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Small open reading frame genes (smORFs) are a new class of genes, which emerged from the revision of the idea that open reading frames have to be longer than 100 codons to be protein coding and functional. Although bio-informatics evidence suggests that thousands of smORF genes could exist in any given genome, proof of their functional relevance can only be obtained through their functional characterization. This work represents such a study for a Drosophila smORF (pncr003;2L), which was initially misannotated as a non-coding RNA because of its lack of a canonical long open reading frame. Here I show that pncr003;2L codes for two small peptides of 28 and 29 aa, expressed in somatic and cardiac muscles. After generating a null condition for this gene, I use the adult Drosophila heart as a system to assess the function of pncr003;2L. With this system, I show that the small pncr003;2L peptides regulate heart contractions by modulating Ca2+ cycling in cardiac muscles, with either lack or excess of function of these peptides leading to cardiac arrhythmias, and abnormal calcium dynamics. Finally, through an extensive homology study, I show that these small peptides share a great amount of structural and functional homology with the peptides encoded by the vertebrate smORFs sarcolipin (sln) and phospoholamban (pln), which act as major regulators of the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), the channel responsible for calcium uptake into the ER following muscle contraction. These results highlight the importance of the pncr003;2L smORF and the Drosophila system, for the study of cardiac pathologies, but most importantly, they show that this family of peptides, conserved across evolution, represent an ancient system for the regulation of calcium trafficking in muscles. This work corroborates the prevalence, and relevance of this novel class of genes, and shows that closer attention should be given to smORFs in order to determine the full extent of their biological contribution.

During the formation of the insect and mammalian nervous system the embryo activates specific programs of cellular differentiation along the main body axis so that the specification and organization of neural cells is set in coordination with axial level. At the genetic level such cellular specification programs rely on the regulated expression of a family of transcription factors encoded by the Hox genes. However, the precise molecular mechanisms controlling Hox expression in the nervous system are not well understood. In this thesis we investigate the molecular mechanisms underlying Hox gene expression within the Drosophila central nervous system (CNS) with a focus on post-transcriptional control via RNA binding proteins (RBP) and microRNAs (miRNAs). Much of the work is centred on the analysis of the Hox gene Ultrabithorax (Ubx) as this is the Hox gene for which post-transcriptional regulation is currently best understood. Through the combination of genetic, molecular and imaging methods we first show that the pan-neural RBP ELAV regulates Ubx RNA processing and protein expression during the embryonic development of the CNS. Secondly, using a suite of genetic and behavioural methods we report that Ubx repression by miRNAs encoded within the iab-4/iab-8 locus (miR-iab4/iab8) is required for the coordination of a specific larval behaviour: self-righting behaviour. Third, we explore the cellular basis of larval self-righting behaviour in the context of miRNA-dependent Ubx regulation and find that: (i) removal of miR-iab4/iab8 does not lead to major anatomical defects in the CNS or muscles; (ii) artificial increase in UBX protein expression in cholinergic interneurons disrupts self-righting behaviour; and (iii) UBX protein expression in cholinergic interneurons is regulated by miR-iab4/iab8. These observations imply that UBX regulation by miR-iab4/iab8 in cholinergic interneurons controls self-righting behaviour. Altogether our work adds to the current understanding of the molecular mechanisms underlying Hox gene expression during CNS formation and gives new insights on the role of RBP and miRNA regulation on the control of gene expression and behaviour.

The Hox genes encode a family of transcriptional regulators that activate distinct developmental programs along the anterior-posterior (AP) axis of animals. Recent observations in Drosophila demonstrate that at least two miRNAs can repress Hox gene expression during development suggesting that miRNA-based regulation might be a general mechanism of Hox gene regulation. Here explore this possibility by applying a comprehensive genetic approach to identify miRNAs able to repress Hox gene expression during development. Given that the reduction of Drosophila Hox gene Ultrabithorax (Ubx) expression leads to easily tractable homeotic transformations in haltere, I use Ubx to test the repressive effects of dozens of miRNAs in an overexpression screen. Scoring over 10,000 halteres showed that out of 106 miRNAs tested, ~28% produced Ubx mutant phenotypes suggesting that miRNA-dependent Hox regulation might be a pervasive mechanism controlling Hox gene function during development. I classify phenotypes into four major categories: Ubx mutant effects (Class I and II) and others (Class III and IV). Through the combination of RNA-Seq data and TaqMan RT-PCR approaches, I confirm that there is no correlation between the phenotypic strength and miRNA expression level indicating that haltere phenotypes emerge from miRNA qualitative roles. Furthermore, using protein expression analysis and Ubx 3' UTR fluorescent reporters, I confirmed that at least nine miRNAs affect Ubx protein expression and that six of these directly target Ubx 3' UTR in vivo. Lastly, I explore the nature and effects of miRNA regulation of Ubx at the cellular level in the Drosophila embryonic CNS and find that miR-252 is sufficient and necessary to repress Ubx expression in specific neural lineages. Our work thus contributes to the understanding of miRNA-mediated Hox gene regulation and, more generally, to the study of miRNA-target interactions within the physiological context of metazoan development.

by Yin Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 146-164). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iv / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axes of Drosophila --- p.3 / Chapter 1.2.1 --- Oogenesis --- p.5 / Chapter 1.2.2 --- Embryogenesis --- p.15 / Chapter 1.2.3 --- Segmentation --- p.16 / Chapter 1.2.4 --- Life cycle --- p.20 / Chapter 1.3 --- Egg-polarity genes are essential for development --- p.22 / Chapter 1.4 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.24 / Chapter 1.4.1 --- Phenotypes of bicoid mutant --- p.24 / Chapter 1.4.2 --- Transplantation experiment --- p.26 / Chapter 1.4.3 --- Establishment of an anterior to posterior bicoid protein gradient --- p.26 / Chapter 1.4.4 --- Localization step of bicoid mRNA --- p.27 / Chapter 1.4.5 --- Formation of bicoid protein gradient --- p.28 / Chapter 1.4.6 --- The bicoid protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.31 / Chapter 1.4.6.1 --- Bicoid protein acts as transcriptional regulators --- p.31 / Chapter 1.4.6.2 --- Bicoid protein acts as transcriptional activators --- p.31 / Chapter 1.4.6.3 --- Bicoid protein acts as translational repressor --- p.34 / Chapter 1.5 --- Components required for the localization of bicoid mRNA --- p.35 / Chapter 1.5.1 --- Cis-acting elements --- p.35 / Chapter 1.5.1.1 --- Bicoid mRNA localization element (BLE1) at 3、UTR directs localization of bicoid mRNA --- p.36 / Chapter 1.5.2 --- Trans-acting elements --- p.37 / Chapter 1.5.2.1 --- exuperantia --- p.40 / Chapter 1.5.2.2 --- swallow --- p.41 / Chapter 1.5.2.3 --- staufen --- p.42 / Chapter 1.5.2.4 --- cytoskeleton --- p.44 / Chapter 1.6 --- Aim of project --- p.48 / Chapter CHAPTER 2 --- Characterization of exuperantia protein --- p.50 / Chapter 2.1 --- Introduction --- p.50 / Chapter 2.1.1 --- Localization step of exuperantia protein in wild type --- p.50 / Chapter 2.1.2 --- Phenotype of exuperantia mutant --- p.51 / Chapter 2.1.3 --- exuperantia gene in both female and male flies --- p.52 / Chapter 2.2 --- Materials and Methods --- p.59 / Chapter 2.2.1 --- General characteristic of exuperantia protein --- p.59 / Chapter 2.2.1.1 --- Preparation of total ovary protein from the female and male flies --- p.59 / Chapter 2.2.1.2 --- Analysis of exuperantia protein by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS - PAGE) and Western blotting --- p.60 / Chapter 2.2.2 --- Determination of the type of phosphorylation residues in exuperantia protein --- p.61 / Chapter 2.2.2.1 --- Preparation of immunoprecipitated exuperantia protein from ovary and testis --- p.61 / Chapter 2.2.2.2 --- Dephosphorylation of exuperantia protein --- p.62 / Chapter 2.2.3 --- Two-dimensional gel electrophoresis analysis of exuperantia protein --- p.63 / Chapter 2.3 --- Results --- p.65 / Chapter 2.3.1 --- General characteristic of exuperantia protein --- p.65 / Chapter 2.3.2 --- Determination of the type of phosphorylation residues in exuperantia protein --- p.67 / Chapter 2.3.3 --- Resolving the multiple phosphorylated isoforms of exuperantia protein by two-dimensional gel electrophoresis --- p.69 / Chapter 2.4 --- Discussion --- p.72 / Chapter CHAPTER 3 --- Determination of the type of kinase(s) phosphorylate exuperantia protein --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.2.1 --- Phosphorylation of recombinant exuperantia protein --- p.83 / Chapter 3.2.1.1 --- Immunoprecipitation of recombinant exuperantia protein and phosphorylation reaction --- p.83 / Chapter 3.2.1.2 --- Sequential phosphorylation reaction --- p.84 / Chapter 3.2.2 --- Inhibitory effect(s) of protein kinase inhibitors on phosphorylation of native exuperantia protein --- p.85 / Chapter 3.2.2.1 --- Incubation of ovaries with protein kinase inhibitors --- p.85 / Chapter 3.2.3 --- Phosphorylation of native exuperantia protein by endogenous protein kinase(s) --- p.86 / Chapter 3.2.3.1 --- Preparation of total tissue homogenate --- p.86 / Chapter 3.2.3.2 --- Endogenous kinase assay --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Phosphorylation of recombinant exuperantia protein by exogenous kinase(s) --- p.88 / Chapter 3.3.2 --- Inhibitory effect(s) of protein kinase inhibitors on phosphorylation of native exuperantia protein --- p.92 / Chapter 3.3.3 --- Phosphorylation of native exuperantia protein by endogenous protein kinase(s) --- p.94 / Chapter 3.3.3.1 --- Phosphorylation of native exuperantia protein by endogenous kinase(s) with addition of protein kinase activators --- p.94 / Chapter 3.3.3.2 --- Phosphorylation of native exuperantia protein by endogenous kinase(s) with addition of protein kinase inhibitor --- p.98 / Chapter 3.4 --- Discussion --- p.101 / Chapter CHAPTER 4 --- Spatial and temporal distribution of exuperantia protein in DCO83 and exuPJ egg chambers --- p.107 / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Initiation of establishment of the two body axes by one single signal --- p.107 / Chapter 4.1.2 --- Stage-specific phosphorylation of exuperantia protein --- p.111 / Chapter 4.2 --- Materials and Methods --- p.113 / Chapter 4.2.1 --- Immunohistochemical distribution of exuperantia protein --- p.113 / Chapter 4.2.2 --- Stage-specific phosphorylation of exuperantia protein --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- Immunohistochemical distribution of exuperantia protein in DCOB3 mutant --- p.119 / Chapter 4.3.2 --- Immunohistochemical distribution of exuperantia protein in exuPJ mutant --- p.121 / Chapter 4.3.3 --- Stage-specific phosphorylation of exuperantia protein in DCOB3 mutant --- p.125 / Chapter 4.3.4 --- Stage-specific phosphorylation of exuperantia protein of exuPJ mutant --- p.127 / Chapter 4.4 --- Discussion --- p.128 / Appendix A --- p.135 / Appendix B --- p.143 / References --- p.146

by Pui-Ki Kwan. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 134-143). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Table of content --- p.v / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- The formation of body axes --- p.1 / Chapter 1.2 --- Maternal genes are essential for development --- p.8 / Chapter 1.3 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.9 / Chapter 1.4 --- Establishment of an anterior to posterior bcd protein gradient --- p.12 / Chapter 1.5 --- The bcd protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.12 / Chapter 1.6 --- bcd protein acts as transcriptional regulators --- p.14 / Chapter 1.7 --- The anterior localization of bcd mRNA --- p.17 / Chapter 1.8 --- Components required for the localization of bcd mRNA --- p.17 / Chapter 1.8.1 --- Cis-acting elements --- p.17 / Chapter 1.8.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.19 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.8.2.1 --- "exuperantia, swallow and staufen are necessary for localization of bcd mRNA" --- p.21 / Chapter 1.8.2.2 --- exu protein is an absolute requirement for the localization --- p.24 / Chapter 1.8.2.3 --- Potential functions of exu based on the coding sequence --- p.25 / Chapter 1.8.2.4 --- Microtubules dependence of the localization --- p.26 / Chapter 1.8.2.5 --- Microtubules polarity directs localization of bcd RNAs --- p.27 / Chapter 1.9 --- Functions of exu in localization of bcd mRNA --- p.27 / Chapter CHAPTER 2 --- Characterization of deletion mutants of exu / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Construction of deletion mutants of exu --- p.31 / Chapter 2.2.1 --- Materials and Methods --- p.31 / Chapter 2.2.2 --- Results --- p.33 / Chapter 2.3 --- Analysis of exu protein in deletion mutants --- p.35 / Chapter 2.3.1 --- Materials and Methods --- p.35 / Chapter 2.3.1.1 --- Preparation of total ovary protein from the transgenic flies --- p.35 / Chapter 2.3.1.2 --- Analysis of protein content by SDS Polyacrylamide Gel Electrophoresis and immunoblotting --- p.35 / Chapter 2.3.2 --- Results --- p.36 / Chapter 2.4 --- Localization of bcd mRNA and exu protein in oogenesis --- p.39 / Chapter 2.4.1 --- Introduction --- p.39 / Chapter 2.4.2 --- Spatial and temporal distribution of exu protein in the deletion mutants --- p.41 / Chapter 2.4.2.1 --- Materials and Methods --- p.41 / Chapter 2.4.2.2 --- Results --- p.43 / Chapter 2.4.3 --- Spatial and temporal distribution of bcd mRNA in the deletion mutants --- p.56 / Chapter 2.4.3.1 --- Materials and Methods --- p.56 / Chapter 2.4.3.1.1 --- Principles of DIG-labeling and in situ hybridization --- p.56 / Chapter 2.4.3.1.2 --- Synthesis of DIG-labeled bcd DNA probe --- p.59 / Chapter 2.4.3.1.3 --- in situ hybridization of bcd mRNA in egg chambers using DIG-labeled DNA probe --- p.59 / Chapter 2.4.3.2 --- Results --- p.62 / Chapter 2.5 --- Discussion --- p.70 / Chapter CHAPTER 3 --- Determination of the interactions between exu and microtubules / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Localization of bcd mRNA and exu protein in the presence of drugs which destabilize cytoskeleton --- p.81 / Chapter 3.2.1 --- Materials and Methods --- p.81 / Chapter 3.2.2 --- Results --- p.82 / Chapter 3.3 --- Analysis of interactions between exu and microtubules by immunoprecipitation --- p.88 / Chapter 3.3.1 --- Materials and Methods --- p.88 / Chapter 3.3.1.1 --- Immunoprecipitation of exu protein and binding of microtubules --- p.88 / Chapter 3.3.1.2 --- Purification of tubulin from bovine or rat brains --- p.89 / Chapter 3.3.1.3 --- Determination of protein concentration of the tubulin stock by Folin-Lowry method --- p.90 / Chapter 3.3.1.4 --- Taxol-stabilized microtubules --- p.90 / Chapter 3.3.2 --- Results --- p.91 / Chapter 3.4 --- Analysis of interactions between exu and microtubules by cosedimentation --- p.94 / Chapter 3.4.1 --- Materials and Methods --- p.94 / Chapter 3.4.2 --- Results --- p.97 / Chapter 3.5 --- Analysis of interactions between exu and microtubules using detergent extracted ovary extract for co sedimentation --- p.100 / Chapter 3.5.1 --- Materials and Methods --- p.100 / Chapter 3.5.2 --- Results --- p.101 / Chapter 3.6 --- Analysis of intracellular distribution of exu protein and Release of exu protein by sodium carbonate treatment for cosedimentation with microtubules --- p.104 / Chapter 3.6.1 --- Materials and Methods --- p.104 / Chapter 3.6.1.1 --- Subcellular fractionation of ovary extracts --- p.104 / Chapter 3.6.1.2 --- Release of contents from fractions by sodium carbonate treatment --- p.105 / Chapter 3.6.1.3 --- Co sedimentation of exu protein with microtubules --- p.105 / Chapter 3.6.2 --- Results --- p.108 / Chapter 3.6.2.1 --- Intracellular distribution of exu protein --- p.108 / Chapter 3.6.2.2 --- Cosedimentation of exu protein with microtubules using Na2CO3 released extracts --- p.108 / Chapter 3.7 --- Cosedimentation of exu protein and microtubules in high ATP concentration --- p.113 / Chapter 3.7.1 --- Materials and Methods --- p.113 / Chapter 3.7.1.1 --- Preparation of ovary extracts and microtubules sedimentation --- p.113 / Chapter 3.7.1.2 --- Western blot using a chemiluminescent detection system --- p.114 / Chapter 3.7.2 --- Results --- p.115 / Chapter 3.8 --- Discussion --- p.122 / Chapter CHAPTER 4 --- Future Prospects --- p.125 / Appendix A Supplementary protocols --- p.126 / Appendix B Reagents --- p.131 / References --- p.134

Invadolysin is a novel metalloprotease, which is conserved amongst metazoans and was first identified in the Heck laboratory. Proteases play a variety of roles in normal physiology. Invadolysin is essential for life in Drosophila. Invadolysin has been shown to be essential for cell division and cell migration. Invadolysin is the only metalloprotease that we know of which localizes to lipid droplets, the lipid storage cell organelle. Previous studies have also shown that invadolysin mutants have a lower triglyceride to protein ratio and reduced fat body thickness and cross sectional area. Fat body in Drosophila is the functionally homolog of adipose tissue in higher organisms. Further suggesting a role of invadolysin in metabolism. In the Heck laboratory, invadolysin is studied using model organisms such as Drosophila melanogaster, Danio rerio (zebrafish) and cultured cell lines. During my PhD, my aim was to study the biosynthesis, activity and function of invadolysin and investigate its role in metabolism and adipogenesis. Invadolysin has a conserved metalloprotease motif ‘HEXXH’ and a potential lipase motif ‘GXSXS’. One of the aims of my PhD was to generate mutant versions of the conserved motifs to study their role on the activity of the proteins. I have generated transgenic flies that express wild type or E258A (protease dead) or S266A (lipase dead) versions of invadolysin. These transgenic flies would help in the study of the importance of the metalloprotease ‘HEILH’ and the lipase ‘GFSVS’ motifs in invadolysin’s activity. Transgenic flies overexpressing wild type and lipase dead form of invadolysin accumulate significantly higher levels of triglycerides as compared to control flies and transgenic flies overexpressing protease dead form of invadolysin. Suggesting a role of the protease motif in lipid accumulation. The other aim of my PhD was to study the role of invadolysin in metabolism. I followed up on previous observations in the laboratory that the insulin-signalling pathway is impaired in invadolysin mutant animals – with the hypothesis that invadolysin plays a role in metabolism and adipogenesis. I used Drosophila to study the effect on downstream targets of the insulin-signalling pathway such as triglyceride synthesis, glycogen synthesis and autophagy in invadolysin mutants. Results suggest that the insulin-signalling pathway and the ability to accumulate lipids are impaired in invadolysin mutants. Insulin also regulates adipogenesis by regulating the expression of PPARγ. I used SGBS cells, a human preadipocyte cell line to study the role of invadolysin in adipogenesis. Increase in protein levels of invadolysin during adipogenesis indicates a potential role of invadolysin in adipogenesis. Invadolysin has a predicted N-terminal signal sequence and also a predicted Cterminus GPI anchor site that suggests invadolysin can either be secreted or anchored to a membrane. Also, leishmanolysin, the closest homolog of invadolysin exists in a secreted and membrane bound form apart from a cytosolic form. This encouraged me to investigate the presence of a secreted form of invadolysin. Analysis of vertebrate and invertebrate plasma fractions of blood and hemolymph led to the identification of a novel secreted form of invadolysin. This novel discovery places invadolysin alongside a small group of metalloproteases, which are secreted into the extracellular environment and which play multiple roles in normal physiology and disease states.

Cellularisation is a specialised form of cytokinesis in Drosophila melanogaster. Cellularisation occurs after the first 13 syncytial cell cycles of the embryo and involves targeted insertion of membrane to form the blastoderm, which represents a polarised epithelium made out of about 6000 cells. The molecular machinery driving cellularisation is complex and not well understood. In this work a novel gene regulating this process is identified and characterised. The mutation drop out causes defects in intracellular transport, cell polarity and nuclear positioning. Previous work provided evidence that dop1 is an allele of the RNA silencing gene argonaute2 (ago2). However, results presented in this thesis showed that ago2 functions are unimpaired in dop mutant embryos using genetic and biochemical tools. Moreover genetic and molecular mapping revealed that dop mutants carry a mutation in a gene within close proximity to ago2.This work demonstrates that dop encodes the sole Drosophila homolog of the mammalian MAST (microtubule associated serine/threonine) kinase family. The molecular lesion in the dop1 allele of dop leads to an amino acid exchange in the kinase domain and results in a significant reduction of Dop protein levels. A detailed investigation of the mutant phenotype indicated that dop1 affects microtubule rigidity and Dynein-dependent microtubule associated transport. Search for possible Dop targets revealed reduced phosphorylation of the Dynein intermediate chain (DIC). DIC is a subunit of Dynein and has been shown to be involved in the binding of cargo to the Dynein complex. Therefore, a possible function for Dop might be the phosphorylation of DIC to regulate microtubule dependent transport by controlling Dynein-cargo interaction.

O elemento transponível Galileo foi encontrado em Drosophila buzzatii e é apontado como o responsável pela formação de três inversões descritas para esta espécie: 2j, 2z3 e 2q7, através de recombinação ectópica. Análises in silico dos 12 genomas de Drosophila disponíveis constatou que ele é distribuído no gênero, estando presente também nos genomas de D. ananassae, D. pseudoobscura, D. persimilis, D. mojavensis, D. willistoni, as quais são reconhecidamente polimórficas, e D. virilis. Drosophila willistoni destacou-se nestas análises devido à presença de 495 cópias defectivas do elemento transponível Galileo encontradas em seu genoma, uma quantidade maior do que nas demais espécies. Estes achados somados com o fato de D. willistoni figurar entre as espécies mais polimórficas de Drosophila abrem um estimulante campo para os estudos da associação do elemento transponível Galileo com seu polimorfismo. O presente estudo analisou através de hibridização in situ a presença do elemento transponível Galileo nos cromossomos politênicos de três linhagens de D. willistoni oriundas de locais distintos da distribuição geográfica da espécie: Gd-H4-1 (América Central), a linhagem utilizada no sequenciamento; WIP-4 (nordeste do Brasil) e 17A2 (sul do Brasil), e estabeleceu as coincidências de inserções deste elemento com pontos de quebra descritos para a espécie e as coincidências com sítios de inserções do elemento P. Para tal, utilizou-se uma sonda com 2441 pb, baseada na maior variante do elemento transponível Galileo encontrada in silico, a qual não engloba suas repetições terminais invertidas (TIRs), portanto somente hibridiza com cópias mais completas do elemento. Nossas análises encontraram 100 sítios de inserções do elemento transponível Galileo distribuídos em todos os cromossomos de D. willistoni. Destes, 20 coincidem com pontos de quebra para inversões paracêntricas descritas, dois com pontos de quebra de uma rara inversão pericêntrica descrita e 10 com sítios de inserção do elemento P. O padrão de distribuição dos sinais de hibridização foi altamente similar entre as três linhagens. Também foram encontrados sinais de hibridizações nos cromocentros das linhagens analisadas. Associações estatisticamente significativas de sítios de inserção do elemento transponível Galileo com pontos de quebra ocorreram nos braços cromossômicos XR e IIL em Gd-H4-1 e IIL em WIP-4. Quando o reuso (compartilhamento) dos pontos de quebras para as diferentes inversões foi considerado, o cromossomo III também apresentou associações estatisticamente significativas em Gd-H4-1 e WIP-4. As análises quanto à distribuição geográfica das inversões mostrou que houve maior coincidência de sítios de inserções do elemento transponível Galileo que correspondem a pontos de quebra de inversões com distribuição restrita, ou seja, provavelmente mais recentes na história evolutiva da espécie. Com base em nossos resultados, é possível inferir que o elemento transponível Galileo é um elemento transponível antigo no genoma de Drosophila willistoni, e que a sua maior prevalência com pontos de quebra de inversões mais recentes pode estar associada à sua característica de formar estruturas secundárias quando desnaturado e assim promover rearranjos cromossômicos, mesmo se tratando de uma cópia defectiva. / The Galileo transposable element was discovered in Drosophila buzzatii and was appointed as responsible for the formation of three chromosomal inversions: 2j, 2z3 e 2q7 by ectopic recombination. In silico analyses of the 12 Drosophila genomes available showed that it is widely distributed in the genus, being present in the genomes of D. ananassae, D. pseudoobscura, D. persimilis, D. mojavensis, D. willistoni, recognized as chromosomally polymorphic, and in D. virilis. Drosophila willistoni was particularly interesting among the others, because of the finding of 495 defective copies of the Galileo transposable element in its genome, the highest amount found among all the studied species. Those results, and the fact that D. willistoni is one of the most polymorphic species of Drosophila, opened a stimulating field of study for the probable relationship between the Galileo transposable element and its chromosomal polymorphism. In the present study we analyzed the presence and the polytene chromosomal localization of the Galileo transposable element in three strains of D. willistoni from different geographical origins, through in situ hybridization: Gd-H4-1 (from Guadaloupe Island, Central America), the strain sequenced; WIP-4 (from Northeast Brazil) and 17A2 (from South Brazil), and detected coincidences of the Galileo transposable element insertion sites with break points of inversions known in this species and with sites of P element insertions. For this, we used a 2441 pb probe, drawn according to the larger variant of the Galileo transposable element found in silico, not including its inverted terminal repetitions (TIRs), thus only hybridizing with more complete copies of the element. We registered 100 sites of the Galileo transposable element insertions, distributed in all chromosomes of Drosophila willistoni. Among them, 20 coincide with break points of described paracentric inversions, two with those of a rare pericentric inversion and 10 with insertion sites of the P transposable element. The pattern of distribution of the hybridization signals was highly similar among the three strains. We also found hybridization signals in the chromocenters of all the strains analyzed. Statistically significant associations between insertion sites of the Galileo transposable element insertions with break points of inversions were detected in the chromosomal arms XR and IIL in Gd-H4-1 and IIL in WIP-4 samples. When the reuse (sharing) of the break points of different inversions was evaluated, we observed significant associations in the data of the third chromosome (III) of the Gd-H4-1 and WIP-4 samples. The analyses of the geographical distribution of the inversions showed higher coincidences of the Galileo transposable element insertions and break points of inversions with narrower distribution, i.e., apparently more recent in the evolutionary history of the species. Considering all our findings, we suggest Galileo as an ancient transposable element in the genome of D. willistoni, and that its apparent preference for break points of more recent inversions, can be due to its molecular characteristic of forming secondary structures when denatured, so promoting chromosomal rearrangements, even if it is a defective copy.

Esta Tese possui dois enfoques como objetivo geral. Primeiramente, nossos dados contribuem para o conhecimento do padrão de evolução do elemento de transposição micropia dentro do gênero Drosophila, também verificando a atuação desses elementos como fonte de variabilidade genética. Em segundo lugar geramos dados citogenéticos e moleculares inéditos a respeito de espécies do grupo cardini de Drosophila, bastante freqüente nas assembléias de Drosophilidae na região Neotropical e que havia sido muito pouco estudado do ponto de vista genético até então. Com isso, no Capítulo II verificamos a presença e a alta similaridade de sequências deste retroelemento em diferentes populações de D. cardinoides, D. neocardini e D. polymorpha. Ainda, quando comparadas com a sequência presente em uma espécie do grupo repleta, D. hydei, as sequências daquelas três espécies também apresentaram alta similaridade (97%). O grupo repleta e o grupo cardini do gênero Drosophila parecem ter divergido há 45 milhões de anos atrás. Portanto, para explicar os dados obtidos sugerimos a atuação de transmissão horizontal entre as espécies analisadas. Ampliando as análises, no Capítulo III a presença de micropia foi identificada nas demais espécies do grupo cardini com exceção das espécies do grupo que apresentam distribuição geográfica restrita às ilhas caribenhas. As comparações com sequências presentes em outras espécies do grupo repleta, assim como nos 12 genomas de espécies do gênero Drosophila disponíveis em bancos de dados públicos, verificamos que a história evolutiva do elemento micropia provavelmente inclui polimorfismo ancestral e transmissão tanto vertical quanto horizontal com mecanismos de introgressão entre as espécies potencialmente atuando na geração desses padrões. 11 Com a finalidade de estudar a possível atuação deste retroelemento como fonte de variabilidade genética através da geração de inversões nas espécies do grupo cardini, o primeiro passo foi a identificação de potenciais sítios de inserção nos cromossomos politênicos dessas espécies. No Capítulo IV estimamos o número de cópias de micropia no genoma de seis espécies do grupo cardini (D. cardini, D. cardinoides, D. neocardini, D. neomorpha, D. parthenogenetica e D. polymorpha) verificando que varia entre seis e 18 cópias. Ainda, observamos a presença de potenciais cópias em pontos de quebra para inversões cromossômicas em três espécies. No Capítulo IV discutimos o significado desses dados com base nos dados já obtidos para outras espécies. O polimorfismo cromossômico dos cromossomos politênicos e a análise comparada dessas estruturas para as seis espécies do grupo cardini previamente citadas, foi estudado no Capítulo V. Neste capítulo identificamos a presença de uma inversão nas populações estudadas de D. cardini e D. neocardini, duas inversões nas populações de D. cardinoides e D. parthenogenetica e quatro inversões nas populações de D. polymorpha. Das 10 inversões identificadas, sete são descritas pela primeira vez neste capítulo. Nenhuma inversão heterozigota foi encontrada na população de D. neomorpha aqui analisada, portanto apresentando um padrão homocariotípico. Ainda no Capítulo V apresentamos os primeiros fotomapas para as espécies D. cardini e D. parthenogenetica, assim como a reconstrução dos fotomapas das espécies D. cardinoides, D. neocardini e D. polymorpha. Também foi realizada a comparação par a par de cada cromossomo politênico entre todas as espécies com a finalidade de encontrar regiões similares úteis no estabelecimento de relações de parentesco através deste marcador. No Capítulo V esses dados são discutidos baseados no enfoque evolutivo para o grupo cardini. / This Thesis possesses two approaches as mainly objectives. First, our data contribute for the knowledge of the evolutionary pattern of the transposable element micropia in the Drosophila genus, and also to the role of these elements as source of genetic variability. Second, we generate cytogenetic and molecular new data regarding the cardini group species of Drosophila genus; these species are frequent in the Neotropical assemblies of Drosophilidae, and they had been very little studied of the genetic point of view until then. In Chapter II we verify the presence and the high sequence similarity of this retroelement in different populations of D. cardinoides, D. neocardini and D. polymorpha. Furthermore, when these sequences are compared with the sequence present in the genome of a repleta group species, D. hydei, they show also a high similarity (97%) among them. The repleta group and the cardini group of the Drosophila genus seem to have diverged 45 million years ago. Therefore, to explain the obtained data we suggest the action of horizontal transmission events between the species. Extending this analysis, in Chapter III the presence of micropia was identified in other species of the cardini group, with exception of the group species with geographic distribution restricted to the Caribbean islands. The comparisons with sequences present in other species of the repleta group as well as in the 12 Drosophila genomes available in public data bases, we verify that the evolutionary history of the micropia element probably includes ancestral polymorphism, vertical and horizontal transmission with introgression mechanisms among species potentially acting in the generation of this evolutionary pattern. 13 Aiming the analysis of micropia as source of genetic variability through the inversions generation in the cardini group species, the first step was the identification of potential insertion sites in the polytene chromosomes of these species. In Chapter IV we estimate the micropia copies number in the genome of six cardini group species (D. cardini, D. cardinoides, D. neocardini, D. neomorpha, D. parthenogenetica and D. polymorpha) verifying that it varies between six and 18 copies. Furthermore, we observed the presence of copies in the break points of chromosomal inversions for three species. In Chapter IV we argue the meaning of these data on the basis of the data already obtained for other species. The chromosomal polymorphism observed in the polytene chromosomes and the comparative analysis of these structures for the six species of the cardini group previously cited were studied in Chapter V. In this chapter we identified the presence of only one chromosomal inversion in the analyzed populations of D. cardini and D. neocardini, two inversions in the D. cardinoides and D. parthenogenetica populations, and four inversions in the D. polymorpha. From these 10 identified inversions, seven are described for the first time in this chapter. No heterozygous inversion was found in the D. neomorpha population analyzed here, therefore presenting a homokaryotipic pattern. Besides this, in Chapter V we also present the first reference photomaps for Drosophila cardini and D. parthenogenetica, as well as the photomaps reconstruction of D. cardinoides, D. neocardini and D. polymorpha. We also performed a pairwise analysis of the polytene chromosomes between the species aiming to find useful regions for the establishment of evolutionary relationships based in this marker. In Chapter V these data are argued based on evolutionary approaches for the cardini group.