Dissertations / Theses on the topic 'Two-Hybrid screening'

Na+/H+ exchangers (NHE) are integral membrane proteins that catalyze the electroneutral exchange of Na+ (or K+) for H+. The nine human isoforms identified to date share the same topology of twelve membrane-spanning domains and a cytoplasmic regulatory tail, and diverge in their tissue expressions and subcellular localizations. This thesis focused on the identification and characterization of proteins interacting with the ubiquitous NHE6 isoform, which resides predominantly in recycling endosomes. Recent yeast two-hybrid (Y2H) screening of a human brain cDNA library using the regulatory tail of NHE6 as a probe resulted in the tentative identification of about 250 partial or full-length NHE6-interacting proteins. Thirty other potential NHE6-interacting partners were identified by isolating NHE6 complexes from embryonic kidney 293 cells by tandem affinity purification (TAP) and subjecting them to mass spectrometry analysis. The interaction between NHE6 and eight of these proteins (four from each Y2H and TAP analyses) was tested by co-immunoprecipitation, co-localization by immunofluorescence microscopy and in vitro GST-fusion protein pull-down assays. We show that apolipoprotein D interacts weakly with NHE6, while myosin light chain kinase and the mitochondrial ATP synthase subunit-alpha are true NHE6-binding partners. The remaining five proteins exhibited poor and/or non-reproducible association with NHE6.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

La plupart des virus ont développé des mécanismes de résistance ou de suppression du système immunitaire pour parvenir à infecter durablement leur hôte. Ces mécanismes demeurent encore imparfaitement connus. Un domaine immunosuppresseur (IS) a été identifié au niveau de la région transmembranaire des protéines d’enveloppe des rétrovirus endogènes ou infectieux. Ce domaine hautement conservé a été décrit par exemple comme inhibant l’activation lymphocytaire. Dans le laboratoire, il a été caractérisé en particulier via des expériences de rejet de cellules tumorales in vivo, ce qui a permis de définir des mutations inactivatrices. Afin de mieux comprendre les mécanismes de résistance des rétrovirus au système immunitaire, mes travaux de thèse ont porté sur l’identification de la ou des protéines capables d’interagir avec le domaine IS. Plusieurs approches cellulaires et moléculaires ont été développées, basées pour la plupart sur l’utilisation de sondes fluorescentes obtenues par synthèse chimique, constituées des domaines IS provenant de différents rétrovirus. Dans un premier temps, les cellules du système immunitaire qui lient les protéines virales ont été identifiées : les lymphocytes B et les cellules myéloïdes (monocytes, cellules dendritiques et macrophages). Dans un second temps, des expériences de co-immunoprécipitation et de chromatographie d’affinité couplées à la spectrométrie de masse ont été réalisées dans le but d’identifier sur ces cellules les protéines membranaires responsables de ces liaisons. Plusieurs agents de couplages chimiques ont été utilisés afin de maintenir les liaisons domaine IS - protéine de faibles affinités. En raison de résultats non-reproductibles obtenus au cours de ces expériences, des tests de liaison du domaine IS sur des cellules transfectées avec des banques d’ADNc, ou lors d’expériences de double hybride ont été réalisées. Ces deux approches ont permis d’identifier des protéines membranaires potentiellement impliquées dans la liaison du domaine IS : les protéines X1 et X2. Les co-transfections de vecteurs d’expression du domaine IS et de X2 ont mis en évidence des interactions protéiques au cours d’expériences de co-immunoprécipitation et de microscopie confocale, en particulier avec le domaine IS du rétrovirus HIV-1. Concernant X1, sa transfection induit la liaison cellulaire des domaines IS de HERV-W et MLV. En revanche, aucune interaction directe entre X1 et le domaine IS n’a pu être démontrée, notamment dans des expériences de co-immunoprécipitation et de microscopie confocale.La découverte des protéines membranaires qui interagissent avec le domaine IS demeure un enjeu critique pour la compréhension des voies de signalisation et de transcription qui permettent aux rétrovirus d’exercer leur effet sur le système immunitaire, l’objectif de ces travaux étant d’identifier à terme des nouvelles cibles thérapeutiques.En conclusion, même si des travaux complémentaires demeurent nécessaires, les protéines X1 et X2 pourraient contribuer à l’immunosuppression rétrovirale<br>Most viruses have developed mechanisms of resistance or suppression of the immune system to achieve lasting infection of their host. These mechanisms are still imperfectly known. An immunosuppressive (IS) domain has been identified in the transmembrane region of envelope proteins of endogenous or infectious retroviruses. This highly conserved domain has been described, for example, as inhibiting lymphocyte activation. In the laboratory, it has been characterized by tumor cell rejection experiments in vivo, which has made it possible to define inactivating mutations. In order to better understand the mechanisms of resistance of retroviruses to the immune system, my thesis focused on the identification of the protein(s) interacting with the IS domain. Several cellular and molecular approaches have been developed, based for the most part on the use of fluorescent probes obtained by chemical synthesis, consisting of IS domains from different retroviruses. At first, immune system cells that bind viral proteins have been identified: B cells and myeloid cells (monocytes, dendritic cells and macrophages). In a second step, co-immunoprecipitation and affinity chromatography coupled to mass spectrometry were performed to identify on these cells the membrane proteins responsible for these bonds. Several chemical coupling agents have been used to prevent detachment of low affinity binding between proteins and the IS domain. Due to non-reproducible results obtained during these experiments, IS domain binding assays on cells transfected with cDNA libraries, or in double hybrid experiments were performed. These two approaches made it possible to identify membrane proteins potentially involved in the binding of the IS domain: the X1 and X2 proteins. Co-transfections of IS domain and X2 expression vectors demonstrated protein interactions in co-immunoprecipitation and confocal microscopy experiments, particularly with the IS domain of the HIV-1 retrovirus. Concerning X1, its transfection induces binding of the IS domains of HERV-W and MLV on cells membrane. On the other hand, no direct interaction between X1 and the IS domain could be demonstrated, especially in co-immunoprecipitation and confocal microscopy experiments.The discovery of membrane proteins that interact with the IS domain remains a critical issue for understanding the signaling and transcription pathways that allow retroviruses to exert their effect on the immune system, the aim of this work being to identify new therapeutic targets.In conclusion, although further work is still needed, the X1 and X2 proteins may contribute to retroviral immunosuppression

Chez les eucaryotes, la voie de protéolyse Ub/ protéasome 26S est responsable de la dégradation sélective de la plupart des protéines intracellulaires. Cette dégradation par le protéasome 26S est initiée par une polyubiquitination de la protéine réalisée grâce à l’action d’une cascade enzymatique impliquant 3 types d'enzymes nommées « ubiquitin-activating enzyme » (E1), « ubiquitin-conjugating enzyme » (E2) et « ubiquitin-protein ligase » (E3). Il existe différentes classes d’ubiquitines ligases (E3), parmi lesquelles la plus connue est le complexe SCF (Skp1-Cullin-F-box). La protéine SKP1 fixe à la fois la Culline et la F-box qui va reconnaitre spécifiquement la protéine cible. Contrairement aux protistes, les champignons et certains vertébrés qui possèdent un unique gène SKP1 fonctionnel, de nombreux animaux et espèces de plantes présentent plusieurs SKP1 homologues. Vingt et un et trente deux gènes SKP1 ont été décrits respectivement chez Arabidopsis thaliana et Oryza sativa. En dépit de l’importance du complexe SCF, chez le riz, peu de travaux décrivent les interactions entre les dizaines de protéines « SKP1-like » et les centaines de protéines F-box. Dans un premier temps, nous avons collecté et analysé les séquences de 288 gènes « SKP1-like » appartenant à 17 espèces, dont la mousse Physcomitrella patens, cinq monocotylédones et 11 eudicotylédones. Les analyses structurales et phylogénétiques de ces gènes indiquent qu’ils peuvent être divisés en différentes sous-familles. Nos analyses ont montré qu’OSK1 et OSK20 chez le riz constituent une classe de gènes SKP1 à intron unique conservé. Dans un deuxième temps, nous avons étudié le profil d’expression des gènes « SKP1-like » chez le riz. Notre investigation sur le nombre d’EST a montré que les gènes OSK1 et OSK20 sont les plus largement représentés dans les bases de données EST publiques. La méta-analyse de l’expression des gènes « SKP1-like » chez le riz, indique que les gènes OSK présentent des profils d'expression hétérogènes selon les tissus et les conditions physiologiques. Les résultats des intearctions protéine-protéine en double hybride ont révélé que les protéines OSK présentent différentes capacités d’interactions avec les protéines F-box. Cependant, OSK1 et OSK20 semblent interagir avec la plupart des protéines F-box testées. Les études de localisation subcellulaire ont indiqué que OSK1 et OSK20 sont des protéines nucléaires et cytosoliques. En se basant sur les divers résultats obtenus dans ce travail, nous pouvons suggérer que chez le riz, les gènes OSK1 et OSK20 sont fonctionnellement équivalents aux gènes ASK1 et ASK2 chez Arabidopsis thaliana. Nous pouvons également proposer les équivalents de ces gènes chez les autres espèces végétales dont le génome a été séquencé<br>In eukaryotes, the ubiquitin Ub/26S proteasome pathway is responsible for the selective degradation of most intracellular proteins. This cellular process is initiated by protein polyubiquitination mediated by a three-step cascade involving: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin-protein ligase (E3). The E3 ubiquitin ligases contain several classes, among which the best-known are Skp1-Cullin-F-box (SCF) complexes. The SKP1 protein binds both Cullin and F-box which recognizes specifically the target proteins. Whereas protists, fungi and some vertebrates have a single functional SKP1 gene, many animal and plant species possess multiple SKP1 homologues. Twenty one and thirty-two SKP1-related genes have been described respectively in the Arabidopsis and Oryza sativa genome. Despite the importance of the SCF complex, there have been a few reports of systematic surveys of interactions between the dozens of SKP1-like proteins and the hundreds of F-box proteins in rice. In a first step, we retrieved and analyzed 288 SKP1-like genes belonging to 17 species including the moss Physcomitrella patens, five monocots and 11 eudicots. Structural and phylogenetic analysis of rice OSK genes and other plant SKP1-like genes have indicated that the different members of the plant SKP1 can be split into different subfamily. Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position. In a second step, we studied expression profiles of the rice Skp1-like genes. Our EST survey indicated that OSK1 and OSK20 are the most widely represented genes in public EST databases. Meta-analysis of the expression of rice SKP1-like genes indicated that OSK genes exhibit an expression profile that was heterogeneous in terms of tissues, conditions and overall intensity. Yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most F-box proteins tested. Subcellular localization studies indicated that OSK1 and OSK20 are nuclear and cytosolic proteins. Based on the results obtained in this study, we can suggest that rice OSK1 and OSK20 are likely to have similar functions as do the Arabidopsis ASK1 and ASK2 genes. Similarly, we suggest a list of functional equivalent in the other sequenced plant genomes

L'auxine est une hormone végétale qui coordonne plusieurs processus de développement des plantes à travers la régulation d'un ensemble spécifique de gènes. Les Auxin Response Factors (ARF) sont des régulateurs transcriptionnels qui modulent l'expression de gènes de réponse à l’auxine. Des données récentes montrent que les membres de la famille des ARF sont impliqués dans la régulation du développement des fruits de la nouaison à la maturation. L'objectif principal de la thèse est d’étudier la part qui revient aux ARF dans le contrôle du développement et de la maturation des fruits et d’en comprendre les mécanismes d’action. L’analyse des données d’expression disponibles dans les bases de données a révélé que, parmi tous les ARF de tomates, SlARF2 affiche le plu haut niveau d'expression dans le fruit avec un profil distinctif d’expression associé à la maturation. Nous avons alors entrepris la caractérisation fonctionnelle de SlARF2 afin d’explorer son rôle dans le développement et la maturation des fruits. Deux paralogues, SlARF2A et SlARF2B, ont été identifiés dans le génome de la tomate. Nous avons montré que l’expression de SlARF2A dans le fruit est régulée par l'éthylène tandis que celle de SlARF2B est induite par l'auxine. La sous-expression de SlARF2A, comme celle de SlARF2B, entraine un retard de maturation alors que l’inhibition simultanée des deux paralogues conduit à une inhibition plus sévère de la maturation suggérant une redondance fonctionnelle entre les deux paralogues lors de la maturation des fruits. Les fruits présentant une sous-expression des gènes SlARF2 produisent de faibles quantités d'éthylène, montrent une faible accumulation de pigments et une plus grande fermeté. Le traitement avec de l'éthylène exogène ne peut pas inverser les phénotypes de défaut de maturation suggérant que SlARF2 pourrait agir en aval de la voie de signalisation de l'éthylène. L'expression des gènes clés de biosynthèse et de signalisation de l'éthylène est fortement perturbée dans les lignées sous-exprimant SlARF2 et les gènes majeurs qui contrôlent le processus de maturation (RIN, CNR, NOR, TAGL1) sont sensiblement sous-régulés. Les données suggèrent que SlARF2 est essentiel pour la maturation des fruits et qu’il pourrait agir au croisement des voies de signalisation de l'auxine et de l'éthylène. Dans le but de mieux comprendre les mécanismes moléculaires par lesquels les ARF régulent l'expression des gènes de réponse à l'auxine, nous avons étudié l'interaction des SlARFs avec des partenaires protéiques ciblés, principalement les co-répresseurs de type Aux/IAA et Topless (TPL) décrits comme les acteurs clés dans la répression des gènes dépendant de la signalisation auxinique. Une fois les gènes codant pour les membres de la famille TPL de tomate isolés, une approche double hybride dans la levure a permis d’établir des cartes exhaustives d'interactions protéine-protéine entre les membres des ARFs et des Aux/IAA d’une part et les ARFs et les TPL d’autre part. L'étude a révélé que les Aux/IAA interagissent préférentiellement avec les SlARF activateurs et qu’à l’inverse les Sl-TPL interagissent uniquement avec les SlARF répresseurs. Les données favorisent l'hypothèse que les ARF activateurs recrutent les Sl-TPL via leur interaction avec les Aux/IAA, tandis que les ARF répresseurs peuvent interagir directement avec les Sl-TPL. Les études d’interactions ont permis également d’identifier de nouveaux partenaires comme les protéines VRN5 et LHP1, composantes des complexes Polycomb PRC impliqués dans la repression par voie épigénétique de la transcription par modification de l'état de méthylation des histones. Au total, le travail de thèse apporte un nouvel éclairage sur le rôle et les mécanismes d'action des ARF et identifie SlARF2 comme un nouvel élément du réseau de régulation contrôlant le processus de maturation des fruits chez la tomate<br>The plant hormone auxin coordinates plant development through the regulation of a specific set of auxin-regulated genes and Auxin Response Factors (ARFs) are transcriptional regulators modulating the expression of auxin-response genes. Recent data demonstrated that members of this gene family are able to regulate fruit set and fruit ripening. ARFs are known to act in concert with Aux/IAA to control auxin-dependent transcriptional activity of target genes. However, little is known about other partners of ARFs. The main objective of the thesis research project was to gain more insight on the involvement of ARFs in fruit development and ripening and to uncover their interaction with other protein partners beside Aux/IAAs. Mining the tomato expression databases publicly available revealed that among all tomato ARFs, SlARF2 displays the highest expression levels in fruit with a marked ripening-associated pattern of expression. This prompted us to uncover the physiological significance of SlARF2 and in particular to investigate its role in fruit development and ripening. Two paralogs, SlARF2A and SlARF2B, were identified in the tomato genome and transactivation assay in a single cell system revealed that the two SlARF2 proteins are nuclear localized and act as repressors of auxin-responsive genes. In fruit tissues, SlARF2A is ethylene-regulated while SlARF2B is auxin-induced. Knock-down of SlARF2A or SlARF2B results in altered ripening with spiky fruit phenotype, whereas simultaneous down-regulation of SlARF2A and SlARF2B leads to more severe ripening inhibition suggesting a functional redundancy among the two SlARF2 paralogs during fruit ripening. Double knock-down fruits produce less climacteric ethylene and show delayed pigment accumulation and higher firmness. Exogenous ethylene treatment cannot reverse the ripening defect phenotypes suggesting that SlARF2 may act downstream of ethylene signaling. The expression of key ethylene biosynthesis and signaling genes is dramatically disturbed in SlARF2 down-regulated fruit and major regulators of the ripening process, like RIN, CNR, NOR, TAGL1, are under-expressed. The data support the notion that SlARF2 is instrumental to fruit ripening and may act at the crossroads of auxin and ethylene signaling. Altogether, while ethylene is known as a key hormone of climacteric fruit ripening, the ripening phenotypes associated with SlARF2 down-regulation bring unprecedented evidence supporting the role of auxin in the control of this developmental process. To further extend our knowledge of the molecular mechanism by which ARFs regulate the expression of auxin-responsive genes we sought to investigate interactions SlARF and putative partners, mainly Aux/IAAs and Topless co-reppressors (TPLs) reported to be key players in gene repression dependent on auxin signaling. To this end, genes encoding all members of the tomato TPL family were isolated and using a yeast-two-hybrid approach comprehensive protein-protein interaction maps were constructed. The study revealed that Aux/IAA interact preferentially with activator SlARFs while Sl-TPLs interact only with repressor SlARFs. The data support the hypothesis that activator ARFs recruit Sl-TPLs co-repressors via Aux/IAAs as intermediates, while repressor ARFs can physically interact with Sl-TPLs. Further investigation indicated that SlARFs and Sl-TPLs can interact with polycomb complex PRC1 PRC2 components, VRN5 and LHP1, known to be essential players of epigenetic repression of gene transcription through the modification of histones methylation status. These data establish a potential link between ARFs and epigenetic regulation and thereby open new and original perspectives in understanding the mode of action of ARFs. Altogether, the thesis work provides new insight on the role of ARFs and their underlying action mechanisms, and defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato

碩士<br>國立中山大學<br>生物科學系研究所<br>93<br>Tumor Susceptibility Gene, TSG101, has been identified as a tumor susceptibility gene with multiple functions. TSG101 encodes a 46 kDa protein composed of 390 amino acids. As previous studies reported, TSG101 participates in cell-cycle control, membrane proteins’ trafficking, and transcriptional regulation. To identify the proteins that mediated function involved TSG101, we perform yeast two-hybrid cDNA library screening to search for TSG101-interacting proteins. A construct pAS2-1-TSG101 was used as a bait to screen a human testis cDNA library. This screening selected 68 TSG101 interacting clones, including 17 known proteins. These proteins were functionally classified as participating in cell-cycle alteration, protein sorting, transcriptional regulation, modification, signal transduction and other functions. Our results provide the evidences which not only confirm the results of previous studies, but also provide further information related to TSG101 biological functions worth intensive study. Among these clones, we choose KLIP1 gene, which encodes a transcription factor, for further study to elucidate the functional role of TSG101 in nucleus. In vitro GST pull-down assay and in vivo co-immunoprecipitation assay were performed using GST-KLIP1 and HA-tagged KLIP1, respectively, have demonstrated that TSG101 and KLIP1 indeed interact with each other within mammalian cells. Detailed biological function mediated through this TSG101 and KLIP1 interaction awaits further investigation.

碩士<br>臺灣大學<br>微生物與生化學研究所<br>98<br>RING proteins are defined as quite a big family in Arabidopsis, and their functions are varied. Many of RING proteins are involved in ubiquitin/26S proteasome system, some of them are ubiquitin ligases (E3s) which can interact directly with ubiquitin-conjugating enzyme (E2) by their RING domains. These RING E3s can also specifically indentify substrates that are going to be ubiquitinated by E2. In this study, we chose RING E3s related to abiotic stress in Arabidopsis based on microarray database and use them as baits to screen Arabidopsis library cDNA. Employing Yeast-two hybrid technique for cDNA (CD4-30) library screening from influoroscence meristem, floral meristem and floral buds up to stage 8 or 9, we indentified a RING E3, R2 (at5g27420), may have interaction with a NAC family protein, NTL11 (At5g04410). NTL11 also showed self-activation when it was fused to GAL4-DNA binding domain, implicating NTL11 may be a transcription factor as previous predictions from other studies. In further test, R2 can interact with NTL11 truncation without NAC domain and so does AtMAPR4 from previous study. Their roles in Arabidopsis physiological pathway need more investigations.

碩士<br>國立陽明大學<br>醫學生物技術研究所<br>91<br>Abstract The androgen receptor(AR), a member of the nuclear receptor superfamily, play a central role in male sexual differentiation and hepatocarcinoma cell proliferation. Like other nuclear receptors, AR exerts its transcriptional function by binding to cis-elements upstream of promoters and interacting with other transcription factors. The yeast two-hybrid assay was employed to identify androgen receptor(AR) protein partners in liver library. By using three functional domain, N-terminal transactivation, DNA binding and ligand binding, as baits, we identified 38 novel clones and 48 known clones. We had domonstrated that ARD1-10-7 and ARN-21-6 was AR interacting proteins. The yeast two hybrid assay indicated that ARD1-10-7 and ARN-21-6 interated with AR in vivo, and GST pull down assay showed that interacted with AR in vitro. RT-PCR experiments revealed that ARD1-10-7 expressed in the tissues of HCC but no difference between tumor part and adjacent part. ARD1-10-7 was found abundantly in prostate cancer. The ARN-21-6 did not express in the normal liver tissue, but highly in some HCC, and in prostae cancer. Both ARD1-10-7 and ARN-21-6 are predominantly located in cytoplasma of the 293T cell. These results indicate that ARD1-10-7 and ARN-21-6 are new AR-interacting proteins and capable of modulating AR activity. They could play significant roles in regulating AR-dependent genes in liver cells. Howere, the mechansims of regulating AR transcativational activity is still not clear.

碩士<br>國立陽明大學<br>生物化學研究所<br>93<br>The Rictor gene is 5124 bp in length, encoding a protein of 1708 amino acids. In mammalian cells, rictor accompanies mTOR and G�浘 to form the mTORC2 complex. Signaling through mTORC2 is rapamycin-insensitive and regulates phosphorylation of Akt/PKB and the organization of cytoskeleton in mammalian cell lines. However, the upstream components and the mechanism of mTORC2 signaling remain unclear. Aiming at further understanding the molecular function of rictor, this thesis research employs yeast two-hybrid screening to identify the rictor-interacting proteins. We have thus identified Tpr2 as one of the rictor-interacting proteins。Tpr2 is 484 amino acids in length, containing two TPR domains and one J domain. Yeast two-hybrid and coimmunoprecipitation analyses have indicated that Tpr2 and rictor interact through the two TPR domains. Immunofluorescence studies revealed that Tpr2 and rictor colocalized in the cytoplasm. Results of our analyses also suggest that the interaction of Tpr2 and rictor might be regulated by the J domain and mTORC1 activity.

碩士<br>國立清華大學<br>生命科學系<br>89<br>Abstract In this study, we first generated a panel of mouse monoclonal antibodies against extracellular domain of human CD63, a novel member of a recently identified protein family, tetraspanins. One of these monoclonal antibodies is found to specifically bind to mouse CD63, suggesting a self-responsive immune response. We also tried to investigate the function of CD63 in two ways: (1) an inhibition of CD63 expression by transfecting inverted CD63 extracellular domain expression vector into mouse macrophage cell line, Raw264.7; (2) Yeast Two Hybrid System to screen the possible protein associations with CD63 extracellular domain from spleen cDNA library. The rationale design is based on our assumptions that CD63 may be related to the basic cell physiology of macrophages-say phagocytosis, antigen presenting, and cytokines secretion. On the other hand, we speculate that extracellular domain of CD63 can play some important role on the plasma membrane, and these functions may not easily revealed by the approaches that have been previously used to understand CD63 function (effects of CD63 antibody binding, and co-immunoprecipitation). Actually, through yeast two hybrid system, the identified proteins interacting with CD63 are totally different from those reported previously: some lysosome (such asα-mannosidase ) or phagocytosis (such as macrophage receptor with collagenous structure and C1q beta chain )-related proteins were found. We propose that CD63 extracellular domain may play an important role in the process of phagocytosis. 中文摘要Chinese Abstract……………………………….……1 英文摘要 English Abstract…………………………………….2 簡介 Introduction………………………………………………3 目的 Specific Aims……………………..………………….…19 材料與方法 Materials and Methods…………..……………..19 結果與討論 Results and Discussions…………….…………..36 參考資料 References……………………..…………………..57 附錄一 Appendix I 附錄二 Appendix II 附錄三 Appendix III 附錄四 Appendix IV

碩士<br>國立臺灣大學<br>生化科技學系<br>107<br>Hairy root, a syndrome induced by Agrobacterium rhizogenes, can be a potential platform for phytochemical production. rolB on Agrobacterium rhizogenes TL-DNA is crucial for hairy root growth and phytochemical accumulation. However, how rolB affect hairy root physiology remains elusive. In this study, a split-ubiquitin based membrane yeast two-hybrid system (MYTH) was constructed to screen the possible interacting proteins of RolB in Arabidopsis thaliana. Yeast co-transformants were constructed, and the known interaction between Nt14-3-3 ωII and RolB is reproducible as a positive control for protein-protein interaction in control test. RolB can express in MYTH abundantly without strong self-activation of HIS3 reporter gene, and the screening stringency was optimized by adjusting the 3-AT concentration. The transformation efficiency reached 1.46 x 105 /μg of plasmid. Total 130 yeast transformants were found by β-galactosidase assay after two 104-scale screening reactions. Two plastid-localized protein, LIL3:1 and AT1G44920 were identified, and the β-galactosidase activity of LIL3:1 and AT1G44920 yeast candidates were as higher as positive controls. LIL3:1 has been shown to promote the biosynthesis of chlorophyll, α-tocopherol and phytol, and AT1G44920 is a transmembrane protein with unknown function. However, the RolB expression site in yeast needs verification, and the transformation efficiency should reached 106/μg of plasmid. To examine if RolB can interact with LIL3:1 and AT1G44920 in plant cell, the isolation procedure for Nicotiana tabacum hairy root protoplast were established, and the cyan fluorescent protein (CFP) expression vector for LIL3:1 and AT1G44920 were constructed. Several crucial parameters for isolation including the tissue processing, pre-treatments, enzyme ratio and incubation time have been optimized. The modified peeled method has been developed, and the yield of hairy root protoplast reached 2.02 * 105 / g of hairy root tip.

碩士<br>淡江大學<br>生命科學研究所碩士班<br>95<br>In part I, high efficiency transformation by electroporation is developed. Factors such as medium preparation, pretreatment of plasmid, quantities of plasmid, are investigated with six different genes YJR129C, YHR209W, EBNA2, shy A, NBS1 and KPNA2. The purpose of our protocol is to achieve ideal efficiency so that the number of transformants can saturate the library used in two-hybrid screening. This protocol has reached a efficiency of more than 2.5 x 106 /μg DNA. In part II, high efficiency transformation by electroporation is applied to yeast two-hybrid screening. Using a “fish-bait” concept, known genes YJR129C, YHR209W, EBNA2 are ligated to the BD vector as “the bait”. YJR129C and YHR209W are from Saccharomyces cerevisiae BY4742, both a putative methyltransferase gene. EBNA2 is one of the early expressed genes during EB virus infection, important in promoting the B cell immortalization. Appropriate DNA library constructed in the AD vector was screened for “the fish” when the library was transformed into yeast, PJ69-4A. The interacting proteins expressed from the library would be selected in the specific medium. Afterwards, more functional analysis may be done to understand role of the genes being selected and the characteristics of the protein product. In the EBNA2-interacting protein using the yeast two-hybrid screening, 421 positive clones were obtained.

碩士<br>國立臺灣大學<br>生化科技學系<br>106<br>Hairy roots are root-like tissues, which are resulted from the infection of Agrobacterium rhizogenes. During infection, Agrobacterium would transfer the T-DNA from its Ri plasmid to plant cell and randomly insert into plant chromosome. Hairy roots are characterized by high growth rate, genetic stability, and high accumulation of plant secondary metabolite, and they could be maintained in hormone-free medium. Due to these features, hairy roots are widely applied in production of high-value pharmaceutical molucules and recombinant proteins. However, still many plants are resistant to the infection of A. rhizogenes, the utilization of hairy root culture in these plants is limited. Understanding the molecular mechanism during hairy root formation increases applicability of medical herbs hairy root. Our previous study found that RolB protein can interact with ORF13a, NtbZIP, and PHI-2 by using yeast two-hybrid assay. But when applying the same method to screen the interacting proteins of RolC, we found that RolC would autoactivate the the reporter genes in Y2HGold, resulting too many false positive. In this study, we randomly mutated rolC gene by error-prone PCR to reduce autoactivity of RolC, and used these mutant RolC as bait to screen the interactors in tobacco hairy roots. Besides, we applied new mating method to increase the mating efficiency of yeasts. Using the new mating method, we found 46 potential interacting proteins of RolC.

Protein-Protein-Interaktionen haben eine wesentliche Bedeutung bei der Regulierung verschiedenster Zellfunktionen. Sie spielen u.a. bei der Funktionssteuerung von Kanälen, Transportern und Ionenpumpen eine wesentliche Rolle. Ein PDZ-Motiv am C- terminalen Ende des ATP-abhängigen Kaliumkanals ROMK ließ mögliche Inter-aktionen mit zellulären und membran-assoziierten Proteinen erhoffen. Nach Durch-führung dreier „Yeast-Two-Hybrid“-Screens zur Identifizierung möglicher Interakt-ionspartner von ROMK kamen 17, von ihrer Funktion schon bekannte, aussichtsreiche Proteine, in die enge Auswahl. Nach weiterer Charakterisierung und Autoaktivierungs-tests blieben 13 Proteine zur weiteren Abklärung übrig. GST-Pulldown-Experimente und Immunfluoreszenz brachten weitere Aufschlüsse und Erkenntnisse zur Interaktion zwischen ROMK und seinen Partnern. Folgende Erkenntnisse konnten aus den Versuchen gewonnen werden: *) 174 positive Klone interagierten bei drei „Yeast-Two-Hybrid“-Screens mit dem zytoplasmatischen Teil von ROMK. *) der zytoplasmatische Teil des ATP-abhängigen Kaliumkanals der Niere, ROMK, ist an Protein- Protein- Interaktionen beteiligt. *) Proteine des Aktin-Zytoskeletts und Tyrosinkinase-assoziierte Proteine binden an den zytoplasmatisch Teil von ROMK. Daher könnten beide in Punkt 1.5.4. erwähnten Theorien der Aktivitätsänderung ROMKs durch a) Stimulierung ruhender Kanäle bzw. b) Einbau von in Vesikel gespeicherten Kanälen in die Membran vertreten werden. *) Shank3a, Calponin2, NHERF2, NUMB2 und Antiquitin1 binden an den C-terminalen Teil von ROMK in den GST-Pull-Down-Experimenten. *) Shank3a und ArgBP2 verändern das Verteilungsmuster von ROMK in der Zelle. *) Shank3a scheint für eine Interaktion mit ROMK am bedeutungsvollsten zu sein. Hypothetische Modelle und Gedankenspiele über den möglichen Einfluss der Interaktionspartner auf ROMK wurden in der Diskussion erstellt und näher erläutert. Es ist davon auszugehen, dass einige dieser Proteine, speziell diese, die mit Tyrosinkinase und dem Aktin-Zytokeletts assoziiert sind, auf ROMK Einfluss nehmen. Weitere Studien werden hoffentlich bald Aufschlüsse über Aktivitätsänderungen des ATP-ab-hängigen K+-Kanal, ROMK, offenbaren.

碩士<br>淡江大學<br>生命科學研究所碩士班<br>96<br>In part I, high efficiency transformation by electroporation is applied to yeast two¬- hybrid screening. JHD1/YER051W is a demethylase gene from Saccharomyces cerevisiae BY4742. The JHD1/YER051W ORF is ligated to the BD vector as the “bait”. Appropriate DNA library constructed on the AD vector was screened for “fish” when the library was transformed into yeast PJ69-4A. The interacting proteins expressed from the AD vector would be selected in the specific medium. Afterwards, more functional analysis will be done to further understand the functional role of the interacting proteins. In this study, JHD1 was found to interact with CIN5/YOR028C gene product using the yeast two-hybrid screening. In part II, a nattokinase from Bacillus subtilis TKU007 was investigated. Propeptide, which is 77 amino acids fragment at the N terminus of the nattokinase, is required to convert pronattokinase to the mature active nattokinase. The propeptide is then cleaved off by the mature nattokinase. In this research, we used PCR (polymerase chain reaction) to clone pronattokinase and nattokinase sequence, respectively. Both of them are linked to His•Tag coding sequence at their C termini, and ligated to the expression vector pYES2. Heterologous expression in yeast was induced by galactose, and the over-expressed protein was purified .

碩士<br>國立陽明大學<br>生物藥學研究所<br>89<br>Telomeres are the specialized structure at the very ends of eukaryotic chromosomes. They are essential for the maintenance of chromosome integrity and protect chromosomes from degradation by nucleases. Loss of telomeres could lead to cell death. In Saccharomyces cerevisiae, the telomere sequence is composed of a ~300 bp of C1-3A/TG1-3 repeats plus a short single-stranded TG1-3 tail. Cdc13p is a single strand TG1-3 telomeric DNA-binding protein. It protects telomeres and regulates telomere length. The relative molecular mass of Cdc13p is about 105 kDa that contains 924 amino acids. Here, we analyszed CDC13 deletion mutants and found that the N-terminal 1-252 amino acids of Cdc13p were involved in telomere maintenance and cell growth. We screened for proteins that interacted with Cdc13(1-252)p with two-hybrid analysis and found Cdc13(1-252)p interacted with Imp4p and the catalytic subunit of DNA polymeraseα(Pol1p). We showed that Cdc13(1-252)p interacted directly with Pol1p or Imp4p in vitro. Also, using co-immuno-precipitation assay, we demonstrated that Cdc13p interacted with Imp4p in vivo. The possible roles of Pol1p and Imp4p in telomere function and cell growth are discussed.