Fábio Siviero

Molecular characterization of the B-2 DNA puff gene of Rhynchosciara americana

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.

Kinetics of the pH-independent hydrolysis of 4-nitrophenyl chloroformate in aqueous micellar solutions: effects of the charge and structure of the surfactant

The pH-independent hydrolysis of 4-nitrophenyl chloroformate in the presence of aqueous micelles of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, alkyltrimethylammonium chlorides, alkyldimethylbenzylammonium chlorides (alkyl group = cetyl and dodecyl) and polyoxyethylene (9) nonylphenyl ether was studied spectrophotometrically. The observed rate constants, kobs, decrease in the following order: cationic micelles > bulk water > non-ionic micelles > anionic micelles. Surfactant–substrate association constants, Ks, were determined from the dependence of kobs on surfactant concentration, and were found to be only slightly dependent on the charge of the surfactant and, for similarly charged micelles, on the length of their hydrophobic tail. A 1H NMR study of the solubilization of a model compound, 4-nitrophenyl chloroacetate, showed that all surfactant segments are affected by the solubilizate and the effect is more pronounced toward the middle of the hydrocarbon chain. The average solubilization site of the acetate ester does not depend on the charge of the micelle or the length of the surfactant hydrophobic tail. Micellar effects on observed rate constants are analyzed in terms of a ‘medium’ effect and an ‘electrostatic’ effect. The lower microscopic polarity at the reaction site retards the reaction, whereas electrostatic interactions of the polar transition state with the charged interface result in a rate decrease by anionic micelles and a rate enhancement by cationic micelles. Copyright

Analysis of expressed sequence tags from Rhynchosciara americana salivary glands.

The diptera Rhynchosciara americana (sciaridae) is an important model organism in polyteny and gene amplification research, but up to now a limited amount of data regarding DNA sequences and molecular aspects of this species is available. Considering the importance of going further on the DNA puffs biological meaning, we proposed to generate EST sequences from a DNA library constructed from salivary glands. After their categorization in gene ontology terms, they were used to construct an ‘electronic Northern’ that represents a general view of the salivary gland metabolic status in an important phase of larval development: the spinning of communal cocoon. In this phase occurs the last polytene DNA replication cycle concomitantly with the specific loci amplification related to protein secretion.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5′-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Mariner-like elements in Rhynchosciara americana (Sciaridae) genome: molecular and cytological aspects

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Beyond DNA puffs: What can we learn from studying sciarids?

Summary: Members of the Sciaridae family attracted the interest of researchers because of the demonstration that the DNA puff regions, which are formed in the salivary gland polytene chromosomes at the end of the fourth larval instar, constitute sites of developmentally regulated gene amplification. Besides contributing to a deeper understanding of the process of gene amplification, the study of sciarids has also provided important insights on other biological processes such as sex determination, programmed cell death, insect immunity, telomere maintenance, and nucleolar organizing regions (NOR) formation. Open questions in sciarids include among others, early development, the role of noncoding RNAs in gene amplification and the relationship between gene amplification and transcription in DNA puff forming regions. These and other questions can now be pursued with next generation sequencing techniques and experiments using RNAi experiments, since this latter technique has been shown to be feasible in sciarids. These new perspectives in the field of sciarid biology open the opportunity to consolidate sciarid species as important emerging models. genesis 54:361–378, 2016.

A shortcut in phage screening technique

A simple modification of the traditional Benton & Davis technique for phage screening is presented that avoids the tedious sample dilutions of putative spots/phages towards the second screening. With the use of a sole agar plate and nylon filter, the modification distinguishes a true positive recombinant from a false positive, with high probability of success.