain as template provided evidence for a unique insertion in the I-CreII structure that disrupted a catalytic α-helix; the insertion is predicted to be a positively charged, hairpinlike loop anchored by two antiparallel β-strands. I propose that this insertion can explain the evolutionary conversion of this catalytic endonuclease domain into a DNA-binding domain. These findings should also help to understand other dual-motif H-N-H/GIY-YIG endonucleases, such as I-CmoeI.

DNA nucleases are essential for various biological functions such as replication, recombination, and repair. Restriction endonucleases (REs) are excellent model system for the investigation of DNA recognition and specificity. SgrAI is a type IIF RE that cuts an 8 base pair primary sequence. In addition to its primary cleavage activity it also cleaves secondary sequences, but only appreciably in the presence of the primary sequence. The longer flanking DNA exhibits much greater activated DNA cleavage by SgrAI (>1000 fold activation by secondary site). Interestingly, the asymmetric cleavage seen in one of the two types of secondary site DNA is lost upon activation of SgrAI, suggesting a loss of communication between DNA recognition and activity upon specificity expansion. The structure of SgrAI bound to 22-1HT supports the cryoelectron microscopy structure of activated, oligomeric SgrAI highlighting the significance of the contacts made by the flanking DNA and the role played by N-terminal domain contacts in forming the run-on oligomer. The biological study suggests that the run-on oligomer formation sequesters the host DNA from being cleaved by the activated SgrAI complex. The DNA sequence binding, cleavage preference, and the structure of K96A SgrAI were determined. Unexpectedly, this mutation did not alter the structure of the enzyme, nor did it result in an enzyme lacking sequence preference at the 7ᵗʰ position. Instead, the largest effect of the mutation appears to be in making the enzyme more specific such that it fails to cleave either type of secondary site. It may be that the K96 side chain is required to distort the non YG sequences (specifically GG and TC) of secondary site DNA for proper positioning in the enzyme active site upon activation and specificity expansion. The crystal structure of Mk0566, XPG homologue from M. kandleri, was solved to 2.48 Å resolution and was found to be very similar to that of human FEN-1 and to other archaeal FEN-1/XPG homologues. These results suggest that the main biological role of Mk0566 is in DNA replication; however, they do not preclude involvement in a modified form of nucleotide excision repair.