Project description:The nematode Caenorhabditis elegans (C. elegans) is often used as a model organism to study cell and developmental biology. Quantitative mass spectrometry has only recently been performed in C. elegans and, so far, most studies have been done on adult worm samples. Here we use quantitative mass spectrometry to characterise protein level changes across the four larval developmental stages (L1-L4) of C. elegans, in biological triplicate. In total, we identify 4,130 proteins and quantify 1,541 proteins that were identified across all four stages in all three biological repeats with at least 2 unique peptides per protein. Using hierarchical clustering and functional ontological analyses, we identify 21 protein groups containing proteins with similar protein profiles across the four stages, and highlight the most overrepresented biological functions in each of these protein clusters. In addition, we use the dataset to identify putative larval stage specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides a system-wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the worm development research community.
Project description:Reports that low-intensity microwave radiation can induce heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by very slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg-1 for 6-well plates) that minimises the temperature differential between sham and exposed conditions to ≤ 0.1°C. Comparable measurement and simulation studies of SAR distribution within this exposure system are presented. We compared 5 Affymetrix gene-arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against 5 gene-arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). Few genes showed consistent expression changes across all 5 comparisons, and all such expression changes appeared modest after applying standard normalisation procedures (≤ 30% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). As one example, an apparent up-regulation of the vit-3 vitellogenin gene by microwave exposure was not mirrored by similar changes affecting the other co-regulated members of the same vit gene family. We conclude that the pattern of gene expression in L4/adult C elegans is not substantially perturbed by low-intensity microwave radiation, and that the minor changes observed in this study may well be explicable as false positives. As a check on the sensitivity of the Affymetrix gene-arrays used, we also compared RNA samples from N2 worms subjected to a sub-heat-shock treatment (28ºC) against controls kept at 26 ºC (but using only 2 gene arrays per condition). After similar normalisation, many more genes (3712) showed substantial expression changes (i.e. > 2-fold at p < 0.05), including a group of six heat-shock genes which were strongly but unexpectedly down-regulated (by > 10-fold). However, further replication and confirmation by real-time RT-PCR would be needed to establish how many of these changes might also be false positives. Experimenter name: Adam Dawe Experimenter phone: +27 21 959 2364 adam@sanbi.ac.za Experimenter institute: South African National Bioinformatics Institute Experimenter address: University of Western Cape, Old Chemistry Building, University of Western Cape, Modderdam Road, Bellville 7530, Capetown Experimenter zip/postal_code: 7530 Experimenter country: South Africa Keywords: Microwave radiation, gene expression, gene arrays, Caenorhabditis elegans
Project description:Reports that low-intensity microwave radiation can induce heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by very slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg-1 for 6-well plates) that minimises the temperature differential between sham and exposed conditions to ≤ 0.1°C. Comparable measurement and simulation studies of SAR distribution within this exposure system are presented. We compared 5 Affymetrix gene-arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against 5 gene-arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). Few genes showed consistent expression changes across all 5 comparisons, and all such expression changes appeared modest after applying standard normalisation procedures (≤ 30% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). As one example, an apparent up-regulation of the vit-3 vitellogenin gene by microwave exposure was not mirrored by similar changes affecting the other co-regulated members of the same vit gene family. We conclude that the pattern of gene expression in L4/adult C elegans is not substantially perturbed by low-intensity microwave radiation, and that the minor changes observed in this study may well be explicable as false positives. As a check on the sensitivity of the Affymetrix gene-arrays used, we also compared RNA samples from N2 worms subjected to a sub-heat-shock treatment (28ºC) against controls kept at 26 ºC (but using only 2 gene arrays per condition). After similar normalisation, many more genes (3712) showed substantial expression changes (i.e. > 2-fold at p < 0.05), including a group of six heat-shock genes which were strongly but unexpectedly down-regulated (by > 10-fold). However, further replication and confirmation by real-time RT-PCR would be needed to establish how many of these changes might also be false positives. Experimenter name: Adam Dawe; Experimenter phone: +27 21 959 2364; adam@sanbi.ac.za; Experimenter institute: South African National Bioinformatics Institute; Experimenter address: University of Western Cape, Old Chemistry Building, University of Western Cape, Modderdam Road, Bellville 7530, Capetown; Experimenter zip/postal_code: 7530; Experimenter country: South Africa Experiment Overall Design: 14 samples were used in this experiment
Project description:microRNAs (miRNAs) constitute a class of small non-coding RNAs (~22nt). They are thought to be generally stable with half-lives of many hours or even days. However, several miRNAs have been reported to decay rapidly in specific situations. In order to examine miRNA stability on a global scale, we quantify relative decay rates of miRNA in first larval stage C. elegans worms that are treated with a transcription inhibitor alpha-amanitin by deep sequencing. Several miRNAs including members of the miR-35 and miR-51 families exhibit accelerated decay. Moreover, biogenesis of miRNAs involves generation of a miRNA duplex intermediate consisting of the miRNA guide strand (miR) and the miRNA passenger strand (miR*). miR and miR* names were originally assigned based on the relative abundance of each strand, with the less abundant strand presumed to be inactive, and thus the miR*. However, subsequent research showed that at least individual miR*s can have biological activity. Our sequencing data reveal that miR*s, operationally defined on the basis of their relative abundance at time point t=1h, are substantially less stable than miRs. This would appear to support the notion that miR*s mainly constitute processing byproducts rather than a less abundant class of functional miRNAs. Examination of microRNA decay rates in the first larval stage C. elegans worms.
Project description:We compare whole-animal RNA-seq transcriptomes for C. elegans males and hermaphrodites from the late L3 larval stage to young adulthood. During this interval, male sexual structures develop, including extensive neurogenesis and synaptogenesis that nearly doubles the size of the nervous system. Previous genome-wide expression studies in C. elegans have usually focused on only one sex â the hermaphrodite, and there are a relatively large number of predicted genes that still remain without meaningful annotation. In the present study, differential expression analysis of the RNA-seq data revealed 1,751 genes expressed at a higher level in the male. By differential expression analysis, unbiased gene correlation analysis, and a guilt-by-association approach, we identified new transcription factors required for differentiation of male genital structures, semen proteins, and candidates for previously-unknown components for synapse function. The results validate the dataset as a rich resource for future gene discovery in C. elegans. To analyze gene expression during sexual maturation in C. elegans, we performed RNA-seq for five samples for each sex ranging at 6 hr intervals from late L3 to young adult stages