Genome-wide screen for genes involved in Caenorhabditis elegans developmentally timed sleep

In Caenorhabditis elegans, Notch signaling regulates developmentally timed sleep during the transition from L4 larval stage to adulthood (L4/A) . To identify core sleep pathways and to find genes acting downstream of Notch signaling, we undertook the first genome-wide, classical genetic screen focused on C. elegans developmentally timed sleep. To increase screen efficiency, we first looked for mutations that suppressed inappropriate anachronistic sleep in adult hsp::osm-11 animals overexpressing the Notch coligand OSM-11 after heat shock. We retained suppressor lines that also had defects in L4/A developmentally timed sleep, without heat shock overexpression of the Notch coligand. Sixteen suppressor lines with defects in developmentally timed sleep were identified. One line carried a new allele of goa-1; loss of GOA-1 Gαo decreased C. elegans sleep. Another line carried a new allele of gpb-2, encoding a Gβ5 protein; Gβ5 proteins have not been previously implicated in sleep. In other scenarios, Gβ5 GPB-2 acts with regulators of G protein signaling (RGS proteins) EAT-16 and EGL-10 to terminate either EGL-30 Gαq signaling or GOA-1 Gαo signaling, respectively. We found that loss of Gβ5 GPB-2 or RGS EAT-16 decreased L4/A sleep. By contrast, EGL-10 loss had no impact. Instead, loss of RGS-1 and RGS-2 increased sleep. Combined, our results suggest that, in the context of L4/A sleep, GPB-2 predominantly acts with EAT-16 RGS to inhibit EGL-30 Gαq signaling. These results confirm the importance of G protein signaling in sleep and demonstrate that these core sleep pathways function genetically downstream of the Notch signaling events promoting sleep

A hydrazine coupled cycling assay validates the decrease in redox ratio under starvation in Drosophila.

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD(+)/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP(+) and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD(+) and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation

Comparison of three different extraction methods.

<p>Three sets of 15 <i>D. melanogaster</i> adult males were subjected to different treatments. Homogenized in buffer with additional 6 M guanidine-HCl or: homogenized and treated with equal volume phenol-chloroform or chloroform only. The samples were then assayed for NADx in duplicates.</p

Slopes between the rate of absorbance increase (<i>V_mean</i>) and the concentration of NAD⁺ (10⁻⁴·OD·min⁻¹·µM⁻¹) in either homogenate or homogenization buffer.

<p><i>N.S.</i>: non-significant; **, <i>p</i><0.01; ***, <i>p</i><0.001. Fly homogenate is generated by homogenizing 15 male <i>D. melanogaster</i> adults in 250 ul homogenization buffer. Particles were removed by 5 min 16000×g centrifugation.</p

The optimal concentration of hydrazine is around 0.02%.

<p>A: In a reaction containing 25 µM NAD⁺, <i>V_mean</i> increases with hydrazine concentration log-linearly up to 0.02%. B: The rate of absorbance increase in no NAD⁺ blank control is affected by hydrazine concentration higher than 0.02%.</p

Comparison of reaction kinetics of NADPx, NADx assay with and without hydrazine.

<p>A: Standard curve of NADPx assay. B: Standard curve of NADx assay without hydrazine. C: Standard curve of NADx assay with hydrazine. D: reaction kinetic of NADPx assay. E: NADx assay kinetic, showing additional hydrazine increases <i>V_mean</i>. F: the increase of <i>V_mean</i> by addition of hydrazine is dependent on the concentration of NAD⁺. All assays were performed in duplicates and hydrazine concentration is 0.02%.</p

Phenol-Chloroform extraction is efficient for extracting NADH.

<p>Six samples of 15 male <i>D. melanogaster</i> adults were homogenized in 250 ul homogenization buffer. Following a 5 min 16000×g centrifugation, the supernatant was then divided into 3 parts. One part was kept as control. The other two parts were treated with equal volume of chloroform and phenol-chloroform respectively. Three parts were then assayed for NAD⁺ and NADH in duplicates. The concentration of NAD⁺ and NADH, with S.E.M, are standardized by the concentration of soluble protein measured from the control group. The difference in NAD⁺ and NADH concentration is tested using two sample <i>t</i>-test. See supplementary material for the method of testing redox ratio difference between groups.</p

Changes in the concentrations of protein and metabolites following starvation.

<p>Comparisons are between flies held on food (‘Fed’) and on 2% agar (starved, ‘Stv’ on x-axes). Unit: µmol/g protein For NADH, NAD⁺, NADPH and NADP⁺. mg/g protein for TAG, glycogen and glucose. µg/ml for protein. n = 8 and measured in duplicates. The between group differences are tested by 2 sample <i>t</i>-test.</p

NUMTs Can Imitate Biparental Transmission of mtDNA&mdash;A Case in Drosophila melanogaster

mtDNA sequences can be incorporated into the nuclear genome and produce nuclear mitochondrial fragments (NUMTs), which resemble mtDNA in their sequence but are transmitted biparentally, like the nuclear genome. NUMTs can be mistaken as real mtDNA and may lead to the erroneous impression that mtDNA is biparentally transmitted. Here, we report a case of mtDNA heteroplasmy in a Drosophila melanogaster&nbsp;DGRP line, in which the one haplotype was biparentally transmitted in an autosomal manner. Given the sequence identity of this haplotype with the mtDNA, the crossing experiments led to uncertainty about whether heteroplasmy was real or an artifact due to a NUMT. More specific experiments revealed that there is a large NUMT insertion in the X chromosome of a specific DGRP line, imitating biparental inheritance of mtDNA. Our result suggests that studies on mtDNA heteroplasmy and on mtDNA inheritance should first exclude the possibility of NUMT interference in their data