Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Assessing the validity of a self-administered food-frequency questionnaire (FFQ) in the adult population of Newfoundland and Labrador, Canada

Background: The Food- Frequency Questionnaire (FFQ) is a dietary assessment tool frequently used in large-scale nutritional epidemiology studies. The goal of the present study is to validate a self-administered version of the Hawaii FFQ modified for use in the general adult population of Newfoundland and Labrador (NL). Methods: Over a one year period, 195 randomly selected adults completed four 24-hour dietary recalls (24-HDRs) by telephone and one subsequent self-administered FFQ. Estimates of energy and nutrients derived from the 24-HDRs and FFQs were compared (protein, carbohydrate, fibre, fat, vitamin A, carotene, vitamin D, and calcium). Data were analyzed using the Pearson’s correlation coefficients, cross-classification method, and Bland–Altman plots. Results: The mean nutrient intake values of the 24-HDRs were lower than those of the FFQs, except for protein in men. Sex and energy-adjusted de-attenuated Pearson correlation coefficients for each nutrient varied from 0.13 to 0.61. Except for protein in men, all correlations were statistically significant with p < 0.05. Cross-classification analysis revealed that on average, 74% women and 78% men were classified in the same or adjacent quartile of nutrient intake when comparing data from the FFQ and 24-HDRs. Bland–Altman plots showed no serious systematic bias between the administration of the two instruments over the range of mean intakes. Conclusion: This 169-item FFQ developed specifically for the adult NL population had moderate relative validity and therefore can be used in studies to assess food consumption in the general adult population of NL. This tool can be used to classify individual energy and nutrient intakes into quartiles, which is useful in examining relationships between diet and chronic disease

ESICM LIVES 2016: part two : Milan, Italy. 1-5 October 2016.

Meeting abstrac