Nadia Monesi

The DNA puff gene BhC4-1 of Bradysia hygida is specifically transcribed in early prepupal salivary glands of Drosophila melanogaster

Abstract. The BhC4-1 gene of the sciarid Bradysia hygida is located at DNA puff C4 and is amplified and actively transcribed in the salivary gland at the end of the last larval instar. We show here that a 3.6 kb fragment from the upstream region of the BhC4-1 gene is able to drive transcription in transgenic Drosophila specifically in prepupal salivary gland in a temporally regulated manner. The mRNA is present in maximal amounts in prepupae +3 h; in prepupae +9 h the levels of BhC4-1 mRNA decline, and it is no longer detected in pupae +24 h. Taken together these results suggest that most, if not all, of the key promoter regulatory elements were included in the DNA fragments employed to transform Drosophila. Moreover, strong expression of the transgenes implies conservation of the regulatory elements involved, since Drosophila transcription factors appear to recognize B. hygida regulatory DNA sequences. Quantitative Southern blot hybridization indicates that the sequences from DNA puff C4 are not amplified at detectable levels in salivary glands of transgenic prepupae when the BhC4-1 gene is transcribed. Transcription of a DNA puff in the absence of amplification indicates that the induction of these processes involves distinct mechanisms.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, Sciaridae)

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly α-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5′ end, and beyond the 3′ end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Alternative Method for Direct DNA Probe Labeling and Detection Using the Checkerboard Hybridization Format

Molecular diagnostic methods using genetic material probes have been employed in the health care field for the detection and quantitation of several species of microorganisms ([2][1], [5][2]). These methods are faster and more suitable than traditional culture methods. In addition, the possibility

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BackgroundThe ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers.ResultsOf the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes.ConclusionsThe versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

The DNA puff BhB10-1 gene encodes a glycine-rich protein secreted by the late stage larval salivary glands of Bradysia hygida

We present the molecular characterization of a gene of Bradysia hygida DNA puff B10 whose temporal expression in the salivary gland correlates with the puff expansion. The transcription unit of this gene, named BhB10-1, was mapped in a 2-kb EcoRI genomic fragment that is amplified in the salivary gland of late fourth instar larvae. Its 1.3-kb transcript undergoes poly-A tail shortening during development, indicating that post-transcriptional controls as well as transcription activation are involved in the temporal regulation of the BhB10-1 gene. Analysis of the deduced amino acid sequence from the cDNA indicates that the BhB10-1 protein is a glycine-rich secretory protein. A BhB10-1-fusion protein expressed in bacteria was used to raise polyclonal antibodies. Using an immunopurified antibody, we identified the product of the DNA puff BhB10-1 gene as a 23-kDa polypeptide that is produced mainly by the salivary gland regions S1 and S3 and is present in the saliva of late larvae. This is the first direct identification of a protein encoded by a DNA puff amplified gene.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

Microbiome of titanium and zirconia dental implants abutments

OBJECTIVES This study employed culture-independent molecular techniques to extend the characterization of the microbial diversity of biofilm associated with either titanium or zirconia implant-abutments, including not-yet-cultivated bacteria species, and to identify and quantify species recovered from peri-implantar/periodontal sulci, supragingival biofilm and the internal parts of implants. Probing depth, clinical attachment level, bleeding on probing, and marginal bone level were also evaluated over time and correlated with biofilm formation. METHODS Twenty healthy participants were analyzed. DNA-Checkerboard and 16S-rDNA-Pyrosequencing were used to quantify and determine species identity. RESULTS 161 bacterial taxa representing 12 different phylotypes were found, of which 25% were non-cultivable. Species common to all sites belonged to genera Fusobacterium, Prevotella, Actinomyces, Porphyromonas, Veillonella and Streptococcus. While some species were subject-specific and detected in most sites, other species were site-specific. Moderate to higher levels of unclassified species were found colonizing titanium-related sites. Pathogenic and non-pathogenic species were detected colonizing oral sites in both materials. Titanium-related sites presented the highest total microbial count and higher counts of pathogenic species. CONCLUSIONS Our results revealed differences regarding microbial diversity and microorganisms counts in oral biofilm associated with titanium or zirconia. The obtained data suggests a possible relation between microbiological findings and clinical outcomes. SIGNIFICANCE Next-generation methods of detection have provided new insights on complex microbiota colonizing different sites of oral cavity. The present study demonstrates relevant differences in the communities and microbial counts colonizing different tested substrates with consequent significant differences in the clinical-outcomes, suggesting a probably different mechanism for specific bacterial adhesion.

DNA puff C4 ofBradysia hygida (Diptera: Sciaridae) contains genes unequally amplified and differentially expressed during development

We report here the isolation and characterization of a 2.3 kb genomicEcoRl fragment that co-localizes in the DNA puff C4 ofBradysia hygida with a 4 kbEcoRl fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when puff C4 expands. Verification of the relative amount of DNA complementary to these two genomic fragments shows that they are unequally amplified in the salivary gland. The fragment containing part of the gene expressed when puff C4 expands amplifies about eight times more than the 2.3 kb fragment. This 2.3 kb fragment also carries sequences complementary to RNA species present in the gland in a period when puff C4 has already receded. Based on these data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites.

Bacterial diversity of periodontal and implant-related sites detected by the DNA Checkerboard method

The aim of this study was to compare the microbial composition of the subgingival biofilm from teeth and implant sulci in relation to contents originating from internal parts of the implant, abutment and implant prosthesis. Twenty subgingival biofilm samples from the mesial and distal aspects of each tooth/implant and 29 samples from the internal parts of titanium implants, abutments and implant prostheses were evaluated for the presence of 18 bacterial species using DNA Checkerboard and the differences between samples from teeth and implants were assessed with Pearson’s correlation analysis. The periodontal and peri-implantar sulci presented significantly higher bacterial counts than the implant-related sites (p < 0.05 and p < 0.01, respectively). The highest counts were observed for Capnocytophaga gingivalis, Prevotella intermedia, P. nigrescens and P. micra. The correlation between the counts in the periodontal and peri-implantar sulci was r = 0.66 (p < 0.001). Weaker correlations between samples from the internal parts of the implants and periodontal sulcus (r = 0.49; p < 0.001) or peri-implant sulcus (r = 0.42; p < 0.001) were found. All 18 bacterial species were detected to be colonising the subgingival sulcus of teeth and implants, and implant components in the evaluated patients. Significant correlations between the microbiota were found, the strongest being between the periodontal and peri-implantar sulci.

Identification of regulatory regions in the DNA puff BhC4-1 promoter

The mechanisms that control DNA puff BhC4‐1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila. By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4‐1 promoter fragment (−3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4‐1‐lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (−186/+40). Also, interestingly, this study identified a 67 bp fragment (−253/−187) that activates BhC4‐1‐lacZ expression specifically in the ring gland.