Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

High Frequency Ultrasound for In Vivo Pregnancy Diagnosis and Staging of Placental and Fetal Development in Mice

Background

Ultrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases.

Methodology/Principal Findings

On each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant.

Conclusions/Significance

The study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.     

Phylogenetic analysis based on full genome sequencing of Italian tomato spotted wilt virus isolates identified in “Roggianese” sweet pepper and chilli pepper

A study aimed at defining population structure of Italian tomato spotted wilt virus (TSWV) isolates was performed. Full genome sequencing of six TSWV isolates found in two Italian regions (two from Latium: Lazio 17 and Tarquinia; and four from Calabria: PepCal 10, 12, 22 and 24) were assembled. Identity percentages in nucleotide sequence among these TSWV isolates are here provided. The six full length genome sequences were compared with other two Italian isolates (p105 and p202/3WT) already fully sequenced, as well as full TSWV genomes that could be retrieved from GenBank. Phylogenetic analysis, performed in concatenated sequences and for each gene of each genome segment (L, M and S), confirmed the presence of two clades, namely A-like and D-like. In particular, the phylogenetic tree based on segment L grouped all the newly sequenced TSWV isolates in D-like clade. In the M segment phylogenetic tree, all our TSWV isolates shifted in the A-like clade. Isolates separation was not correlated to their geographical origin in phylogenetic study of distinct ORFs encoded by the RNA S segment. In fact, in nucleocapsid-encoding phylogenetic tree, PepCal 10 and 22 grouped in an A-like clade with p105, PepCal 12 and 24 in a D-like clade with p202/3WT, whereas Lazio 17 and Tarquinia in a third well distinct group. NSs tree displayed only PepCal 10 with p105 in A-like clade, whereas PepCal 12, 22, 24 with p202/3WT in D-like subclade; and isolates from Latium grouped a separated clade adjacent to D-like isolates. Additional analysis on putative reassortment events showed that TSWV Calabrian isolates likely originated from a reassortment event in M RNA and other in S RNA with p105 as major parent. Recombination events were detected in isolates from Latium in L and S RNAs with Chinese isolates as putative major parent.     

Dynamic changes in microbiota and mycobiota during spontaneous ‘Vino Santo Trentino’ fermentation

Vino Santo is a sweet wine produced from late harvesting and pressing of Nosiola grapes in a small, well‐defined geographical area in the Italian Alps. We used metagenomics to characterize the dynamics of microbial communities in the products of three wineries, resulting from spontaneous fermentation with almost the same timing and procedure. Comparing fermentation dynamics and grape microbial composition, we show a rapid increase in a small number of wine yeast species, with a parallel decrease in complexity. Despite the application of similar protocols, slight changes in the procedures led to significant differences in the microbiota in the three cases of fermentation: (i) fungal content of the must varied significantly in the different wineries, (ii) Pichia membranifaciens persisted in only one of the wineries, (iii) one fermentation was characterized by the balanced presence of Saccharomyces cerevisiae and Hanseniaspora osmophila during the later phases. We suggest the existence of a highly winery‐specific ‘microbial‐terroir’ contributing significantly to the final product rather than a regional ‘terroir’. Analysis of changes in abundance during fermentation showed evident correlations between different species, suggesting that fermentation is the result of a continuum of interaction between different species and physical–chemical parameters.