<p>Post-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of <italic>Drosophila melanogaster</italic> is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the <italic>Drosophila </italic> embryo. This thesis addresses the actions of the translational repressor, Pumilio, in regulating two mRNAs during early embryogenesis. First, we examined the ability of Pumilio to regulate the mRNA stability of <italic>bicoid</italic>, a gene required for <italic>Drosophila </italic> head development. <italic>bicoid</italic> mRNA contains the canonical Pumilio recognition site, termed the Nanos response element (NRE), within the 3'UTR. Interestingly, we show that Pumilio binds to the NRE both in vitro and in vivo; however, no physiological significance is associated with this interaction. Furthermore, in <italic> pumilio</italic> mutant embryos <italic>bicoid</italic> mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate <italic>bicoid</italic> mRNA. Second, Pumilio has been shown to negatively regulate <italic>Cyclin B</italic>, the cyclin necessary for mitotic entry, in the somatic cytoplasm of the embryo and this repression is alleviated by the PNG Kinase complex through currently unidentified mechanisms. We further investigated the actions of Pumilio in regulating <italic>Cyclin B</italic> and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic <italic>Cyclin B</italic>. Furthermore, we show that the 3'UTR of <italic>Cyclin B</italic> is not required for the regulation by Pumilio and the PNG Kinase complex. Lastly, through genetic analyses, we conclude that Pumilio may actually act upstream of the PNG Kinase complex to regulate <italic>Cyclin B</italic>.</p> / Dissertation

<p>Transcription initiation is a key component in the regulation of gene expression. Recent high-throughput sequencing techniques have enhanced our understanding of mammalian transcription by revealing narrow and broad patterns of transcription start sites (TSSs). Transcription initiation is central to the determination of condition specificity, as distinct repertoires of transcription factors (TFs) that assist in the recruitment of the RNA polymerase II to the DNA are present under different conditions. However, our understanding of the presence and spatiotemporal architecture of the promoter patterns in the fruit fly remains in its infancy. Nucleosome organization and transcription initiation have been considered hallmarks of gene expression, but their cooperative regulation is also not yet understood.</p> <p>In this work, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs), and developed an improved paired-end sequencing strategy to explore the transcription initiation landscape of the D.melanogaster genome. We distinguished three initiation patterns: 'Peaked or Narrow Peak TSSs&#8219;, 'Broad Peak TSSs&#8219;, and 'Broad TSS cluster groups or Weak Peak TSSs&#8219;. The promoters of peaked TSSs contained the location specific sequence elements, and were bound by TATA Binding Protein (TBP), while the promoters of broad TSS cluster groups were associated with non-location-specific elements, and were bound by the TATA-box related Factor 2 (TRF2).</p> <p>Available ESTs and a tiling array time series enabled us to show that TSSs had distinct associations to conditions, and temporal patterns of embryonic activity differed across the majority of alternative promoters. Peaked promoters had an association to maternally inherited transcripts, and broad TSS cluster group promoters were more highly associated to zygotic utilization. The paired-end sequencing strategy identified a large number of 5' capped transcripts originating from coding exons that were unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications.</p> <p>We applied an innovative search program called FREE to embryo, head, and testes specific core promoter sequences and identified 123 motifs: 16 novel and 107 supported by other motif sources. Motifs in the embryo specific core promoters were found at location hotspots from the TSS. A family of oligos was discovered that matched the Pause Button motif that is associated with RNA pol II stalling.</p> <p>Lastly, we analyzed nucleosome organization, chromatin structure, and insulators across the three promoter patterns in the fruit fly and human genomes. The WP promoters showed higher associations with H2A.Z, DNase Hypersensitivity Sites (DHS), H3K4 methylations, and Class I insulators CTCF/BEAF32/CP190. Conversely, NP promoters had higher associations with polII and GAF binding. BP promoters exhibited a combination of features from both promoter patterns. Our study provides a comprehensive map of initiation sites and the conditions under which they are utilized in D. melanogaster. The presence of promoter specific histone replacements, chromatin modifications, and insulator elements support the existence of two divergent strategies of transcriptional regulation in higher eukaryotes. Together, these data illustrate the complex regulatory code of transcription initiation.</p> / Dissertation

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

Chez les organismes pluricellulaires, la prolifération, la différenciation ou encore la<br />mort des cellules reposent en partie sur l'activation et la répression de gènes spécifiques. Les<br />profils d'expressions géniques ainsi mis en place sont maintenus d'une génération cellulaire à<br />l'autre par des mécanismes épigénétiques stables qui altèrent la structure de la chromatine au<br />niveau des gènes cibles. Les protéines des groupes Polycomb (PcG) et Trithorax (TrxG)<br />établissent une mémoire cellulaire au cours des mitoses en maintenant l'état transcriptionnel<br />de nombreux gènes par une réorganisation de la structure chromatinienne. Les protéines PcG<br />maintiennent la répression des gènes cibles en créant des domaines chromatiniens compacts<br />où l'ADN est peu accessible. Les protéines TrxG assurent le maintien de l'activation des gènes<br />en bloquant la propagation des complexes PcG et en déplaçant les nucléosomes. Chez la<br />drosophile, ces protéines agissent au sein de complexes multimèriques qui se lient à la<br />chromatine au niveau de modules communs appelés modules de mémoire cellulaire (CMM).<br />Les PcG et TrxG régulent l'expression de nombreux gènes dont les plus étudiés sont les gènes<br />homéotiques. Aujourd'hui, si les différents acteurs de la mémoire cellulaire commencent à<br />être bien connus, plusieurs questions restent posées. Quels sont les moyens de recruter<br />spécifiquement les PcG et TrxG sur leurs cibles ? Quels sont les mécanismes moléculaires qui<br />permettent le maintien de cette mémoire ? Quels sont les gènes cibles ? Est-ce que les<br />mécanismes d'action que nous étudions au niveau des gènes homéotiques sont communs à<br />l'ensemble des gènes régulés par les PcG et TrxG ?<br />Notre équipe travaille sur une protéine à boîte HMG de drosophile : DSP1 (Dorsal<br />Switch Protein 1). L'étude d'un mutant nul dsp11 a montré que la protéine intervient dans la<br />régulation des gènes homéotiques. Par ailleurs, des études d'interactions génétiques et des<br />expériences de digestion à la DNase I montrent que DSP1 est un facteur de remodelage de la<br />chromatine qui agit suivant le locus considéré en synergie avec les protéines des groupes PcG<br />ou TrxG. DSP1 fait donc partie des protéines qui participent à la mémoire cellulaire.<br />Le travail de thèse que nous présentons ici a pour objectif la recherche des gènes cibles<br />de DSP1 et du rôle de cette protéine dans la régulation de l'expression de ces gènes. Nous<br />avons identifié des cibles préférentielles de DSP1 sur l'ensemble du génome par la technique<br />d'immunoprécipitation de la chromatine pontée (ChIP). Nous avons étudié le rôle de DSP1<br />sur l'une de ces cibles, le CMM Fab-7 dont l'activité régule le gène homéotique<br />Ultrabithorax. Au cours de cette étude nous montrons par des expériences de transgenèse que<br />DSP1 est indispensable au fonctionnement du CMM. Le recrutement des protéines PcG et<br />TrxG sur les CMM reste mal connu. La fixation de DSP1 sur le CMM Fab-7 est essentielle au<br />recrutement des PcG. Ainsi nous montrons pour la première fois que DSP1 est un recruteur<br />précoce de ces protéines suivant le locus considéré au cours de l'embryogenèse. Au contraire,<br />nous montrons que DSP1 intervient tardivement dans l'activation du gène homéotique Sex<br />combs reduced. Dans ce cas, la protéine n'est essentielle qu'au cours de la vie larvaire. Enfin,<br />nous montrons que DSP1 régule l'expression du gène de segmentation knirps et est impliquée<br />dans l'établissement de son profil d'expression plutôt que dans son maintien.<br />161<br />Les protéines des groupes Polycomb (PcG) et Trithorax (TrxG) établissent une<br />mémoire cellulaire en maintenant l'état transcriptionnel de nombreux gènes par un<br />remodelage de la chromatine. Chez la drosophile, ces protéines agissent au sein de complexes<br />multimériques qui se lient à des unités fonctionnelles appelées Modules de Mémoire<br />Cellulaire (CMM). Quels sont les gènes cibles des protéines PcG/TrxG ? Quels sont les<br />moyens de recruter spécifiquement ces protéines ? Sont deux questions auxquelles nous avons<br />voulu répondre. Notre équipe travaille sur une protéine à boîtes HMG de drosophile : DSP1.<br />L'objectif du travail que nous présentons ici est de rechercher des gènes cibles de DSP1 et son<br />rôle dans la régulation de l'expression de ces gènes. Plusieurs cibles ont été identifiées par la<br />technique d'immunoprécipitation de la chromatine pontée (ChIP). Nous avons étudié le rôle<br />de DSP1 sur le CMM Fab-7 qui régule le gène Ultrabithorax. Nous montrons par des<br />expériences de transgenèse que la fixation de DSP1 est indispensable à l'activité du CMM.<br />Par ailleurs, nous montrons pour la première fois que DSP1 est un recruteur précoce des<br />protéines PcG. Nous montrons également que DSP1 intervient en tant que protéine du groupe<br />TrxG dans l'activation du gène Sex combs reduced. Dans ce cas, DSP1 est essentielle au cours<br />de la vie larvaire. Enfin, nous montrons que DSP1 régule l'expression du gène knirps et<br />participe à l'établissement de son profil d'expression plutôt qu'à son maintien.

Thesis (Ph. D.)--University of Chicago, Department of Organismal Biology and Anatomy, June 2001. / Includes bibliographical references. Also available on the Internet.

Phenotypic differences between the sexes evolve largely because selection favours a different complement of traits in either sex. Theory suggests that, despite its frequency, sexual dimorphism should be generally constrained from evolving because the sexes share much of their genome. While selection can lead to adaptation in one sex, correlated responses to selection can be maladaptive in the other. In this thesis I use Drosophila to examine the extent to which the shared genome constrains the evolution of sexual dimorphism and whether the sex chromosomes might play a special role in resolving intralocus sexual conflict. Gene expression data shows that intersexual genetic correlations are generally high, suggesting that genes often affect both sexes. The intersexual genetic correlation is negatively associated with sex-bias in expression in D. melanogaster, and the rate of change in sex-bias between D. melanogaster and six closely related species, showing that a sex-specific genetic architecture is a prerequisite for the evolution of sex difference. In further studies I find that genetic variance affecting lifespan is found in the male-limited Y chromosome within a population, which could offer a route to the evolution of further sexual dimorphism in lifespan, though the amount of variance was small suggesting adaptive potential from standing genetic variance is limited. Genetic variance on the X chromosome is also expected to be depleted once the sex chromosomes evolve, but here I find no evidence of depletion in either sex. Dosage compensation does not appear to double the male X-linked genetic variance, but this effect may be complex to detect. Finally, the X chromosome appears to be enriched for sex-specific genetic variance, and the consequences of this are explored using a variety of analytical methods to test biologically meaningful aspects of G-matrix structure. In summary, this thesis suggests that the evolution of sexual dimorphism is generally constrained by the shared genome, but intralocus sexual conflict could be resolved by novel mutations on the Y chromosomes, and by standing sex-specific genetic variance on the X chromosome. It highlights a special role for the X chromosome in the evolution of sexual dimorphism.

The Drosophila dorsal group gene pipe provides the crucial link that transmits dorsal-ventral (DV) polarity information from the ovary to the embryo. Females homozygous for mutations in pipe produce dorsalized embryos. pipe encodes ten protein isoforms with amino acid sequence similarity to vertebrate glycosaminoglycan 2-O-sulfotransferases, suggesting that Pipe functions by modifying a carbohydrate-bearing molecule that controls embryonic DV patterning. Two major components of my project have been to examine the functional specificities of different Pipe isoforms and to identify Pipe's enzymatic substrate and learn how it participates in DV pattern formation. I have used two approaches to investigate whether the various Pipe isoforms share the same functional specificities. In one approach, I expressed each isoform in the follicle cells and found that the expression of only one of them was able to rescue the pipe mutant phenotype or ventralize progeny embryos. In a second set of transgenic studies, three of the other isoforms were individually shown to restore the production of a pipe-dependent sulfated epitope when expressed in the salivary glands of otherwise pipe null mutant embryos. These data indicate that distinct functional specificities are associated with the various Pipe protein isoforms. In addition, these studies allowed me to determine that embryos from females lacking endogenous pipe expression nevertheless retain polarity along their dorsal-ventral axis, suggesting the existence of a second polarizing signal in addition to the ventral transcription of pipe. To identify Pipe’s substrate, I developed a technique for metabolic labeling which enabled me to identify a molecule exhibiting Pipe-dependent sulfation. This molecule was identified as the protein Vitelline Membrane-Like (VML), a putative component of the vitelline membrane layer of the eggshell. The involvement of VML in dorsalventral patterning was demonstrated on the basis of the enhancing effects of a vml mutation on the severity of dorsalization of embryos from females of a sensitized genetic background. Thus, VML represents a bona fide substrate of Pipe that participates in the establishment of dorsal-ventral polarity. In these studies I was also able to show Pipedependent sulfation of other vitelline membrane components which may also influence embryonic dorsal-ventral patterning. / text

Η μελέτη της φυλοσύνδετης μετάλλαξης wisertsl (1-21.7, 7E) της Drosophila melanogaster αποκάλυψε ότι υπεύθυνο για τους φαινότυπους φαγωμένα/τσιμπημένα φτερά, ελαφρώς ανώμαλα μάτια και τη θερμοευαισθησία είναι το γονίδιο CG32711, που ονομάσαμε wiser (wings scalloped-eyes rough). Το γονίδιο wiser είναι απαραίτητο για τη σωστή ανάπτυξη της Drosophila melanogaster. Η μετάλλαξη wisertsl χαρτογραφείται στη 5΄ ρυθμιστική περιοχή του γονιδίου wiser. Στην ίδια περιοχή χαρτογραφείται και η θανατογόνος μετάλλαξη wiserPL26. Παραπέρα μελέτη του γονιδίου wiser με τη χρήση αυτών των δυο μεταλλάξεων και του διαγονιδίου UAS-wiser αποκάλυψε ότι: α) Οι μεταλλάξεις wisertsl και wiserPL26 ενισχύουν το φαινότυπο των μεταλλάξεων Beadex1 και Serrate1. Το γονίδιο wiser αλληλεπιδρά με τα γονίδια Beadex και Serrate, τα οποία εμπλέκονται στην ενεργοποίηση του Notch μονοπατιού σηματοδότησης κατά μήκος του ραχιοκοιλιακού άξονα του φτερού. Η παρατήρηση αυτή δείχνει, ότι το wiser εμπλέκεται στον προσδιορισμό του ραχιοκοιλιακού άξονα. β) Η μετάλλαξη wisertsl σε ομοζυγωτία μειώνει σημαντικά την έκφραση των διαγονιδίων fringe-lacZ, m8-lacZ, wingless-lacZ, vestigial-lacZ και Distalless-lacZ, αλλάζει το πρότυπο έκφρασης του mβ-lacZ και δεν επηρεάζει την έκφραση του apterous-lacZ στους εμβρυικούς δίσκους του φτερού προνυμφών 3ου σταδίου. Τα αποτελέσματα αυτά έδειξαν, ότι το γονίδιο wiser δρα μετά το γονίδιο apterous και πριν το γονίδιο fringe, που είναι τροποποιητής του υποδοχέα Notch. Επομένως, η δράση του Notch εξαρτάται και από το wiser. γ) Εκτοπική έκφραση του διαγονιδίου UAS-wiser με οδηγό το ap-Gal4, έδειξε ότι διασώζει μερικώς το φαινότυπο apterous- αλλά όχι το φαινότυπο Serrate1. δ) Εκτοπική έκφραση του UAS-wiser με οδηγό το dpp-Gal4, επηρεάζει την έκφραση του wingless-lacZ αλλά όχι των apterous, fringe, mβ, m8, vestigial και Distalless στους εμβρυικούς δίσκους φτερού. ε) Η δημιουργία μιτωτικών κλώνων με το σύστημα FRT/FLP, σε θηλυκά άτομα wiserPL26/+ οδήγησε στη δημιουργία κλώνων +/+ και wiserPL26/wiserPL26 διαφορετικού μέγεθος στους εμβρυικούς δίσκους του φτερού. Οι πρώτοι (+/+), έχουν σημαντικά μεγαλύτερο μέγεθος από τους δεύτερους όταν συμβαίνουν στη περιοχή του εμβρυικού δίσκου που θα δώσει το φτερό του ακμαίου ατόμου. Στα ακμαία θηλυκά οι σωματικοί κλώνοι εκδηλώνονται με το φαινότυπο φαγωμένα φτερά. Σωματικοί κλώνοι παρατηρήθηκαν και στα μάτια των ακμαίων. Τα αποτελέσματα των μιτωτικών κλώνων δείχνουν ότι το γονίδιο wiser εμπλέκεται στον πολλαπλασιασμό των κυττάρων. Όλα τα παραπάνω αποτελέσματα, δείχνουν ότι το γονίδιο wiser είναι απαραίτητο για την ανάπτυξη του φτερού, καθώς εμπλέκεται στο σχηματισμό του ραχιοκοιλιακού άξονα και επηρεάζει τον πολλαπλασιασμό των κυττάρων. / The analysis of the X-linked wisertsl (1-21.7, 7E) mutation in Drosophila melanogaster has shown that responsible for the scalloped phenotype and the temperature sensitivity is the CG32711 gene, which we name wiser (wings scalloped-eyes rough). The gene wiser is essential for Drosophila development. The wisertsl mutation is mapped at the 5′ regulatory region of the gene CG32711. The wiserPL26 lethal mutation is mapped in the same region. Using these two mutations and a UAS-wiser transgene we have shown that: a) The wisertsl and wiserPL26 mutations increase the wing scalloping (phenotype) of the mutations Beadex1 and Serrate1. The genes Beadex and Serrate are implicated in the activation of Notch signaling pathway along the dorsal-ventral axis of the wing. This observation indicates that the wiser gene is involved in determination of dorsal-ventral axis. b) The wisertsl mutation in homozygous condition reduces substantially the expression of fringe-lacZ, m8-lacZ, wingless-lacZ, vestigial-lacZ and Distalless-lacZ transgenes, alters the expression pattern of mβ-lacZ and does not affect the expression of apterous-lacZ transgene in the wing imaginal disc. This indicates that the expression of fringe (a modifier of Notch receptor) is regulated by wiser too. c) Ectopic expression of UAS-wiser by the ap-Gal4 driver partially rescues apterous- but not Serrate1 phenotype. d) Ectopic expression of UAS-wiser by the dpp-Gal4 driver affects the expression of wingless and does not affects the expression of apterous, fringe, mβ, m8, vestigial and Distalless in the wing imaginal disc (revealed by the corresponding -lacZ strains). e) Induction of somatic clones with the FRT/FLP system in wiserPL26/+ mutants led to mitotic +/+ and wiserPL26/wiserPL26 clones of different sizes. The first clones were much larger than the second ones in the territory of wing pouch. Adult females with scalloped wings were also produced. These results indicate that the wiser gene is involved in cell proliferation. All the above findings suggest that the wiser gene is essential for wing development and cell proliferation.

The vast variety of long-chain fatty acid amides identified in biological systems is intriguing. The general structure of a fatty acid amide is R-CO-NH-X, where R is an alkyl group and X is derived from an immense variety of biogenic amines. Although structurally simple, the bioactivities of these molecules as signaling lipids are very diverse and have just recently begun to emerge in the literature. Interest in the long-chain fatty acid amides dramatically increased following the identification and characterization of one specific N-acylethanolamine, N-arachidonoylethanolamine (anandamide), as the endogenous ligand for the cannabinoid receptors in the mammalian brain. Since this discovery, the details of N-acylethanolamine metabolism have been elucidated. However, a lesser extent of progress has been made in the last twenty years to identify and study the non-N-acylethanolamine long-chain fatty acid amides. The focus of this dissertation is the elucidation of the biosynthetic pathways for long-chain fatty acid amides, including N-acylglycines, primary fatty acid amides, N-acylarylalkylamides, and N-acylethanolamines. The details of long-chain fatty acid amide metabolism will lead to the determination of possible therapeutic targets. We identified mammalian glycine N-acyltransferase like 3 as the enzyme that catalyzes the formation of long-chain N-acylglycines in mouse N18TG2 neuoblastoma cells, identified and quantified a panel of long-chain fatty acid amides in Drosophila melanogaster extracts by LC/QTOF-MS, established Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism, and identified arylalkylamine N-acyltransferase like 2 as the enzyme that catalyzes the formation of long-chain N-acylserotonins and N-acyldopamines in Drosophila melanogaster.

Το φυλοσύνδετο γονίδιο wiser (CG32711) είναι απαραίτητο για την ανάπτυξη της Drosophila melanogaster. Η μελέτη μιας θερμοευαίσθητης, θανατογόνου μετάλλαξης που ονομάζεται wisertsl αποκάλυψε ότι το γονίδιο wiser εμπλέκεται μεταξύ άλλων στην ανάπτυξη των φτερών. Η μετάλλαξη wisertsl οφείλεται σε ένα P στοιχείο (7E P) που βρίσκεται 490 bp ανοδικά του σημείου έναρξης της μεταγραφής του γονιδίου wiser. 95 bp καθοδικά του 7E P στοιχείου υπάρχει μια P{lacW} ένθεση υπεύθυνη για τη θανατογόνο μετάλλαξη PL26. Οι μεταλλάξεις wisertsl και PL26 είναι αλληλόμορφα του ίδιου γονιδίου ενώ 12000 περίπου βάσεις ανοδικά του γονιδίου wiser και 490 bp ανοδικά του γονιδίου trf2 υπάρχει μια άλλη P{lacW} ένθεση που είναι υπεύθυνη για τη θανατογόνο μετάλλαξη PL28. Οι PL26 και PL28 δεν δείχνουν συμπληρωματικότητα με τη μετάλλαξη wisertsl όσον αφορά το θανατογόνο φαινότυπο στους 29ºC. Όμως το διαγονίδιο UAS-wiser δε διασώζει το θανατογόνο φαινότυπο του PL28. Τα αποτελέσματα της παρούσας εργασίας αποκάλυψαν ότι: 1) Το γονίδιο wiser αλληλεπιδρά με το γονίδιο dpp. Εκτοπική έκφραση του διαγονιδίου (UAS wiser) υπό τον έλεγχο του οδηγού στελέχους apGAL4, μειώνει την έκφραση του dpp στην περιοχή του εμβρυικού δίσκου που θα δώσει τμήμα του θώρακα (notum). 2) Σε ομόζυγα wisertsl άτομα η έκφραση των γονιδίων dpp, dad, omb και salm (όπως αποκαλύπτεται από την έκφραση των αντίστοιχων –lacZ διαγονιδίων) μειώνεται στον εμβρυικό δίσκο του φτερού. Τα παραπάνω γονίδια είναι απαραίτητα για την ανάπτυξη του προσθοπίσθιου άξονα του εμβρυικού δίσκου του φτερού που σημαίνει ότι και το γονίδιο wiser εμπλέκεται στο σχηματισμό του. 3) Το γονίδιο wiser αλληλεπιδρά με το γονίδιο Notch (N) καθώς N wisertsl /wisertsl θηλυκά έχουν εντονότερα φαγωμένα φτερά. 4) Οι μεταλλάξεις wisertsl και PL28 είτε αφορούν και οι δύο το γονίδιο wiser ή η PL28 αφορά το γονίδιο trf2 που σημαίνει ότι και αυτό εμπλέκεται στο σχηματισμό του φτερού. / The X- linked wiser (CG32711) gene is a vital gene for the development of Drosophila melanogaster. The study of a temperature sensitive lethal mutation, named wisertsl, revealed that the wiser gene is implicated among others in the development of wings. The wisertsl mutation is due to a wild P element (7E P) located 490 bp upstream of the presumed transcription start site of the gene wiser at the region 7Ε. 95 bp downstream of the 7E P element is located a P{lacW} responsible for the lethal mutation PL26 and ~ 12000 bp upstream of the gene wiser and 490 bp upstream of the gene trf2 exists another P{lacW} insertion which is responsible of the lethal mutation PL28. The mutations PL26 and PL28 do not show complementation with the wisertsl mutation as regards the lethal phenotype at 29°C. However, while the transgene UAS-wiser saves the lethal phenotypes of wisertsl and PL26 it does not save the lethal phenotype of the mutation PL28. The present data study revealed that: 1) The wiser gene interacts with the dpp gene. Ectoping expression of the UAS wiserCDS construct under the control of apGAL4 driver, reduced the dpp expression (revealed by dpp-lacZ) in the notum territory of the wing imaginal disc. 2) In the homozygous wisertsl individuals the expression of dpp, dad, salm and omb genes (revealed by the corresponding -lacZ strains) is reduced in the wing imaginal disc. The above genes are implicated in the development of the anterior-posterior (A/P) axis of the wing imaginal disc. 3) The wiser gene interacts with the Notch (N) gene. N wisertsl/wisertsl females have stronger notching phenotype. 4) The induction of mitotic clones revealed that the mutation PL28 either concerns an enhancer of the wiser gene or the gene trf2. At the late case the gene trf2 must affect the development of the wings as well.