The problem of identifying genes in eukaryotic genomic sequences by computational methods has attracted considerable research attention in recent years. Many early approaches to the problem focused on prediction of individual functional elements and compositional properties of coding and non coding deoxyribonucleic acid (DNA) in entire eukaryotic gene structures. More recently, a number of approaches has been developed which integrate multiple types of information including structure, function and genetic properties of proteins. Knowledge of the structure of a protein is essential for describing and understanding its function. In addition, subcellular localization of a protein can be used to provide some amount of characterization of a protein. In this study, a method for the prediction of protein subcellular localization based on primary sequence data is described. Primary sequence data for a protein is based on amino acid sequence. The frequency value for each amino acid is computed in one given position. Assigned frequencies are used in a new encoding scheme that conserves biological information based on point accepted mutations (PAM) substitution matrix. This method can be used to predict the nuclear, the cytosolic sequences, the mitochondrial targeting peptides (mTP) and the signal peptides (SP). For clustering purposes, other than well known traditional techniques, principle component analysis (PCA)&quot / and self-organizing maps (SOM)&quot / are used. For classication purposes, support vector machines (SVM)&quot / , a method of statistical learning theory recently introduced to bioinformatics is used. The aim of the combination of feature extraction, clustering and classification methods is to design an acccurate system that predicts the subcellular localization of proteins presented into the system. Our scheme for combining several methods is cascading or serial combination according to its architecture. In the cascading architecture, the output of a method serves as the input of the other model used.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

In this study, the focus is on predicting the subcellular localization of a protein, since subcellular localization is helpful in understanding a protein&rsquo / s functions. Function of a protein may be estimated from its sequence. Motifs or conserved subsequences are strong indicators of function. In a given sample set of protein sequences known to perform the same function, a certain subsequence or group of subsequences should be common / that is, occurrence (frequency) of common subsequences should be high. Our idea is to find the common subsequences through clustering and use these common groups (implicit motifs) to classify proteins. To calculate the distance between two subsequences, traditional string edit distance is modified so that only replacement is allowed and the cost of replacement is related to an amino acid substitution matrix. Based on the modified string edit distance, spectral clustering embeds the subsequences into some transformed space for which the clustering problem is expected to become easier to solve. For a given protein sequence, distribution of its subsequences over the clusters is the feature vector which is subsequently fed to a classifier. The most important aspect if this approach is the use of spectral clustering based on modified string edit distance.

The focus of this study is on predicting the subcellular localization of a protein. Subcellular localization information is important for protein function annotation which is a fundamental problem in computational biology. For this problem, a classification system is built that has two main parts: a predictor that is based on a feature mapping technique to extract biologically meaningful information from protein sequences and a client/server architecture for searching and predicting subcellular localizations. In the first part of the thesis, we describe a feature mapping technique based on frequent patterns. In the feature mapping technique we describe, frequent patterns in a protein sequence dataset were identified using a search technique based on a priori property and the distribution of these patterns over a new sample is used as a feature vector for classification. The effect of a number of feature selection methods on the classification performance is investigated and the best one is applied. The method is assessed on the subcellular localization prediction problem with 4 compartments (Endoplasmic reticulum (ER) targeted, cytosolic, mitochondrial, and nuclear) and the dataset is the same used in P2SL. Our method improved the overall accuracy to 91.71% which was originally 81.96% by P2SL. In the second part of the thesis, a client/server architecture is designed and implemented based on Simple Object Access Protocol (SOAP) technology which provides a user-friendly interface for accessing the protein subcellular localization predictions. Client part is in fact a Cytoscape plug-in that is used for functional enrichment of biological networks. Instead of the individual use of subcellular localization information, this plug-in lets biologists to analyze a set of genes/proteins under system view.

Positive strand RNA viruses, inculding tombusviruses, are known to utilize cellular membranes to assemble their replicase complexes (VRCs). Two tombusviruses , Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), replicate on different organellar membranes, peroxisomes or endoplasmic reticulum (ER) for TBSV and mitochodria outer membranes in case of CIRV. I showed that both TBSV and CIRV replicase proteins could assemble VRCs and replicate viral RNA on purified microsomes (ER) and mitochondria. Different efficiencies of assembly was shown determined by multiple domains on TBSV or CIRV replication proteins. To study why VRC assembly could occur on an alternative organellar membranes, I focused on the phospholipids, key lipid components in ER or mitochondria membranes. Phospholipids directly interact with viral replicases, however, their specific roles during (+)RNA virus replication are far less understood. I used TBSV as a model (+) RNA virus, and established a cell-free TBSV replication system using artificial membranes prepared from different phospholipids. I showed that phosphatidylethanolamine (PE) is required for full cycle replication of the viral RNA.Moreover, PE is enriched at the sites of TBSV replication in plant and yeast cells, and was up-regulated during TBSV replication. Furthermore, up-regulation of total cellular PE content in yeast due to deletion of CHO2 leads to dramatically stimulated TBSV replication. Overall, I identified PE as the key lipid component of membranes required for TBSV replication, and my data highlighted that PE, an abundant phospholipid in all eukaryotic cells, not only serves as a structural component of membrane bilayers, its interaction with the viral replication proteins also stimulates (+)RNA virus replication. Further experiments indicated both early secretory pathway and endocytic pathway are involved in PE re-distribution to site of replication. In addition to lipids and subcellular membranes, certain host proteins are also involved in (+) RNA virus replication and VRC assembly. I identified Hop-like stress- inducible protein 1 (Sti1p), which interacts with heat shock protein 70, is required for the inhibition of CIRV replication. My findings indicate that Hop/Sti1 co-chaperone could act as a virus restriction factor in case of mitochondrial CIRV, but not against peroxisomal tombusvirus.

Understanding microscale biological processes as cells develop into tissues is one of the most important, yet most difficult, problems in modern biology. Cells encounter a dynamic chemical and physical environment and delineating the myriad of variables proves daunting with even the most sophisticated experiments. This dissertation focuses on the development and application of unique enabling technologies designed to sample and control biological microenvironments. By developing two approaches – one aimed at intracellular biochemistry and another for extracellular targets – based on photochemistry and optical force generation, research presented here will allow new areas of subcellular dynamics to be addressed. On the intracellular side, enzyme-immobilized polymeric microspheres or enzyme microstructures are placed into the cell cytosol via optical tweezers for sustained and localized chemical modification of the intracellular environment. This approach is complemented by the use of extracellular guidance barriers formed from photo-induced crosslinking of proteins. Through the use of minimally toxic photosensitizers and femtosecond (fs) near infrared (NIR) light, it is possible to fabricate three-dimensional protein structures in a living cell’s environment. Moreover, this work explores the ability to form protein structures with enzymatic activity as well as with high aspect-ratio features at micron resolution. Finally, the photochemical transformation of serotonin into a highly fluorescent visible photoproduct is investigated as a means to overcome problems associated with sample size in neurotransmitter detection during synaptic chemical signaling. Optimization of this multiphoton process entails understanding the mechanism by which the photoproduct is created and experiments towards this goal are presented here. Ultimately, the precision and flexibility of these technologies will allow access to new areas of the biosciences. / text

Για τη διατήρηση της γονιδιωματικής σταθερότητας, είναι θεμελιώδους σημασίας η πραγματοποίηση της αντιγραφής του DNA με ακρίβεια και πιστότητα. Στη σωστή χρονικά έναρξη της αντιγραφής σημαντικό ρόλο κατέχει η «αδειοδότηση» της αντιγραφής, η οποία διασφαλίζει ότι η χρωματίνη είναι ικανή για τον επόμενο κύκλο αντιγραφής μόνο αφότου το κύτταρο έχει διέλθει από τη μίτωση. Η αδειοδότηση λαμβάνει χώρα μέσω της διαδοχικής συγκρότησης στις αφετηρίες της αντιγραφής, ενός πολύ-πρωτεϊνικού συμπλόκου, του καλούμενου προ-αντιγραφικού συμπλόκου. Απαραίτητος για τη συγκρότηση του προ-αντιγραφικού συμπλόκου είναι ο παράγοντας Cdt1. Στα μετάζωα, ένα μικρό μόριο, η Geminin, αναστέλλει τη δράση του Cdt1 κατά τις φάσεις S, G2 και Μ. Η Geminin είναι ένα μόριο με δομή σπειροειδούς σπειράματος, το οποίο συμμετέχει σε ευρύ φάσμα μοριακών αλληλεπιδράσεων και ελέγχει διαδικασίες του κυτταρικού κύκλου και της διαφοροποίησης. Η ρύθμιση της Geminin επιτυγχάνεται κύρια σε μεταγραφικό επίπεδο και μέσω πρωτεόλυσης, ενώ πρόσφατα δείχθηκε ότι μετα-μεταφραστικές τροποποιήσεις του μορίου διασφαλίζουν την απενεργοποίησή του κατά την G1. Για την εκδήλωση της δράσης της Geminin απαιτείται η παρουσία της πρωτεΐνης στον πυρήνα. Για την ενδοκυτταρική μελέτη της συμπεριφοράς της Geminin, δημιουργήσαμε υβριδικές μορφές του μορίου με την πράσινη φθορίζουσα πρωτεΐνη (GFP) και κατόπιν παροδικής διαμόλυνσης μελετήσαμε τον ενδοκυτταρικό του εντοπισμό. Η εξωγενής Geminin εντοπίζεται στον πυρήνα (όπως και το ενδογενές μόριο) σε έναν υποπληθυσμό ασύγχρονων κυττάρων αλλά επιπλέον, στα μισά κύτταρα του πληθυσμού, αποκλείεται από τον πυρήνα και συσσωρεύεται στο κυτταρόπλασμα. Η εμφάνιση της Geminin αποκλειστικά εκτός πυρήνα πραγματοποιείται σε κύτταρα που βρίσκονται στη φάση G1 του κυτταρικού κύκλου και συγκεκριμένα φαίνεται να λαμβάνει χώρα στο τέλος της G1 φάσης, πλησίον της μετάβασης από την G1 στην S. Κατά την είσοδο των κυττάρων στον κυτταρικό κύκλο από τη φάση ηρεμίας G0, το ποσοστό των κυττάρων που εκφράζουν την Geminin στο κυτταρόπλασμα αυξάνεται σημαντικά. Το γεγονός αυτό προσδίδει φυσιολογικό ρόλο στο φαινόμενο του αποκλεισμού της Geminin από τον πυρήνα και το συνδέει με την προαγωγή της αδειοδότησης της αντιγραφής πριν την είσοδο του κυττάρου στην S. Ανάλυση των επιμέρους περιοχών του μορίου με δημιουργία προοδευτικών απαλοιφών περιοχών της Geminin ανέδειξε το ρόλο της αμινοτελικής περιοχής του μορίου (aa 1-30) στον κυτταροπλασματικό εντοπισμό του. Ο πυρηνικός εντοπισμός της Geminin επάγεται από την κεντρική περιοχή του μορίου ενώ ένα αμινοτελικό πιθανολογούμενο NLS δε φαίνεται από μόνο του –τουλάχιστον- στην ανθρώπινη Geminin να αρκεί για τον πυρηνικό εντοπισμό της. Από τους παράγοντες που μελετήθηκαν, οι οποίοι θα μπορούσαν να επηρεάζουν in trans τον εντοπισμό της Geminin, το Cdt1, ο μοριακός συνοδός της Geminin, είναι ικανός να κατευθύνει τον αναστολέα του στον πυρήνα μετά από συνδιαμόλυνση σε ανθρώπινα καρκινικά κύτταρα. Συνδιαμόλυνση με μορφές των δύο μορίων που δεν μπορούν να αλληλεπιδράσουν δεν οδηγεί σε πυρηνικό εντοπισμό της Geminin. Συμπεραίνουμε ότι το Cdt1 προκαλεί διαμετατόπιση της Geminin στον πυρήνα μέσω άμεσης αλληλεπίδρασης με αυτήν. Τα αποτελέσματά μας αναδεικνύουν έναν νέο τρόπο ρύθμισης της Geminin μέσω πυρηνικού αποκλεισμού και παρέχουν πληροφορίες για το πότε και πώς λαμβάνει χώρα αυτή η ρύθμιση. Πρόσφατα, στις ηλεκτρονικές βάσεις δεδομένων εντοπίσαμε έναν γονιδιακό τόπο που κωδικοποιεί για πρωτεϊνικό μόριο με μεγάλη ομολογία με το σπειροειδές σπείραμα (coiled-coil) της Geminin (Geminin cc-like ή Castor). Στην παρούσα μελέτη εξετάζεται η χωρο-χρονική έκφραση της Geminin cc-like (Castor), οι αλληλεπιδράσεις με υποψήφια μόρια και η συμπεριφορά του μορίου σε συνθήκες υπερέκφρασης και αποσιώπησης σε ανθρώπινα κύτταρα. Για τη μελέτη αυτή δημιουργήθηκε υβριδική μορφή της πρωτεΐνης με την GFP. Τα αποτελέσματά μας δείχνουν ότι Geminin cc-like (Castor) είναι πυρηνική πρωτεΐνη, όπως και η ενδογενής Geminin και η ενεργότητα σήματος πυρηνικού εντοπισμού φαίνεται να εδράζεται στην καρβοξυτελική περιοχή του μορίου. Η Geminin cc-like (Castor) αλληλεπιδρά με την Geminin και μάλιστα αποτελεί έναν ακόμη παράγοντα, μαζί με το Cdt1, που μπορεί να κατευθύνει in trans την Geminin στον πυρήνα. Πειράματα υπερέκφρασης παρέχουν ενδείξεις για παρεμπόδιση της εισόδου των κυττάρων στη φάση S, ενώ αποσιώπηση της Geminin cc-like (Castor) με τη χρήση RNAi φαίνεται να αυξάνει το ποσό της φωσφορυλιωμένης μορφής της ιστόνης H2Ax, δείκτη διπλών κατατμήσεων στο DNA. Από εξέταση για την ύπαρξη ορθολόγων σε άλλους οργανισμούς, βρέθηκε ορθόλογη πρωτεΐνη στον ιχθύ Oryzias latipes (medaka). Κλωνοποιήθηκε τμήμα του mRNA της O.l Castor πρωτεΐνης, πραγματοποιήθηκε ανάλυση της έκφρασης της Geminin cc-like (Castor) σε έμβρυα medaka καθώς και πειράματα υπερέκφρασης μετά από ένεση τμήματος του mRNA, τα οποία παρέχουν προκαταρκτικές ενδείξεις για ελαττωματικό σχηματισμό της κεφαλής και των οφθαλμών. Συμπερασματικά, μελετήσαμε μία νέα πρωτεϊνη στον άνθρωπο η οποία εμφανίζει μερική ομολογία προς την Geminin (Geminin cc-like ή Castor), και δείξαμε ότι αποτελεί ένα νέο μοριακό συνοδό της Geminin, με πιθανή συμμετοχή σε κυτταρικό κύκλο και διαφοροποίηση. / For genomic stability to be maintained, accurate regulation of replication has to be achieved. Replication licensing takes places during G1 and involves the stepwise formation of a multimeric complex, the pre-replicative complex (pre-RC), onto origins of replication, ensuring that chromatin will be competent for replication only once per cell cycle. Cdt1, a key component of this complex, is accurately regulated throughout cell cycle, through multiple mechanisms. Geminin, a metazoan-specific inhibitor of licensing, binds and inhibits Cdt1, thus preventing ectopic pre-RC formation during S, G2 and M. Geminin is regulated both at the level of transcription and proteolysis. Recently, it was also demonstrated that inactivation of non-proteolysed Geminin during G1 ensures timely formation of the pre-RC. Through its coiled-coil region, many interactions with binding partners are achieved, thus enabling participation in a variety of cellular processes during the cell cycle and differentiation. In order to study the intracellular behaviour of Geminin, fused forms of the molecule with the fluorescent protein GFP were constructed and transfected in MCF7 cells. Exogenous Geminin is located in the nucleus, similar to the endogenous molecule, in a subpopulation of asynchronously growing cells, but in approximately 50% of cells, Geminin is specifically excluded from the nucleus. Cytoplasmic localization of Geminin takes place during G1 and specifically during late G1, close to the G1 to S transition. In addition, upon exit from quiescence and cell cycle re-entry, cytoplasmic localization of Geminin increases notably. This may reflect a physiological role of Geminin exclusion to the cytoplasm upon cell cycle re-entry, in order to permit a ‘window of opportunity’ for licensing to take place prior to S phase onset. Progressive deletions of the Geminin molecule resulted in a variety of mutated forms which were then analysed for their ability to be localized to the nucleus or the cytoplasm. This analysis showed the importance of the aminoteminal region of Geminin (aa 1-30) in the cytoplasmic localization of the molecule. A putative nuclear localization activity was mapped to the central part of the molecule, while a proposed NLS at the aminoterminal part was shown to be neither sufficient nor required for nuclear localization. Amongst the factors tested, which could affect intracellular localization of Geminin, we show that Cdt1 possesses the ability to direct Geminin to the nucleus. Our data provide evidence for a novel means of regulation of Geminin by cell cycle dependant subcellular localization and offer insight on when and how this regulation takes place. Recently, in silico analysis revealed a gene locus in the human genome, coding for a predicted coiled coil protein with homology to Geminin. The gene product was initially named after this similarity as Geminin coiled coil-like (Geminin cc-like) and then renamed ‘Castor’. Present work examines the spatio-temporal expression of a hybrid Castor-GFP molecule, interactions with candidate molecular partners and the consequences for the cell of Castor overexpression or silencing. Our results show that exogenous Castor-GFP is localized in the nucleus and excluded from the nucleoli, similar to endogenous Geminin, whereas evidence for a putative NLS activity at the carboxy-terminus of the molecule is provided. Castor interacts with Geminin in cellular extracts and is sufficient to translocate Geminin-GFP to the nucleus, similar to Cdt1. Overexpression experiments provided evidence for a delayed G1 to S transition. Silensing Castor by siRNA led to an increase in γ-H2Ax staining, a marker of double strand breaks. Further in silico analysis revealed orthologues of Castor in other organisms, from mammals to fish. Part of the putative Castor mRNA in the fish Oryzias latipes (Medaka) was cloned. In vivo analysis of Castor in medaka involved expression pattern analysis by whole mount in situ hybridization and overexpression amalysis by mRNA injections, providing preliminary evidence for impaired forehead and eye formation. In conclusion, we have studied a novel protein with similarity to the coiled-coil region of Geminin (Geminin cc-like/ Castor) and showed that it constitutes a novel molecular partner of Geminin with possible roles during the cell cycle and in development.

Η παρούσα εργασία είχε ως αντικείμενο, αρχικά, τη δημιουργία υβριδικών μορφών της πρωτεΐνης Geminin- ενός αρνητικού ρυθμιστή του κυτταρικού κύκλου- με το φθορίζον μόριο GFP (Green Fluorescent Protein). Πραγματοποιήθηκε τόσο αμινοτελική όσο και καρβοξυτελική σύντηξη της Geminin με το μόριο-ιχνηθέτη, έτσι ώστε να μελετηθεί η συμπεριφορά του νέου, υβριδικού μορίου και στις δύο περιπτώσεις και να εξεταστεί εάν ο προσανατολισμός της GFP πρωτεΐνης δύναται να μεταβάλλει το χαρακτήρα της σημασμένης πρωτεΐνης. Ακολούθησε ανίχνευση και παρακολούθηση καθενός υβριδικού μορίου σε ανθρώπινα καρκινικά κύτταρα μετά από διαμόλυνση για μικρό (παροδική διαμόλυνση διάρκειας 22 ωρών) ή μεγαλύτερο χρονικό διάστημα (σταθερά διαμολυσμένες κυτταρικές σειρές). Ο χαρακτηρισμός των φθοριζουσών μορφών της Geminin περιελάμβανε μελέτη τόσο του υποκυτταρικού εντοπισμού όσο και της χρονικής έκφρασης των μορίων και σύγκριση με τα αντίστοιχα χαρακτηριστικά του ενδογενούς μορίου. Ανάλυση του ενδοκυτταρικού εντοπισμού τόσο της καρβοξυτελικής όσο και της αμινοτελικής σύντηξης της Geminin με τη GFP, αποκάλυψε, εκτός του πυρηνικού φαινοτύπου, που παρατηρείται φυσιολογικά για το ενδογενές μόριο, σημαντικό ποσοστό παροδικά διαμολυσμένων κυττάρων με τα υβριδικά μόρια εκτός του πυρηνικού διαμερίσματος. Ο φαινότυπος αυτός εμφανίζεται – σε μικρότερο ποσοστό- ακόμη και στην περίπτωση σταθερά διαμολυσμένων κυττάρων, όπου τα επίπεδα έκφρασης της πρωτεΐνης Geminin-GFP είναι παρόμοια με τα αντίστοιχα του ενδογενούς μορίου. Στη σειρά αυτή, το μεγαλύτερο ποσοστό κυττάρων, παρά την ισχυρή μεταγραφική δραστηριότητα που προσδίδει ο CMV υποκινητής, εκφράζει το εξωγενές μόριο σύμφωνα με το πρότυπο που παρουσιάζει η ενδογενής Geminin, δηλαδή κατά τις φάσεις S και G1. Επομένως, η ρύθμιση της Geminin-GFP υβριδικής πρωτεΐνης πραγματοποιείται σε μεγάλο βαθμό σε μετα-μεταγραφικό επίπεδο. Ιδιαίτερο ενδιαφέρον παρουσιάζει το γεγονός ότι η αλλαγή στην ενδοκυτταρική κατανομή του υβριδικού μορίου δεν παρουσιάζει τυχαία χρονική εμφάνιση. Το γεγονός ότι εμφανίζεται αποκλειστικά σε αρνητικά για κυκλίνη Α κύτταρα, φανερώνει την ύπαρξη συσχέτισης με τη φάση του κυτταρικού κύκλου και μάλιστα υποδηλώνει την επιλεκτική έξοδό της από τον πυρήνα κατά τη φάση G1. Πιθανόν, η έξοδος της Geminin από τον πυρήνα κατά τη φάση αυτή, να διασφαλίζει τη διεκπεραίωση της διαδικασίας της αδειοδότησης του γενετικού υλικού –αναστολέας της oποίας είναι η Geminin και κατ’ επέκταση την πρόοδο του κυτταρικού κύκλου Το φαινόμενο αυτό χρήζει περαιτέρω διερεύνησης μιας και η αλλαγή στην ενδοκυτταρική κατανομή πολλών ρυθμιστικών μορίων αποτελεί έναν αποδοτικό τρόπο ελέγχου της γονιδιακής έκφρασης στους ευκαρυωτικούς οργανισμούς, ενώ παράλληλα φαίνεται να επηρεάζει πολλές ζωτικής σημασίας λειτουργίες. / The aim of the present study was the construction of fused forms of Geminin- a negative cell cycle regulator-with GFP (Green Fluorescent Protein). Amino- and carboxy terminal fusions of Geminin and GFP were performed, in order to study the behaviour of the hybrid molecule in both cases and to examine if the orientation of the reporter gene can interfere with the character of the new molecule. Each fused molecule was either transiently or stably transfected in human cancer cells and its behaviour was studied. The characterization of the fluorescent molecules of Geminin was performed at the level of subcellular localization and regulation throughout the cell cycle and was compared to the endogenous characteristics. Analysis of the subcellular distribution of both fused forms, revealed -apart from the nuclear phenotype, normally observed- a significant percentage of transiently transfected cells, where the hybrid molecules were excluded from the nucleus. This phenotype was observed- though less strongly- in the case of stably transfected cells, where the expression levels of Geminin-GFP protein are similar to the endogenous molecule. The majority of these cells, despite the strong CMV driven transcriptional activity, express Geminin-GFP during S and G1 phase, like the endogenous protein, suggesting that the regulation of Geminin-GFP is performed mainly at post-transcriptional level. This nuclear exclusion was observed according to a cell cycle- dependent manner. The phenomenon of nucleocytoplasmic shuttling of many proteins seems to be a very efficient way of controlling gene expression in eukaryotes. In order to elucidate the role of sucellular distribution of Geminin-GFP in cell cycle regulation, further examination of this phenomenon is needed.

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