| Abstract |
This thesis consists of two parts. In the first, we describe the construction of a high resolution, sequence-ready physical map of the gene-rich q23.31-q23.33 region of human chromosome 10 and evaluate the information derived from the in silico analysis of the corresponding DNA sequence. In the second part, we present our work on structural, evolutionary and functional characteristics of the murine Neuralized ( Νeurl ) gene, the human NEURL ortholog located adjacently to the above region. By using as probes 36 STSs (Sequence Tag Sites) mapped to 10q23.31-q23.33, we isolated 22 PAC and 316 BAC chromosomal clones and proceeded to their structural analysis by means of comparative DNA fingerprinting. Our data led to the construction of a well-supported, sequence-ready unique contig of about 4.38 Mb delimited by the microsatellite markers D10S541 and D10S583, located proximal and distal to the centromere, respectively. The contig was used by the collaborating laboratory of Sanger Center, for the selection of a tiling path of 35 BACs, which were then employed in DNA sequence determination. The corresponding finished DNA sequence is without gaps and, as revealed by extensive in silico analysis, contains 51 genes, 35 of which are of unknown function. However, 11 of the latter encode polypeptides with distinctive domains present in proteins of known function. Recent studies from a number of groups including ours, have suggested that the drosophila neuralized (neur) participates in Notch signaling and that the mouse ortholog, Neurl, may play a regulatory role in various cellular functions. In order to further characterize Neurl , we isolated 17 phage clones from a mouse strain 129SV genomic library and determined the chromosomal organization of the gene. In combination with information from various databases, our experimental data suggested that Neurl spans 79 Kb of genomic DNA, consists of 7 exons and appears to be under the control of two distinct, alternatively used promoters, one of which is located in the 47.3 Kb long, intron 1. The gene encodes two Neurl polypeptides, 574 and 557 aa long, differing at their amino-terminus, as a result of the alternative use of exon 1 and the downstream exon 1a. Extensive comparative analysis at the nucleotide level suggested that the chromosomal organization of the gene is quite similar in insects and mammals and demonstrated possible Νeurl regulatory elements in mammals. Moreover, systematic protein sequence comparisons among neuralized orthologs from several organisms, i.e.: C. elegans D. melanogaster, A. gambiae, F. rubripes, D. rerio, X. laevis, R. norvegicus, revealed that the neuralized protein family is well-conserved in multicellular animal species from the worm and insects to mammals, prominently at the characteristic NEUZ 1, 2 and RING domains. In particular, NEUZ1 represents the most conserved domain among vertebrates whereas the RING is the most conserved domain between vertebrate and invertebrate species. The latter, possibly suggests similar biochemical function of the protein in all species and demonstrates the central role of the RING domain. In order to further elucidate the biological role of Neurl, we employed the yeast two-hybrid system to identify proteins that directly interact with it. The screening resulted in the identification of 9 proteins known to participate in significant cell functions, i.e: motor-based intracellular transport, cell movement or stability (Dlc-1, Gas8, Evpl and ankycorbin), cell division or apoptosis (Numa1, Lek1, Dlc-1, Peg3), posttranslational protein modification (Ubc9) and transcriptional regulation (Dnmt3a). Protein interactions were further verified by in vitro and cell culture experiments, using GST pull-downs and co-immunoprecipitation (co-IP) assays, respectively. Supporting evidence for these interactions, also, derived from: i. Demonstrating overlapping expression patterns of the corresponding genes in mouse embryo sections, using comparative RNA in situ hybridization, and ii. detecting subcellular co-localization of the respective endogenous and/or transiently expressed recombinant proteins, using confocal microscopy. As shown by co-IP assays where a number of complimentarily truncated polypeptides were employed, Neurl-protein interactions are in general mediated by NEUZ1 and NEUZ2. However, a number of differencess exist. NEUZ1 is absolutely necessary and sufficient for binding with Dlc-1 and Gas8 but not with Ubc9, whereas the RING domain is required for adequate Neurl binding with Ubc9. In accordance to the general notion about RING containing proteins, our results suggest that Neurl may function as an adaptor molecule bringing, selectively, together various proteins and thus, participating as an essential component, in multi-protein cellular complexes. Accordingly, its interaction with Dlc-1 may explain the axonemal defects manifested in Neurl -/- male mice. In addition, its interaction with Ubc9 may imply either its post-translational modification by SUMO (Small Ubiquitin-like Modifier) or its role as a SUMO E3 ligase. Interestingly, several of the Neurl interactors display appropriate sites for sumoylation and thus could be considered as candidate substrates. In fact one of them, Dnmt3a, is already known to be subjected in sumoylation. Conclusively, we believe that the identification of Neurl interacting proteins is fundamental towards the elucidation of its biological role and motivates the design of a long series of interesting in vitro and in vivo experiments for its further functional analysis.
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Φυσική χαρτογράφηση υψηλής ευκρίνειας στη χρωμοσωμική περιοχή 10q23 του ανθρώπου και συμβολή στη λειτουργική ανάλυση του γονιδίου Neuralized
