RESUMO
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Assuntos
Drosophila melanogaster/genética , Genoma , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Biologia Computacional , Mapeamento de Sequências Contíguas , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Cromossomos Politênicos , Mapeamento por RestriçãoRESUMO
We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.
Assuntos
Proteínas Luminescentes/química , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Animais , Antígenos , Mapeamento Encefálico , Drosophila , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios , Conformação ProteicaRESUMO
Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Memória/fisiologia , Odorantes/análise , Recompensa , Animais , Comportamento Apetitivo/fisiologia , Sinalização do Cálcio , Dendritos/fisiologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Olfato/genética , Olfato/fisiologiaRESUMO
We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.
Assuntos
Encéfalo/citologia , Olho Composto de Artrópodes/inervação , Drosophila/anatomia & histologia , Vias Neurais/citologia , Neurônios/citologia , Coloração e Rotulagem/métodos , Animais , Olho Composto de Artrópodes/citologia , Drosophila/fisiologia , Genótipo , Imuno-Histoquímica , Microscopia ConfocalRESUMO
The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire(ts1), and for the use of cytoplasmic GFP to image the fine processes of neurons.
Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Biossíntese de Proteínas , Transgenes , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Dados de Sequência Molecular , RNA Mensageiro/genéticaRESUMO
In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.
Assuntos
DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Feminino , Genoma de Inseto/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Ligação Proteica/genéticaRESUMO
Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast--KD, B2, B3, and R--are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Genoma/genética , Animais , Células CHO , Cricetinae , Cricetulus , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Expressão Gênica , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética/genética , Saccharomyces cerevisiae/enzimologiaRESUMO
We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
Assuntos
Drosophila melanogaster , Neurônios/metabolismo , Neurociências/métodos , Recombinação Genética/genética , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica/métodos , Encéfalo/citologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de InsetosRESUMO
Threat displays are a universal feature of agonistic interactions. Whether threats are part of a continuum of aggressive behaviors or separately controlled remains unclear. We analyze threats in Drosophila and show they are triggered by male cues and visual motion, and comprised of multiple motor elements that can be flexibly combined. We isolate a cluster of â¼3 neurons whose activity is necessary for threat displays but not for other aggressive behaviors, and whose artificial activation suffices to evoke naturalistic threats in solitary flies, suggesting that the neural control of threats is modular with respect to other aggressive behaviors. Artificially evoked threats suffice to repel opponents from a resource in the absence of contact aggression. Depending on its level of artificial activation, this neural threat module can evoke different motor elements in a threshold-dependent manner. Such scalable modules may represent fundamental "building blocks" of neural circuits that mediate complex multi-motor behaviors.
Assuntos
Agressão/fisiologia , Comportamento Animal/fisiologia , Encéfalo/fisiologia , Atividade Motora/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Sinais (Psicologia) , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , TermogêneseRESUMO
Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.
Assuntos
Agressão/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/citologia , Drosophila/fisiologia , Vias Neurais , Neurônios/fisiologia , Receptores de Neurotransmissores/fisiologia , Comportamento Social , Animais , Animais Geneticamente Modificados , Nível de Alerta/fisiologia , Corte , Proteínas de Drosophila/genética , Interneurônios/fisiologia , Masculino , Receptores de Neurotransmissores/genéticaRESUMO
Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report G: ene targeting during O: ogenesis with L: ethality I: nhibitor and C: RISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.
Assuntos
Drosophila/genética , Marcação de Genes/métodos , Animais , DNA , Feminino , Recombinação Homóloga , Oogênese , Óvulo , TransgenesRESUMO
Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. 33 postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. We devised genetic approaches to define the anatomy of most of these hemilineages and to assessed their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contained local interneurons and their activation caused tonic or phasic leg movements lacking interlimb coordination. The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking. The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior.
Assuntos
Sistema Nervoso Central/anatomia & histologia , Drosophila/anatomia & histologia , Drosophila/fisiologia , Neurônios/fisiologia , Animais , Linhagem da Célula/fisiologiaRESUMO
The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.
Assuntos
Linhagem da Célula , Cérebro/citologia , Proteínas de Drosophila , Drosophila/citologia , Técnicas Genéticas , Células-Tronco Neurais/citologia , Animais , Linhagem da Célula/fisiologia , Cérebro/fisiologia , Drosophila/fisiologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Receptores Notch/biossíntese , Receptores Notch/genética , Recombinação Genética , TransgenesRESUMO
Brain connectivity maps display a delicate balance between individual variation and stereotypy, suggesting the existence of dedicated mechanisms that simultaneously permit and limit individual variation. We show that during the development of the Drosophila central nervous system, mutual inhibition among groups of neighboring postmitotic neurons during development regulates the robustness of axon target choice in a nondeterministic neuronal circuit. Specifically, neighboring postmitotic neurons communicate through Notch signaling during axonal targeting, to ensure balanced alternative axon target choices without a corresponding change in cell fate. Loss of Notch in postmitotic neurons modulates an axon's target choice. However, because neighboring axons respond by choosing the complementary target, the stereotyped connectivity pattern is preserved. In contrast, loss of Notch in clones of neighboring postmitotic neurons results in erroneous coinnervation by multiple axons. Our observations establish mutual inhibition of axonal target choice as a robustness mechanism for brain wiring and unveil a novel cell fate independent function for canonical Notch signaling. DOI:http://dx.doi.org/10.7554/eLife.00337.001.
Assuntos
Encéfalo/fisiologia , Drosophila/fisiologia , Mitose , Inibição Neural , Neurônios/fisiologia , Vias Visuais/fisiologia , Animais , Axônios/fisiologia , Encéfalo/metabolismo , Linhagem Celular , Simulação por Computador , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Vias Visuais/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Sistema Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Bases de Dados Factuais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Imuno-Histoquímica , Microscopia Confocal , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.
Assuntos
Drosophila melanogaster/genética , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Genes Reporter , Engenharia Genética , Marcadores Genéticos , Técnicas Genéticas , Vetores Genéticos , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the "VALIUM" series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.
Assuntos
Drosophila/genética , Técnicas de Silenciamento de Genes/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Proteínas de Transporte/genética , Canais Iônicos/genética , Métodos , Sistema Nervoso , Fatores de Transcrição/genéticaRESUMO
The small GTPase Rab5 has emerged as an important regulator of animal development, and it is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. We show here that Drosophila Rabenosyn (Rbsn) is a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45, and we further identify the syntaxin Avalanche (Avl) as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl, and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and it provides in vivo evidence for a Rbsn-Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery.
Assuntos
Proteínas de Drosophila/fisiologia , Endossomos/metabolismo , Regulação da Expressão Gênica , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/fisiologia , Alelos , Animais , Drosophila , Proteínas de Drosophila/metabolismo , Genes Supressores de Tumor , Humanos , Modelos Biológicos , Modelos Genéticos , Fenótipo , Ligação Proteica , Proteínas de Transporte Vesicular/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt), and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.