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Gene expression analysis of third instar larvae ski3 mutants

ABSTRACT: Purpose: To identify genes that are differentially expressed between control and ski3 mutants Methods: Gene expressions of control and ski3 mutants of third instar larvae were generated by deep sequencing, in two replicates, using Illumina NovaSeq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Bowtie2 and TopHat followed by Cufflinks. Results: We mapped 800,000-1,200,000 sequence reads per each sample to the fruit fly genome (BDGP6) and identified 93,606 transcripts in the whole body of control and ski3[f03251] with TopHat workflow. Analysis of RNA-seq data idnetified 32 differentially expressed genes relevant to mitochondrial function between control and ski3 mutants with FDR <0.05. The mutation affected over 600 genes, which are involved in many different cellular functions. The results were complex: among 32 affected genes, half of them were upregulated, and the remaining half were downregulated. Six genes were related to the TCA cycle. Of these, the downregulated genes include CG9582 (alpha-ketoglutarate transmembrane transporter), CG32832 (mitochondrial pyruvate carrier), and CG7514 (oxoglutarate:malate antiporter). On the other hand, upregulated genes include AcCoAS (Acetyl-CoA synthase), Sirup (Succinate dehydrogenase), and Men-b (malate dehydrogenase), all of which were directly involved in the TCA cycle. Conclusions: Our study represents the first detailed analysis of ski3 mutant transcriptomes, with biologic replicates, generated by RNA-seq technology. In order to examine the effects of ski3 mutations on gene expression, we performed RNA-seq analyses of wild-type and mutant larvae. Downregulation of transporter, carrier and antiporter suggested a chronic undersupply of TCA cycle substrates, while some of the TCA cycle enzymes were upregulated.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE144343 | GEO | 2020/01/29

REPOSITORIES: GEO

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Project description:Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. The bacterium Sinorhizobium meliloti has a NAD(P)-malic enzyme (DME) and a NADP-malic enzyme (TME) and DME is required for symbiotic N2-fixation. To help understand the role of these enzymes, we examined growth, metabolic and transcriptional consequences resulting from the deletion of these enzymes. Few effects were observed upon growth with glucose, whereas growth with the gluconeogenic substrate, succinate, resulted in transcriptional and metabolic effects particularly in the dme mutant strains. When grown with succinate, DME mutant cells accumulated hexose sugar phosphates and trehalose, while TME mutants accumulated putrescine. Succinate-grown DME mutant cells also showed increased transcription of genes for gluconeogenesis and for pathways such as amino acid and fatty acid synthesis that divert metabolites away from the TCA cycle. These data suggested that, DME is required to regulate the levels of TCA cycle intermediates and that the activity of TME is insufficient to prevent the accumulation of TCA cycle intermediates in cells utilizing succinate as carbon source. Consistent with this, in short-1-3 hour incubations with succinate, dme mutant cells excreted large amounts of malate whereas little malate was excreted from tme or wild-type cells. These results support the suggestion that DME is required for N2-fixation in alfalfa because it is required for synthesis of pyruvate and acetyl-CoA and the rapid metabolism of C4-dicarboxylates supplied by the plant. RNA expression was measured for S. meliloti in exponential phase grown in MOPS (morpholinpropanesulfonic acid) buffered minimal media under 2 growth conditions: 1) 15 mM succinate, 2) 15 mM glucose; using wild type cells as well as dme and tme mutant strains (6 experiments, 2 replicates: total 12 samples)

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Gene expression analysis of third instar larvae ski3 mutants

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