scholarly journals Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

2021 ◽  
Author(s):  
Hongying Shen ◽  
Xiaojian Shi ◽  
Bryn Reinstadler ◽  
Hardik Shah ◽  
Tsz-Leung To ◽  
...  
Keyword(s):  
De Novo ◽  
Screening Strategy ◽  
Organelle Transport ◽  
Gxe Interaction ◽  
De Novo Purine Biosynthesis ◽  
Metabolic States ◽  
The Family ◽  
Genetic Perturbations ◽  
Gxe Interactions ◽  
Mitochondrial Glutathione

Abstract The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities are coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells were genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggest SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systematically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

scholarly journals Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

2021 ◽  
Author(s):  
Xiaojian Shi ◽  
Bryn Reinstadler ◽  
Hardik Shah ◽  
Tsz-Leung To ◽  
Katie Byrne ◽  
...  
Keyword(s):  
De Novo ◽  
Screening Strategy ◽  
Organelle Transport ◽  
Gxe Interaction ◽  
De Novo Purine Biosynthesis ◽  
Metabolic States ◽  
The Family ◽  
Genetic Perturbations ◽  
Gxe Interactions ◽  
Mitochondrial Glutathione

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states- glucose, galactose, OXPHOS inhibition, and absence of pyruvate-designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

scholarly journals Carbocyclic substrates for de novo purine biosynthesis.

1991 ◽  
Vol 266 (25) ◽  
pp. 16699-16702
Author(s):  
D.S. Liu ◽  
C.A. Caperelli
Keyword(s):  
De Novo ◽  
Purine Biosynthesis ◽  
De Novo Purine Biosynthesis

scholarly journals De Novo SNP Discovery and Genotyping of Iranian Pimpinella Species Using ddRAD Sequencing

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1342
Author(s):  
Shaghayegh Mehravi ◽  
Gholam Ali Ranjbar ◽  
Ghader Mirzaghaderi ◽  
Anita Alice Severn-Ellis ◽  
Armin Scheben ◽  
...  
Keyword(s):  
De Novo ◽  
Genetic Relationships ◽  
Nucleotide Polymorphisms ◽  
High Quality ◽  
Genomic Resources ◽  
High Quality Snps ◽  
The Family ◽  
Double Digestion ◽  
Flanking Sequences ◽  
Downstream Analysis

The species of Pimpinella, one of the largest genera of the family Apiaceae, are traditionally cultivated for medicinal purposes. In this study, high-throughput double digest restriction-site associated DNA sequencing technology (ddRAD-seq) was used to identify single nucleotide polymorphisms (SNPs) in eight Pimpinella species from Iran. After double-digestion with the enzymes HpyCH4IV and HinfI, a total of 334,702,966 paired-end reads were de novo assembled into 1,270,791 loci with an average of 28.8 reads per locus. After stringent filtering, 2440 high-quality SNPs were identified for downstream analysis. Analysis of genetic relationships and population structure, based on these retained SNPs, indicated the presence of three major groups. Gene ontology and pathway analysis were determined by using comparison SNP-associated flanking sequences with a public non-redundant database. Due to the lack of genomic resources in this genus, our present study is the first report to provide high-quality SNPs in Pimpinella based on a de novo analysis pipeline using ddRAD-seq. This data will enhance the molecular knowledge of the genus Pimpinella and will provide an important source of information for breeders and the research community to enhance breeding programs and support the management of Pimpinella genomic resources.

scholarly journals Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 359 ◽  
Author(s):  
Maria Romero-Gutiérrez ◽  
Carlos Santibáñez-López ◽  
Juana Jiménez-Vargas ◽  
Cesar Batista ◽  
Ernesto Ortiz ◽  
...  
Keyword(s):  
Serine Proteases ◽  
De Novo ◽  
Sequence Similarity ◽  
Scorpion Venom ◽  
Secretory Proteins ◽  
Host Defense Peptides ◽  
The Family ◽  
Pathogenesis Related ◽  
Molecular Masses

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as “other venom components”. A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

scholarly journals Stacks 2: Analytical Methods for Paired-end Sequencing Improve RADseq-based Population Genomics

2019 ◽  
Author(s):  
Nicolas C. Rochette ◽  
Angel G. Rivera-Colón ◽  
Julian M. Catchen
Keyword(s):  
Population Genomics ◽  
De Novo ◽  
Simulated Data ◽  
Natural World ◽  
Massively Parallel ◽  
Genetic Knowledge ◽  
Type Ii ◽  
Short Read Sequencing ◽  
The Family ◽  
Paired End Sequencing

AbstractFor half a century population genetics studies have put type II restriction endonucleases to work. Now, coupled with massively-parallel, short-read sequencing, the family of RAD protocols that wields these enzymes has generated vast genetic knowledge from the natural world. Here we describe the first software capable of using paired-end sequencing to derive short contigs from de novo RAD data natively. Stacks version 2 employs a de Bruijn graph assembler to build contigs from paired-end reads and overlap those contigs with the corresponding single-end loci. The new architecture allows all the individuals in a meta population to be considered at the same time as each RAD locus is processed. This enables a Bayesian genotype caller to provide precise SNPs, and a robust algorithm to phase those SNPs into long haplotypes – generating RAD loci that are 400-800bp in length. To prove its recall and precision, we test the software with simulated data and compare reference-aligned and de novo analyses of three empirical datasets. We show that the latest version of Stacks is highly accurate and outperforms other software in assembling and genotyping paired-end de novo datasets.

Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine

1980 ◽  
Vol 26 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Stephen A. Morse ◽  
Lynne Bartenstein
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Growth Medium ◽  
De Novo ◽  
Defined Medium ◽  
Purine Metabolism ◽  
Purine Biosynthesis ◽  
Free Medium ◽  
De Novo Purine Biosynthesis ◽  
Reduced Rate

Strains isolated from disseminated gonococcal infections often require hypoxanthine for growth. The biochemical bases for the requirement for hypoxanthine in strains isolated from both disseminated (Ile−Val−Arg−Hyx−Ura−phenotype) and non-disseminated (Hyx−phenotype) infections were compared. The requirement for hypoxanthine was dependent upon the composition of the growth medium. In a complete defined medium, hypoxanthine was replaced by a mixture of adenine and guanine but not by either purine alone. The addition of adenine alone inhibited gonococcal growth. This inhibition was reversed by the addition of guanine and most likely resulted from an inhibition of de novo purine biosynthesis. In a histidine-free medium, adenine replaced the hypoxanthine requirement in Ile−Val−Arg−Hyx−Ura− strains. Adenine did not replace the hypoxanthine requirement in Hyx− strains. The Ile−Val−Arg−Hyx−Ura− strains exhibited a markedly reduced rate of de novo purine biosynthesis while Hyx− strains were blocked in this pathway. In vivo concentrations of purines are important factors which may limit the intracellular or extracellular growth of these strains.

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