Position Weight Matrix or Acyclic Probabilistic Finite Automaton: Which model to use? A decision rule inferred for the prediction of transcription factor binding sites.

Prediction of transcription factor binding sites (TFBS) is an example of application of Bioinformatics where DNA molecules are represented as sequences of A, C, G and T symbols. The most used model in this problem is Position Weight Matrix (PWM). Notwithstanding the advantage of being simple, PWMs cannot capture dependency between nucleotide positions, which may affect prediction performance. Acyclic Probabilistic Finite Automata (APFA) is an alternative model able to accommodate position dependencies. However, APFA is a more complex model, which means more parameters have to be learned. In this paper, we propose an innovative method to identify when position dependencies influence preference for PWMs or APFAs. This implied using position dependency features extracted from 1106 sets of TFBS to infer a decision tree able to predict which is the best model - PWM or APFA - for a given set of TFBSs. According to our results, as few as three pinpointed features are able to choose the best model, providing a balance of performance (average precision) and model simplicity.

Global repression by tailless during segmentation.

The orphan nuclear receptor Tailless (Tll) exhibits conserved roles in brain formation and maintenance that are shared, for example, with vertebrate orthologous forms (Tlx). However, the early expression of tll in two gap domains in the segmentation cascade of Drosophila is unusual even for most other insects. Here we investigate tll regulation on pair-rule stripes. With ectopic misexpression of tll we detected unexpected repression of almost all pair-rule stripes of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz). Examining Tll embryonic ChIP-chip data with regions mapped as Cis-Regulatory Modules (CRMs) of pair-rule stripes we verified Tll interactions to these regions. With the ChIP-chip data we also verified Tll interactions to the CRMs of gap domains and in the misexpression assay, Tll-mediated repression on Kruppel (Kr), kni (kni) and giant (gt) according to their differential sensitivity to Tll. These results with gap genes confirmed previous data from the literature and argue against indirect repression roles of Tll in the striped pattern. Moreover, the prediction of Tll binding sites in the CRMs of eve stripes and the mathematical modeling of their removal using an experimentally validated theoretical framework shows effects on eve stripes compatible with the absence of a repressor binding to the CRMs. In addition, modeling increased tll levels in the embryo results in the differential repression of eve stripes, agreeing well with the results of the misexpression assay. In genetic assays we investigated eve 5, that is strongly repressed by the ectopic domain and representative of more central stripes not previously implied to be under direct regulation of tll. While this stripe is little affected in tll-, its posterior border is expanded in gt- but detected with even greater expansion in gt-;tll-. We end up by discussing tll with key roles in combinatorial repression mechanisms to contain the expression of medial patterns of the segmentation cascade in the extremities of the embryo.

Biocatalytic potential of Pseudolycoriella CAZymes (Sciaroidea, Diptera) in degrading plant and fungal cell wall polysaccharides.

Soil biota has a crucial impact on soil ecology, global climate changes, and effective crop management and studying the diverse ecological roles of dipteran larvae deepens the understanding of soil food webs. A multi-omics study of Pseudolycoriella hygida comb. nov. (Diptera: Sciaroidea: Sciaridae) aimed to characterize carbohydrate-active enzymes (CAZymes) for litter degradation in this species. Manual curation of 17,881 predicted proteins in the Psl. hygida genome identified 137 secreted CAZymes, of which 33 are present in the saliva proteome, and broadly confirmed by saliva CAZyme catalytic profiling against plant cell wall polysaccharides and pNP-glycosyl substrates. Comparisons with two other sciarid species and the outgroup Lucilia cuprina (Diptera: Calliphoridae) identified 42 CAZyme families defining a sciarid CAZyme profile. The litter-degrading potential of sciarids corroborates their significant role as decomposers, yields insights to the evolution of insect feeding habits, and highlights the importance of insects as a source of biotechnologically relevant enzymes.

An additive repression mechanism sets the anterior limits of anterior pair-rule stripes 1.

Segments are repeated anatomical units forming the body of insects. In Drosophila, the specification of the body takes place during the blastoderm through the segmentation cascade. Pair-rule genes such as hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) are of the intermediate level of the cascade and each pair-rule gene is expressed in seven transversal stripes along the antero-posterior axis of the embryo. Stripes are formed by independent cis-regulatory modules (CRMs) under the regulation of transcription factors of maternal source and of gap proteins of the first level of the cascade. The initial blastoderm of Drosophila is a syncytium and it also coincides with the mid-blastula transition when thousands of zygotic genes are transcribed and their products are able to diffuse in the cytoplasm. Thus, we anticipated a complex regulation of the CRMs of the pair-rule stripes. The CRMs of h 1, eve 1, run 1, ftz 1 are able to be activated by bicoid (bcd) throughout the anterior blastoderm and several lines of evidence indicate that they are repressed by the anterior gap genes slp1 (sloppy-paired 1), tll (tailless) and hkb (huckebein). The modest activity of these repressors led to the premise of a combinatorial mechanism regulating the expression of the CRMs of h 1, eve 1, run 1, ftz 1 in more anterior regions of the embryo. We tested this possibility by progressively removing the repression activities of slp1, tll and hkb. In doing so, we were able to expose a mechanism of additive repression limiting the anterior borders of stripes 1. Stripes 1 respond depending on their distance from the anterior end and repressors operating at different levels.

Characterizing the embryonic development of B. hygida (Diptera: Sciaridae) following enzymatic treatment to permeabilize the serosal cuticle.

Understanding the evolution of the developmental programs active during dipteran embryogenesis depends on comparative studies. As a counterpoint to the intensively investigated and highly derived cyclorrhaphan flies that include the model organism Drosophila melanogaster, we are studying the basal Diptera Bradysia hygida, a member of the Sciaridae family that is amenable to laboratory cultivation. Here we describe the B. hygida embryogenesis, which lasts 9 days at 22 °C. The use of standard fixation D. melanogaster protocols resulted in embryos refractory to DAPI staining and to overcome this, a new enzyme-based method was developed. Calcofluor-White staining of enzimatically-treated embryos revealed that this method removes chitin from the serosal cuticle surrounding the B. hygida embryo. Chitin is one of the main components of serosal cuticles and searches in a B. hygida embryonic transcriptome database revealed conservation of the chitin synthesis pathway, further supporting the occurrence of chitin biosynthesis in B. hygida embryos. Combining the enzymatic treatment protocol with the use of both DIC and fluorescence microscopy allowed the first complete description of the B. hygida embryogenesis. Our results constitute an important step towards the understanding of early development of a basal Diptera and pave the way for future evo-devo studies.

Repression activity of Tailless on h 1 and eve 1 pair-rule stripes.

We investigated the hypothesis that several transcriptional repressors are necessary to set the boundaries of anterior pair-rule stripes in Drosophila. Specifically, we tested whether Tailless (Tll) is part of a repression mechanism that correctly sets the anterior boundaries of hairy 1 (h 1) and even-skipped 1 (eve 1) stripes. Single mutant tll embryos displayed subtle deviations from the normal positions of h 1 and eve 1 stripes. Moreover, we observed stronger stripe deviations in embryos lacking both Tll and Sloppy-paired 1 (Slp 1), a common repressor for anterior pair-rule stripes. Using h 1 and eve 1 reporter constructs in the genetic assays, we provided further evidence that interference with normal mechanisms of stripe expression is mediated by Tll repression. Indeed, Tll represses both h 1 and eve 1 reporter stripes when misexpressed. Investigating the expression of other anterior gap genes in different genetic backgrounds and in the misexpression assays strengthened Tll direct repression in the regulation of h 1 and eve 1. Our results are consistent with tll being a newly-identified component of a combinatorial network of repressor genes that control pair-rule stripe formation in the anterior blastoderm of Drosophila.

Toward new Drosophila paradigms.

The fruit fly Drosophila melanogaster is a great model system in developmental biology studies and related disciplines. In a historical perspective, I focus on the formation of the Drosophila segmental body plan using a comparative approach. I highlight the evolutionary trend of increasing complexity of the molecular segmentation network in arthropods that resulted in an incredible degree of complexity at the gap gene level in derived Diptera. There is growing evidence that Drosophila is a highly derived insect, and we are still far from fully understanding the underlying evolutionary mechanisms that led to its complexity. In addition, recent data have altered how we view the transcriptional regulatory mechanisms that control segmentation in Drosophila. However, these observations are not all bad news for the field. Instead, they stimulate further study of segmentation in Drosophila and in other species as well. To me, these seemingly new Drosophila paradigms are very challenging ones.

Huckebein is part of a combinatorial repression code in the anterior blastoderm.

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated here, led us to the hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. In this study, we investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods we were able to detect deviations from the wild-type patterns of the anterior-most pair-rule stripes in different genetic backgrounds, which were consistent with Hkb-mediated repression. Moreover, we developed an image processing tool that, for the most part, confirmed our assumptions. Using an hkb misexpression system, we further detected specific repression on anterior stripes. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. We further discuss that Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes.

Investigating giant (Gt) repression in the formation of partially overlapping pair-rule stripes.

Drosophila pair-rule genes are expressed in striped patterns with a precise order of overlap between stripes of different genes. We investigated the role of Giant (Gt) in the regulation of even-skipped, hairy, runt, and fushi tarazu stripes formed in the vicinity of Gt expression domains. In gt null embryos, specific stripes of eve, h, run, and ftz are disrupted. With an ectopic expression system, we verified that stripes affected in the mutant are also repressed. Simultaneously hybridizing gt misxpressing embryos with two pair-rule gene probes, we were able to distinguish differences in the repression of pairs of stripes that overlap extensively. Together, our results showed Gt repression roles in the regulation of two groups of partially overlapping stripes and that Gt morphogen activity is part of the mechanism responsible for the differential positioning of these stripes borders. We discuss the possibility that other factors regulate Gt stripe targets as well.

Ribosomal RNA gene insertions in the R2 site of Rhynchosciara (Diptera: Sciaridae).

Ribosomal RNA genes of most insects are interrupted by R1/R2 retrotransposons. The occurrence of R2 retrotransposons in sciarid genomes was studied by PCR and Southern blot hybridization in three Rhynchosciara species and in Trichosia pubescens. Amplification products with the expected size for non-truncated R2 elements were only obtained in Rhynchosciara americana. The rDNA in this species is located in the proximal end of the X mitotic chromosome but in the salivary gland is associated with all four polytene chromosomes. Approximately 50% of the salivary gland rDNA of most R. americana larval groups analysed had an insertion in the R2 site, while no evidence for the presence of R1 elements was found. In-situ hybridization results showed that rDNA repeat units containing R2 take part in the structure of the extrachromosomal rDNA. Also, rDNA resistance to Bal 31 digestion could be interpreted as evidence for nonlinear rDNA as part of the rDNA in the salivary gland. Insertions in the rDNA of three other sciarid species were not detected by Southern blot and in-situ hybridization, suggesting that rDNA retrotransposons are significantly under-represented in their genomes in comparison with R. americana. R2 elements apparently restricted to R. americana correlate with an increased amount of repetitive DNA in its genome in contrast to other Rhynchosciara species. The results obtained in this work together with previous results suggest that evolutionary changes in the genus Rhynchosciara occurred by differential genomic occupation not only of satellite DNA but possibly also of rDNA retrotransposons.

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae).

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Drosophila brakeless interacts with atrophin and is required for tailless-mediated transcriptional repression in early embryos.

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. We isolated mutations in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, we noted an expanded expression pattern of the Krüppel (Kr) and knirps (kni) genes. We found that Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. We show that both Drosophila and human Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. We discuss the possibility that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA).

Staurosporine induces tyrosine phosphorylation in Dictyostelium discoideum proteins.

The treatment of cells with staurosporine results in inhibition and less frequently activation of protein kinases, in a cell-type specific manner. In the social amoeba Dictyostelium discoideum, staurosporine induces marked changes in cell morphology affecting growth and development. Here we describe that incubation of D. discoideum growing or starved cells with staurosporine results in a rapid and unexpected tyrosine phosphorylation on two polypeptides of approximately 64 and approximately 62 kDa. These proteins emerge as novel substrates for tyrosine phosphorylation opening up new perspectives for the study of cell signalling in D. discoideum.

Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms.

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers.

Clonagem e caracterização da serina/treonina fosfatase tipo 1(PP1) de Dictyostelium discoideum / Cloning and characterization of the serine/threonine phosphatase type 1(PP1) of Dictyostelium discoideum

A serina/treonina fosfatase tipo 1 (PP1) é uma enzima importante para diversos processos celulares nos eucariotos. Apesar disso, a atividade enzimática da PP1 nunca foi detectada em extratos celulares de Dictyostelium discoideum, ao contrário da atividade da enzima PP2A, que é outra proteína fosfatase de ocorrência geral nos eucariotos. Nesse estudo repostamos a clonagem e o sequenciamento de um cDNA codificador para a subunidade catalílitica da PP1 de D. discoideum. Verificamos que esse cDNA tem origem a partir de um gene em cópia única, que expressa uma proteína ao longo de todo o ciclo de vida desse organismo. A dedução da seqüência da proteína a partir do cDNA revela uma identidade de 80 por cento em média com as seqüências de outras PP1, sendo a principal diferença observada a substituição da cisteína 269 por uma fenilalanina...